CN113278052B - Preparation method of recombinant Protein A and affinity chromatography medium - Google Patents

Preparation method of recombinant Protein A and affinity chromatography medium Download PDF

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CN113278052B
CN113278052B CN202110525261.5A CN202110525261A CN113278052B CN 113278052 B CN113278052 B CN 113278052B CN 202110525261 A CN202110525261 A CN 202110525261A CN 113278052 B CN113278052 B CN 113278052B
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CN113278052A (en
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张洪
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Boglong Zhejiang Biotechnology Co ltd
Pinghu Youpu Biotechnology Co ltd
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Abstract

The invention discloses a recombinant ProteinA protein and a preparation method of an affinity chromatography medium, wherein the protein is a mutant of a parent immunoglobulin-binding protein defined by SEQ ID No 1 and SEQ ID No 2; the preparation method of the affinity chromatography medium comprises the following steps: s1, synthesizing genes of recombinant Protein A, S2, respectively constructing expression vectors by using the genes synthesized in S1, S3, culturing, expressing and purifying to obtain a recombinant Protein A solution of SEQ ID corresponding to the sequence, and S4, preparing to obtain an affinity chromatography medium by using the recombinant Protein A. Has the advantages that: the invention greatly improves the chemical stability of Protein A in alkali liquor by redesigning an amino acid sequence, thereby providing the Protein A with specific binding action to an antibody, the Protein A has good chemical stability under an alkaline condition, the binding capacity of a recombinant Protein A affinity chromatography medium is obviously improved, the Protein A can tolerate the in-situ cleaning of 0.5-1.0M NaOH, and the IgG binding capacity is 60-90 mg/ml.

Description

一种重组Protein A蛋白及亲和层析介质的制备方法A kind of preparation method of recombinant Protein A protein and affinity chromatography medium

技术领域technical field

本发明涉及蛋白工程技术领域，具体来说，涉及一种重组Protein A蛋白及亲和层析介质的制备方法。The invention relates to the technical field of protein engineering, in particular to a method for preparing a recombinant Protein A protein and an affinity chromatography medium.

背景技术Background technique

抗体类药物对疾病具有优异的疗效和安全性，已经在肿瘤类、心血管类，特别是自身免疫性疾病的治疗过程中逐步凸显重要作用，是目前生物类药物中增长率最高的一类药物。得益于高表达细胞系的构建、培养基的改良以及反应器规模不断扩大，单克隆抗体药物技术在过去几十年间得到迅猛发展。而单克隆抗体药物的制备过程中，抗体的纯化工艺尤为重要，其中，对抗体的捕获起着关键作用。而Protein A亲和层析介质是目前抗体捕集和纯化过程中使用最为广泛以及技术最为成熟的一种方法，70％以上的抗体药物纯化工艺都运用该项技术。Antibody drugs have excellent efficacy and safety on diseases, and have gradually played an important role in the treatment of tumors, cardiovascular diseases, especially autoimmune diseases, and are currently the class of drugs with the highest growth rate among biological drugs. . Thanks to the construction of high-expressing cell lines, the improvement of media, and the continuous expansion of reactor scale, the technology of monoclonal antibody drugs has developed rapidly in the past few decades. In the preparation of monoclonal antibody drugs, the purification process of the antibody is particularly important, and the capture of the antibody plays a key role. The Protein A affinity chromatography medium is currently the most widely used and most mature method in the process of antibody capture and purification, and more than 70% of antibody drug purification processes use this technology.

但是当前常用Protein A亲和层析介质在抗体药物纯化过程中有以下缺陷，如：载量较低；抗体回收率较低，洗脱过程中，洗脱液的pH值较低，导致部分抗体发生变性，降低了抗体的回收率；配基脱落率高，并且存在成本高、寿命短和操作条件苛刻等问题。另外，为了降低成本，Protein A亲和层析介质的重复使用是必需的。抗体洗脱后，Protein A亲和层析介质需经严格验证的、标准的在位清洗(CIP)以除去介质上的杂蛋白、残留抗体及其聚合物、内毒素等杂质。而在位清洗的条件(0.5mol/L至1.0mol/L氢氧化纳溶液处理)对于Protein A亲和层析介质而言是非常苛刻的，其直接结果就是Protein A高级结构的破坏、介质吸附能力的下降和寿命的降低。现有技术中，Protein A亲和层析介质在碱性条件下稳定性较为不理想，从而影响其应用。因此，对Protein A亲和层析介质性能的提高己成为单抗药物制备的主要技术“瓶颈”和亟待突破的重点研究内容。However, the currently commonly used Protein A affinity chromatography medium has the following defects in the process of antibody drug purification, such as: low loading; low antibody recovery rate, and the pH value of the eluate is low during the elution process, resulting in some antibodies Denaturation occurs, reducing the recovery rate of the antibody; the ligand shedding rate is high, and there are problems such as high cost, short life and harsh operating conditions. In addition, in order to reduce costs, the reuse of Protein A affinity chromatography media is required. After antibody elution, the Protein A affinity chromatography medium needs to undergo rigorously validated, standard cleaning-in-place (CIP) to remove impurities such as impurity proteins, residual antibodies and their polymers, and endotoxins on the medium. The conditions of cleaning in place (0.5mol/L to 1.0mol/L sodium hydroxide solution treatment) are very harsh for Protein A affinity chromatography medium, and the direct result is the destruction of Protein A's advanced structure, medium adsorption Decreased capacity and reduced lifespan. In the prior art, the stability of the Protein A affinity chromatography medium under alkaline conditions is relatively unsatisfactory, thereby affecting its application. Therefore, the improvement of the performance of Protein A affinity chromatography medium has become the main technical "bottleneck" for the preparation of monoclonal antibodies and a key research content that needs to be broken through.

发明内容SUMMARY OF THE INVENTION

针对相关技术中的问题，本发明提出一种配置方法，以克服现有相关技术所存在的上述技术问题。In view of the problems in the related art, the present invention proposes a configuration method to overcome the above-mentioned technical problems existing in the related art.

为此，本发明采用的具体技术方案如下：For this reason, the concrete technical scheme that the present invention adopts is as follows:

根据本发明的一个方面，提供了一种重组Protein A蛋白，所述的重组Protein A蛋白是由包含SEQ ID No 1或SEQ ID No 2指定的E或D结构域的突变体。According to one aspect of the present invention, there is provided a recombinant Protein A protein, which is a mutant comprising the E or D domain specified by SEQ ID No 1 or SEQ ID No 2.

进一步的，所述SEQ ID No 1指定E结构域的突变体中2位，8位，42位的谷氨酰胺已经突变为选自酪氨酸、天冬氨酸、苯丙氨酸、异亮氨酸的氨基酸；Further, the glutamine at position 2, position 8 and position 42 in the mutant of the specified E domain of SEQ ID No 1 has been mutated to be selected from tyrosine, aspartic acid, phenylalanine, isoleucine amino acid;

所述SEQ ID No 1指定E结构域的突变体中16位的天冬酰胺已经突变为选自苏氨酸、丝氨酸、谷氨酸、精氨酸、酪氨酸、亮氨酸、异亮氨酸的氨基酸；The asparagine at position 16 in the mutant of the specified E domain of SEQ ID No 1 has been mutated to be selected from the group consisting of threonine, serine, glutamic acid, arginine, tyrosine, leucine, isoleucine acid amino acids;

所述SEQ ID No 1指定E结构域的突变体中22位，39位的甘氨酸已经突变为选自丙氨酸、丝氨酸的氨基酸。Said SEQ ID No 1 specifies that the glycine at position 22 and position 39 in the mutant of the E domain has been mutated to an amino acid selected from alanine and serine.

进一步的，所述SEQ ID No 2指定D结构域的突变体中6位，9位，26位的天冬酰胺已经突变为选自苏氨酸、丝氨酸、谷氨酸、精氨酸、酪氨酸、亮氨酸、异亮氨酸的氨基酸；Further, the asparagine at position 6, position 9 and position 26 in the mutant of the specified D domain of SEQ ID No 2 have been mutated to be selected from threonine, serine, glutamic acid, arginine, tyrosine Amino acids of acid, leucine, isoleucine;

所述SEQ ID No 2指定D结构域的突变体中12位的谷氨酰胺已经突变为选自酪氨酸、天冬氨酸、苯丙氨酸、异亮氨酸的氨基酸；The glutamine at position 12 in the mutant of the specified D domain of SEQ ID No 2 has been mutated to an amino acid selected from tyrosine, aspartic acid, phenylalanine, and isoleucine;

所述SEQ ID No 2指定D结构域的突变体中18位、56位的谷氨酸已经突变为选自甘氨酸、亮氨酸、精氨酸、天冬氨酸、苏氨酸、组氨酸、丝氨酸、苯丙氨酸的氨基酸；The glutamic acid at positions 18 and 56 in the mutant of the specified D domain of SEQ ID No 2 has been mutated to be selected from glycine, leucine, arginine, aspartic acid, threonine, histidine , amino acids of serine and phenylalanine;

所述SEQ ID No 2指定D结构域的突变体中32位、49位的甘氨酸已经突变为选自丙氨酸、丝氨酸的氨基酸。The glycines at positions 32 and 49 in the mutant of the specified D domain of SEQ ID No 2 have been mutated to amino acids selected from alanine and serine.

进一步的，所述突变选自：Further, the mutation is selected from:

Q2Y,Q2D,Q2F,Q2I,Q8Y,Q8D,Q8F,Q8I,Q42Y,Q42D,Q42F,Q42I,N16T,N16S,N16E,N16R,N16Y,N16L,N16I,G22A,G22S,G39A,G39S；且包含以下序列SEQ ID No 3、SEQ ID No4、SEQ ID No 5、SEQ ID No 6、SEQ ID No 7、SEQ ID No 8、SEQ ID No 9、SEQ ID No 10、SEQ ID No 11、SEQ ID No 12、SEQ ID No 13、SEQ ID No 14、SEQ ID No 15、SEQ ID No16、SEQ ID No 17、SEQ ID No 18、SEQ ID No 19、SEQ ID No 20、SEQ ID No 21、SEQ ID No22。Q2Y,Q2D,Q2F,Q2I,Q8Y,Q8D,Q8F,Q8I,Q42Y,Q42D,Q42F,Q42I,N16T,N16S,N16E,N16R,N16Y,N16L,N16I,G22A,G22S,G39A,G39S; and contains the following sequence SEQ ID No 3, SEQ ID No4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 12 ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21, SEQ ID No22.

进一步的，所述突变选自：Further, the mutation is selected from:

N6T,N6S,N6E,N6R,N6Y,N6L,N6I,N9T,N9S,N9E,N9R,N9Y,N9L,N9I,N26T,N26S,N26E,N26R,N26Y,N26L,N26I,Q12Y,Q12D,Q12F,Q12I,E18G,E18L,E18R,E18D,E18T,E18H,E18S,E18F,E56G,E56L,E56R,E56D,E56T,E56H,E56S,E56F,G32A,G32S,G49A,G49S；且包含以下序列SEQ ID No 23、SEQ ID No 24、SEQ ID No 25、SEQ ID No 26、SEQ ID No 27、SEQID No 28、SEQ ID No 29、SEQ ID No 30、SEQ ID No 31、SEQ ID No 32、SEQ ID No 33、SEQID No 34、SEQ ID No 35、SEQ ID No 36、SEQ ID No 37、SEQ ID No 38、SEQ ID No 39、SEQID No 40、SEQ ID No 41、SEQ ID No 42。N6T,N6S,N6E,N6R,N6Y,N6L,N6I,N9T,N9S,N9E,N9R,N9Y,N9L,N9I,N26T,N26S,N26E,N26R,N26Y,N26L,N26I,Q12Y,Q12D,Q12F,Q12I, E18G, E18L, E18R, E18D, E18T, E18H, E18S, E18F, E56G, E56L, E56R, E56D, E56T, E56H, E56S, E56F, G32A, G32S, G49A, G49S; ID No 24, SEQ ID No 25, SEQ ID No 26, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 34. SEQ ID No 35, SEQ ID No 36, SEQ ID No 37, SEQ ID No 38, SEQ ID No 39, SEQ ID No 40, SEQ ID No 41, SEQ ID No 42.

进一步的，所述突变体包含4-8个重复单元，且包含以下序列SEQ ID No43、SEQ IDNo 44、SEQ ID No 45、SEQ ID No 46、SEQ ID No 47、SEQ ID No48、SEQ ID No 49、SEQ IDNo 50、SEQ ID No 51、SEQ ID No 52。Further, the mutant comprises 4-8 repeating units, and comprises the following sequences: SEQ ID No43, SEQ ID No 44, SEQ ID No 45, SEQ ID No 46, SEQ ID No 47, SEQ ID No48, SEQ ID No 49 , SEQ ID No 50, SEQ ID No 51, SEQ ID No 52.

进一步的，所述重组Protein A蛋白C端包含一个半胱氨酸残基的偶联基团。Further, the C-terminus of the recombinant Protein A protein comprises a coupling group of cysteine residues.

根据本发明的另一个方面，提供了一种亲和层析介质的制备方法，该亲和层析介质的配基为以上所述的重组Protein A蛋白，该亲和层析介质的制备方法包括以下步骤：According to another aspect of the present invention, there is provided a preparation method of an affinity chromatography medium, wherein the ligand of the affinity chromatography medium is the recombinant Protein A protein described above, and the preparation method of the affinity chromatography medium comprises: The following steps:

S1、合成重组Protein A蛋白的基因；S1. Gene for synthesizing recombinant Protein A protein;

S2、利用所述S1中合成的基因分别构建表达载体；S2, utilize the genes synthesized in the S1 to construct expression vectors respectively;

S3、培养并表达纯化得到序列对应的SEQ ID的重组Protein A溶液；S3, cultivate and express and purify the recombinant Protein A solution that obtains the SEQ ID corresponding to the sequence;

S4、利用所述重组Protein A制备得到亲和层析介质。S4, using the recombinant Protein A to prepare an affinity chromatography medium.

进一步的，所述S2中的表达载体为PET23a。Further, the expression vector in S2 is PET23a.

进一步的，所述S3中培养并表达纯化得到对应序列的重组Protein A溶液具体包括以下步骤：Further, the recombinant Protein A solution obtained by culturing and expressing and purifying the corresponding sequence in the S3 specifically includes the following steps:

S31、利用单个重组质粒转化到大肠杆菌，并通过LB液体培养基进行发酵培养并进行诱导表达；S31, transforming into Escherichia coli with a single recombinant plasmid, and fermenting and inducing expression in LB liquid medium;

S32、发酵后，收集菌体并采用热裂解的方式破坏细胞壁将表达产物释放出来并离心分离；S32, after fermentation, collect thalline and destroy the cell wall by means of thermal cracking to release the expression product and separate by centrifugation;

S33、将分离液体经过IgG亲和介质进行纯化。S33, purifying the separation liquid through an IgG affinity medium.

本发明的有益效果为：The beneficial effects of the present invention are:

1)、本发明通过对氨基酸序列重新设计，大大提高Protein A在碱液中的化学稳定性，从而提供一种对抗体具有特异性结合作用的Protein A，其在碱性条件下具有良好的化学稳定性，重组Protein A亲和层析介质结合载量显著提高，可耐受0.5-1.0M NaOH在位清洗，IgG结合载量60-90mg/ml。1), the present invention greatly improves the chemical stability of Protein A in lye by redesigning the amino acid sequence, thereby providing a kind of Protein A with specific binding effect to antibodies, which has good chemical stability under alkaline conditions. Stability, the binding capacity of recombinant Protein A affinity chromatography medium is significantly increased, it can withstand 0.5-1.0M NaOH wash-in-place, and the binding capacity of IgG is 60-90 mg/ml.

2)、本发明还提供了一种利用该突变结构域的Protein A为配基的亲和层析介质制备方法，与以结构域Z及其寡聚体为配基的亲和层析介质相比，本发明制备的亲和层析介质的抗体结合能力更为优异。2) The present invention also provides a method for preparing an affinity chromatography medium using Protein A of the mutant domain as a ligand, which is in phase with the affinity chromatography medium using the domain Z and its oligomer as a ligand. In comparison, the antibody binding ability of the affinity chromatography medium prepared by the present invention is more excellent.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

图1是根据本发明实施例中亲和层析介质的制备方法的流程图；1 is a flow chart of a method for preparing an affinity chromatography medium according to an embodiment of the present invention;

图2是根据本发明实施例中由SEQ ID NO 1、SEQ ID NO 2、SEQ ID NO 53定义的Protein A蛋白结构域的比对示意图；Figure 2 is a schematic diagram of the alignment of the Protein A protein domains defined by SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 53 according to an embodiment of the present invention;

图3是根据本发明中实施例一的对0.5M NaOH碱稳定性的结果示意图；Fig. 3 is the result schematic diagram according to the embodiment one of the present invention to 0.5M NaOH alkali stability;

图4是根据本发明中实施例二的对1.0M NaOH碱稳定性的结果示意图。4 is a schematic diagram of the results of the alkali stability to 1.0M NaOH according to Example 2 of the present invention.

具体实施方式Detailed ways

为进一步说明各实施例，本发明提供有附图，这些附图为本发明揭露内容的一部分，其主要用以说明实施例，并可配合说明书的相关描述来解释实施例的运作原理，配合参考这些内容，本领域普通技术人员应能理解其他可能的实施方式以及本发明的优点，图中的组件并未按比例绘制，而类似的组件符号通常用来表示类似的组件。In order to further illustrate the various embodiments, the present invention provides accompanying drawings, which are part of the disclosure of the present invention, and are mainly used to illustrate the embodiments, and can be used in conjunction with the relevant descriptions in the specification to explain the operation principles of the embodiments. For these, those of ordinary skill in the art will understand other possible implementations and the advantages of the present invention. Components in the figures are not drawn to scale, and similar component symbols are generally used to represent similar components.

根据本发明的实施例，提供了一种重组ProteinA蛋白及亲和层析介质的制备方法。According to the embodiments of the present invention, a preparation method of recombinant Protein A protein and an affinity chromatography medium is provided.

根据本发明的一个方面，提供了一种重组ProteinA蛋白，该蛋白为SEQ ID No 1、SEQ ID No 2所定义的亲本免疫球蛋白-结合蛋白的突变体。According to one aspect of the present invention, a recombinant ProteinA protein is provided, which is a mutant of the parent immunoglobulin-binding protein defined by SEQ ID No 1 and SEQ ID No 2.

Protein A蛋白结构域主要由E、D、A、B、C或者结构域Z及Zvar蛋白结构域的突变体组成，其中SEQ ID No 1(E domain)、SEQ ID No 2(D domain)、SEQ ID No 53(Zvar)见图2所示。本实施例保护对象由SEQ ID No 1(D domain)和SEQ ID No 2(E domain)的突变体构成，突出其耐碱性和结合载量方面的显著效果。Protein A protein domain is mainly composed of E, D, A, B, C or mutants of domain Z and Zvar protein domain, wherein SEQ ID No 1 (E domain), SEQ ID No 2 (D domain), SEQ ID No 2 (D domain), ID No 53 (Zvar) is shown in Figure 2. The protection object of this example is composed of mutants of SEQ ID No 1 (D domain) and SEQ ID No 2 (E domain), which highlight the remarkable effects on alkali resistance and binding capacity.

具体的，所述的重组Protein A蛋白是由包含SEQ ID No 1或SEQ ID No 2指定的E或D结构域的突变体。Specifically, the recombinant Protein A protein is a mutant comprising the E or D domain specified by SEQ ID No 1 or SEQ ID No 2.

其中，所述SEQ ID No 1指定E结构域的突变体中2位，8位，42位的谷氨酰胺已经突变为选自酪氨酸、天冬氨酸、苯丙氨酸、异亮氨酸的氨基酸；Wherein, the glutamine at position 2, position 8 and position 42 in the mutant of the specified E domain of SEQ ID No 1 has been mutated to be selected from tyrosine, aspartic acid, phenylalanine, isoleucine acid amino acids;

所述SEQ ID No 1指定E结构域的突变体中16位的天冬酰胺已经突变为选自苏氨酸、丝氨酸、谷氨酸、精氨酸、酪氨酸、亮氨酸、异亮氨酸的氨基酸；The asparagine at position 16 in the mutant of the specified E domain of SEQ ID No 1 has been mutated to be selected from the group consisting of threonine, serine, glutamic acid, arginine, tyrosine, leucine, isoleucine acid amino acids;

所述SEQ ID No 1指定E结构域的突变体中22位，39位的甘氨酸已经突变为选自丙氨酸、丝氨酸的氨基酸；Said SEQ ID No 1 specifies that the glycine at position 22 and position 39 in the mutant of the E structural domain has been mutated to an amino acid selected from alanine and serine;

根据上述所述的突变体，其中，所述突变选自：The mutant according to the above, wherein the mutation is selected from:

Q2Y,Q2D,Q2F,Q2I,Q8Y,Q8D,Q8F,Q8I,Q42Y,Q42D,Q42F,Q42I,N16T,N16S,N16E,N16R,N16Y,N16L,N16I,G22A,G22S,G39A,G39S；且包含以下序列SEQ ID No 3、SEQ ID No4、SEQ ID No 5、SEQ ID No 6、SEQ ID No 7、SEQ ID No 8、SEQ ID No 9、SEQ ID No 10、SEQ ID No 11、SEQ ID No 12、SEQ ID No 13、SEQ ID No 14、SEQ ID No 15、SEQ ID No16、SEQ ID No 17、SEQ ID No 18、SEQ ID No 19、SEQ ID No 20、SEQ ID No 21、SEQ ID No22。Q2Y,Q2D,Q2F,Q2I,Q8Y,Q8D,Q8F,Q8I,Q42Y,Q42D,Q42F,Q42I,N16T,N16S,N16E,N16R,N16Y,N16L,N16I,G22A,G22S,G39A,G39S; and contains the following sequence SEQ ID No 3, SEQ ID No4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 12 ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 18, SEQ ID No 19, SEQ ID No 20, SEQ ID No 21, SEQ ID No22.

其中，所述SEQ ID No 2指定D结构域的突变体中6位，9位，26位的天冬酰胺已经突变为选自苏氨酸、丝氨酸、谷氨酸、精氨酸、酪氨酸、亮氨酸、异亮氨酸的氨基酸；Wherein, the asparagine at position 6, position 9 and position 26 in the mutant of the specified D domain of SEQ ID No 2 have been mutated to be selected from threonine, serine, glutamic acid, arginine, tyrosine , leucine, isoleucine amino acids;

所述SEQ ID No 2指定D结构域的突变体中12位的谷氨酰胺已经突变为选自酪氨酸、天冬氨酸、苯丙氨酸、异亮氨酸的氨基酸；The glutamine at position 12 in the mutant of the specified D domain of SEQ ID No 2 has been mutated to an amino acid selected from tyrosine, aspartic acid, phenylalanine, and isoleucine;

所述SEQ ID No 2指定D结构域的突变体中18位、56位的谷氨酸已经突变为选自甘氨酸、亮氨酸、精氨酸、天冬氨酸、苏氨酸、组氨酸、丝氨酸、苯丙氨酸的氨基酸；The glutamic acid at positions 18 and 56 in the mutant of the specified D domain of SEQ ID No 2 has been mutated to be selected from glycine, leucine, arginine, aspartic acid, threonine, histidine , amino acids of serine and phenylalanine;

所述SEQ ID No 2指定D结构域的突变体中32位、49位的甘氨酸已经突变为选自丙氨酸、丝氨酸的氨基酸；The glycines at positions 32 and 49 in the mutant of the specified D domain of SEQ ID No 2 have been mutated to amino acids selected from alanine and serine;

根据上述所述的突变体，其中，所述突变选自：The mutant according to the above, wherein the mutation is selected from:

N6T,N6S,N6E,N6R,N6Y,N6L,N6I,N9T,N9S,N9E,N9R,N9Y,N9L,N9I,N26T,N26S,N26E,N26R,N26Y,N26L,N26I,Q12Y,Q12D,Q12F,Q12I,E18G,E18L,E18R,E18D,E18T,E18H,E18S,E18F,E56G,E56L,E56R,E56D,E56T,E56H,E56S,E56F,G32A,G32S,G49A,G49S；且包含以下序列SEQ ID No 23、SEQ ID No 24、SEQ ID No 25、SEQ ID No 26、SEQ ID No 27、SEQID No 28、SEQ ID No 29、SEQ ID No 30、SEQ ID No 31、SEQ ID No 32、SEQ ID No 33、SEQID No 34、SEQ ID No 35、SEQ ID No 36、SEQ ID No 37、SEQ ID No 38、SEQ ID No 39、SEQID No 40、SEQ ID No 41、SEQ ID No 42。N6T,N6S,N6E,N6R,N6Y,N6L,N6I,N9T,N9S,N9E,N9R,N9Y,N9L,N9I,N26T,N26S,N26E,N26R,N26Y,N26L,N26I,Q12Y,Q12D,Q12F,Q12I, E18G, E18L, E18R, E18D, E18T, E18H, E18S, E18F, E56G, E56L, E56R, E56D, E56T, E56H, E56S, E56F, G32A, G32S, G49A, G49S; ID No 24, SEQ ID No 25, SEQ ID No 26, SEQ ID No 27, SEQ ID No 28, SEQ ID No 29, SEQ ID No 30, SEQ ID No 31, SEQ ID No 32, SEQ ID No 33, SEQ ID No 34. SEQ ID No 35, SEQ ID No 36, SEQ ID No 37, SEQ ID No 38, SEQ ID No 39, SEQ ID No 40, SEQ ID No 41, SEQ ID No 42.

在一个实施例中，所述突变体包含4-8个重复单元，且包含以下序列SEQ ID No43、SEQ ID No 44、SEQ ID No 45、SEQ ID No 46、SEQ ID No 47、SEQ ID No 48、SEQ ID No49、SEQ ID No 50、SEQ ID No 51、SEQ ID No 52。In one embodiment, the mutant comprises 4-8 repeat units and comprises the following sequences SEQ ID No 43, SEQ ID No 44, SEQ ID No 45, SEQ ID No 46, SEQ ID No 47, SEQ ID No 48 , SEQ ID No49, SEQ ID No 50, SEQ ID No 51, SEQ ID No 52.

在一个实施例中，所述重组Protein A蛋白C端包含一个半胱氨酸残基的偶联基团。In one embodiment, the C-terminus of the recombinant Protein A protein comprises a coupling group of cysteine residues.

在一些实施方案中，所述重组Protein A蛋白包含选自以下的序列或基本由选自以下的序列组成：In some embodiments, the recombinant Protein A protein comprises or consists essentially of a sequence selected from the group consisting of:

SEQ ID No 3(Q2Y)SEQ ID No 3 (Q2Y)

AYQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KAYQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 4(Q2D)SEQ ID No 4 (Q2D)

ADQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KADQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 5(Q2F)SEQ ID No 5 (Q2F)

AFQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KAFQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 6(Q2I)SEQ ID No 6 (Q2I)

AIQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KAIQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 7(Q2Y,Q8D)SEQ ID No 7 (Q2Y, Q8D)

AYQNAFYDVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KAYQNAFYDVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 8(Q2Y,Q42F)SEQ ID No 8 (Q2Y, Q42F)

AYQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AFKLNDSQAP KAYQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AFKLNDSQAP K

SEQ ID No 9(Q2I,N16T)SEQ ID No 9 (Q2I, N16T)

AIQNAFYQVL NMPNLTADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KAIQNAFYQVL NMPNLTADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 10(Q8F,G22A)SEQ ID No 10 (Q8F, G22A)

AQQNAFYFVL NMPNLNADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP KAQQNAFYFVL NMPNLNADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 11(Q2D,Q42F,N16S)SEQ ID No 11 (Q2D, Q42F, N16S)

ADQNAFYQVL NMPNLSADQR NGFIQSLKDD PSQSANVLGE AFKLNDSQAP KADQNAFYQVL NMPNLSADQR NGFIQSLKDD PSQSANVLGE AFKLNDSQAP K

SEQ ID No 12(Q2I,Q8F,G22A)SEQ ID No 12 (Q2I, Q8F, G22A)

AIQNAFYFVL NMPNLNADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP KAIQNAFYFVL NMPNLNADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 13(Q8D,N16R,G22A)SEQ ID No 13 (Q8D, N16R, G22A)

AQQNAFYDVL NMPNLRADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP KAQQNAFYDVL NMPNLRADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 14(G22S,G39S,Q42D)SEQ ID No 14 (G22S, G39S, Q42D)

AQQNAFYQVL NMPNLNADQR NSFIQSLKDD PSQSANVLSE ADKLNDSQAP KAQQNAFYQVL NMPNLNADQR NSFIQSLKDD PSQSANVLSE ADKLNDSQAP K

SEQ ID No 15(Q8F,N16E,G39A,Q42D)SEQ ID No 15 (Q8F, N16E, G39A, Q42D)

AQQNAFYFVL NMPNLEADQR NGFIQSLKDD PSQSANVLAE ADKLNDSQAP KAQQNAFYFVL NMPNLEADQR NGFIQSLKDD PSQSANVLAE ADKLNDSQAP K

SEQ ID No 16(Q2I,Q8D,Q42F,N16Y)SEQ ID No 16 (Q2I, Q8D, Q42F, N16Y)

AQQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP KAQQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

SEQ ID No 17(Q8Y,N16L,G39A,Q42I)SEQ ID No 17 (Q8Y, N16L, G39A, Q42I)

AQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP KAQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP K

SEQ ID No 18(Q2I,Q8D,N16I,G39A,Q42F)SEQ ID No 18 (Q2I, Q8D, N16I, G39A, Q42F)

AIQNAFYDVL NMPNLIADQR NGFIQSLKDD PSQSANVLAE AFKLNDSQAP KAIQNAFYDVL NMPNLIADQR NGFIQSLKDD PSQSANVLAE AFKLNDSQAP K

SEQ ID No 19(Q2D,Q8F,N16T,G22A,G39S)SEQ ID No 19 (Q2D, Q8F, N16T, G22A, G39S)

ADQNAFYFVL NMPNLTADQR NAFIQSLKDD PSQSANVLSE AQKLNDSQAP KADQNAFYFVL NMPNLTADQR NAFIQSLKDD PSQSANVLSE AQKLNDSQAP K

SEQ ID No 20(Q8Y,N16L,G22A,G39S,Q42I)SEQ ID No 20 (Q8Y, N16L, G22A, G39S, Q42I)

AQQNAFYYVL NMPNLLADQR NAFIQSLKDD PSQSANVLSE AIKLNDSQAP KAQQNAFYYVL NMPNLLADQR NAFIQSLKDD PSQSANVLSE AIKLNDSQAP K

SEQ ID No 21(Q2F,Q8Y,N16R,G22A,G39S,Q42D)SEQ ID No 21 (Q2F, Q8Y, N16R, G22A, G39S, Q42D)

AFQNAFYYVL NMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP KAFQNAFYYVL NMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K

SEQ ID No 22(Q2Y,Q8D,N16L,G22S,G39A,Q42F)SEQ ID No 22 (Q2Y, Q8D, N16L, G22S, G39A, Q42F)

AYQNAFYDVL NMPNLLADQR NSFIQSLKDD PSQSANVLAE AFKLNDSQAP KAYQNAFYDVL NMPNLLADQR NSFIQSLKDD PSQSANVLAE AFKLNDSQAP K

SEQ ID No 23(N6T)SEQ ID No 23 (N6T)

ADAQQTKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQTKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 24(N6S)SEQ ID No 24 (N6S)

ADAQQSKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQSKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 25(N6E)SEQ ID No 25 (N6E)

ADAQQEKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQEKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 26(N6R)SEQ ID No 26 (N6R)

ADAQQRKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQRKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 27(N6T,N9S)SEQ ID No 27 (N6T, N9S)

ADAQQTKFSK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQTKFSK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 28(N6S,N9Y)SEQ ID No 28 (N6S, N9Y)

ADAQQSKFYK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQSKFYK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 29(N6E,N26L)SEQ ID No 29 (N6E, N26L)

ADAQQEKFNK DQQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQEKFNK DQQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 30(N6T,N9S,N26E)SEQ ID No 30 (N6T, N9S, N26E)

ADAQQTKFSK DQQSAFYEIL NMPNLEEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQTKFSK DQQSAFYEIL NMPNLEEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 31(N6S,N9Y,Q12D)SEQ ID No 31 (N6S, N9Y, Q12D)

ADAQQSKFYK DDQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQSKFYK DDQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 32(N6E,Q12F,N26L)SEQ ID No 32 (N6E, Q12F, N26L)

ADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 33(N6T,N9S,N26E,E18L)SEQ ID No 33 (N6T, N9S, N26E, E18L)

ADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 34(N6S,N9Y,Q12D,E18R)SEQ ID No 34 (N6S, N9Y, Q12D, E18R)

ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No35(N6E,N26L,Q12F,E56D)SEQ ID No35 (N6E, N26L, Q12F, E56D)

ADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNDSQAP KADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNDSQAP K

SEQ ID No 36(N6R,N26I,E18G,E56H)SEQ ID No 36 (N6R, N26I, E18G, E56H)

ADAQQRKFNK DQQSAFYGIL NMPNLIEGQR NGFIQSLKDD PSQSTNVLGE AKKLNHSQAP KADAQQRKFNK DQQSAFYGIL NMPNLIEGQR NGFIQSLKDD PSQSTNVLGE AKKLNHSQAP K

SEQ ID No37(N6T,N9S,N26E,E18L,G32A)SEQ ID No37 (N6T, N9S, N26E, E18L, G32A)

ADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NAFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NAFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 38(N6S,N9Y,Q12D,E18R,G32S)SEQ ID No 38 (N6S, N9Y, Q12D, E18R, G32S)

ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 39(N6E,N26L,Q12F,E56D,G49A)SEQ ID No 39 (N6E, N26L, Q12F, E56D, G49A)

ADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLAE AKKLNDSQAP KADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLAE AKKLNDSQAP K

SEQ ID No 40(N6S,N9Y,Q12D,E18R,N26I,G32S)SEQ ID No 40 (N6S, N9Y, Q12D, E18R, N26I, G32S)

ADAQQSKFYK DDQSAFYRIL NMPNLIEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQSKFYK DDQSAFYRIL NMPNLIEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 41(N6E,N26L,Q12F,E18H,E56D,G49A)SEQ ID No 41 (N6E, N26L, Q12F, E18H, E56D, G49A)

ADAQQEKFNK DFQSAFYHIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLAE AKKLNDSQAP KADAQQEKFNK DFQSAFYHIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLAE AKKLNDSQAP K

SEQ ID No 42(N6T,N9S,N26E,E18L,G32A,G49S)SEQ ID No 42 (N6T, N9S, N26E, E18L, G32A, G49S)

ADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NAFIQSLKDD PSQSTNVLGE AKKLNESQAP KADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NAFIQSLKDD PSQSTNVLGE AKKLNESQAP K

SEQ ID No 43(G22S,G39S,Q42D)4SEQ ID No 43 (G22S, G39S, Q42D) 4

AQQNAFYQVL NMPNLNADQR NSFIQSLKDD PSQSANVLSE ADKLNDSQAP KAQQNAFYQVLNMPNLNADQ RNSFIQSLKD DPSQSANVLS EADKLNDSQA PKAQQNAFYQ VLNMPNLNAD QRNSFIQSLKDDPSQSANVL SEADKLNDSQ APKAQQNAFY QVLNMPNLNA DQRNSFIQSL KDDPSQSANV LSEADKLNDSQAPKAQQNAFYQVL NMPNLNADQR NSFIQSLKDD PSQSANVLSE ADKLNDSQAP KAQQNAFYQVLNMPNLNADQ RNSFIQSLKD DPSQSANVLS EADKLNDSQA PKAQQNAFYQ VLNMPNLNAD QRNSFIQSLKDDPSQSANVL SEADKLNDSQ APKAQQNAFY QVLNMPNLNA DQRNSFIQSL KDDPSQSANV LSEADKLNDSQAPK

SEQ ID No 44(Q8Y,N16L,G39A,Q42I)4SEQ ID No 44 (Q8Y, N16L, G39A, Q42I) 4

AQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP KAQQNAFYYVLNMPNLLADQ RNGFIQSLKD DPSQSANVLA EAIKLNDSQA PKAQQNAFYY VLNMPNLLAD QRNGFIQSLKDDPSQSANVL AEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNGFIQSL KDDPSQSANV LAEAIKLNDSQAPKAQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP KAQQNAFYYVLNMPNLLADQ RNGFIQSLKD DPSQSANVLA EAIKLNDSQA PKAQQNAFYY VLNMPNLLAD QRNGFIQSLKDDPSQSANVL AEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNGFIQSL KDDPSQSANV LAEAIKLNDSQAPK

SEQ ID No 45(Q8Y,N16L,G22A,G39S,Q42I)4SEQ ID No 45 (Q8Y, N16L, G22A, G39S, Q42I) 4

AQQNAFYYVL NMPNLLADQR NAFIQSLKDD PSQSANVLSE AIKLNDSQAP KAQQNAFYYVLNMPNLLADQ RNAFIQSLKD DPSQSANVLS EAIKLNDSQA PKAQQNAFYY VLNMPNLLAD QRNAFIQSLKDDPSQSANVL SEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNAFIQSL KDDPSQSANV LSEAIKLNDSQAPKAQQNAFYYVL NMPNLLADQR NAFIQSLKDD PSQSANVLSE AIKLNDSQAP KAQQNAFYYVLNMPNLLADQ RNAFIQSLKD DPSQSANVLS EAIKLNDSQA PKAQQNAFYY VLNMPNLLAD QRNAFIQSLKDDPSQSANVL SEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNAFIQSL KDDPSQSANV LSEAIKLNDSQ

SEQ ID No 46(Q2F,Q8Y,N16R,G22A,G39S,Q42D)4SEQ ID No 46 (Q2F, Q8Y, N16R, G22A, G39S, Q42D) 4

AFQNAFYYVL NMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVLNMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVL NMPNLRADQRNAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVL NMPNLRADQR NAFIQSLKDDPSQSANVLSE ADKLNDSQAP KAFQNAFYYVL NMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVLNMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVL NMPNLRADQRNAFIQSLKDD PSQSANVLSE ADKLNDSQAP K

SEQ ID No 47(Q8Y,N16L,G39A,Q42I)6SEQ ID No 47 (Q8Y, N16L, G39A, Q42I)6

AQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP KAQQNAFYYVLNMPNLLADQ RNGFIQSLKD DPSQSANVLA EAIKLNDSQA PKAQQNAFYY VLNMPNLLAD QRNGFIQSLKDDPSQSANVL AEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNGFIQSL KDDPSQSANV LAEAIKLNDSQAPKAQQNAF YYVLNMPNLL ADQRNGFIQS LKDDPSQSAN VLAEAIKLND SQAPKAQQNAF YYVLNMPNLLADQRNGFIQS LKDDPSQSAN VLAEAIKLND SQAPKAQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP KAQQNAFYYVLNMPNLLADQ RNGFIQSLKD DPSQSANVLA EAIKLNDSQA PKAQQNAFYY VLNMPNLLAD QRNGFIQSLKDDPSQSANVL AEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNGFIQSL KDDPSQSANV LAEAIKLNDSQAPKAQQNAF YYVLNMPNLL ADQRNGFIQS LKDDPSQSAN VLAEAIKLND SQAPKAQQNAF YYVLNMPNLLADQRNGFIQS LKDDPSQSAN VLAEAIKLND SQAPK

SEQ ID No 48(N6S,N9Y,Q12D)4SEQ ID No 48 (N6S, N9Y, Q12D) 4

ADAQQSKFYK DDQSAFYEIL NMPNLNEEQR NGFIQSLKDDPSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYEI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYE ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APK ADAQQSKFYKDDQSAFYE ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSKFYK DDQSAFYEIL NMPNLNEEQR NGFIQSLKDDPSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYEI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYE ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APK ADAQQSKFYKDDQSAFYE ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APK

SEQ ID No 49(N6S,N9Y,Q12D,E18R)4SEQ ID No 49 (N6S, N9Y, Q12D, E18R) 4

ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSK FYKDDQSAFYRILNMPNLNE EQRNGFIQSL KDDPSQSTNV LGEAKKLNES QAPKADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSK FYKDDQSNEQ APKDDNVSTNL

SEQ ID No 50(N6S,N9Y,Q12D,E18R,G32S)4SEQ ID No 50 (N6S, N9Y, Q12D, E18R, G32S) 4

ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNSFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNSFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSK FYKDDQSAFYRILNMPNLNE EQRNSFIQSL KDDPSQSTNV LGEAKKLNES QAPKADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNSFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNSFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSK FYKDDQSAFYRILNMPNL

SEQ ID No 51(N6S,N9Y,Q12D,E18R,N26I,G32S)4SEQ ID No 51 (N6S, N9Y, Q12D, E18R, N26I, G32S) 4

ADAQQSKFYK DDQSAFYRIL NMPNLIEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLIEEQ RNSFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLIEE QRNSFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSK FYKDDQSAFYRILNMPNLIE EQRNSFIQSL KDDPSQSTNV LGEAKKLNES QAPKADAQQSKFYK DDQSAFYRIL NMPNLIEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLIEEQ RNSFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLIEE QRNSFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSK FYKDDQSAF YRILNMPNL

SEQ ID No 52(N6S,N9Y,Q12D,E18R)6SEQ ID No 52 (N6S, N9Y, Q12D, E18R)6

ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSKFYKDDQSAFYRILNMPNLNE EQRNGFIQSL KDDPSQSTNV LGEAKKLNESQAPKADAQQS KFYKDDQSAF YRILNMPNLNEEQRNGFIQS LKDDPSQSTNVLGEAKKLNE SQAPKADAQQ SKFYKDDQSA FYRILNMPNL NEEQRNGFIQSLKDDPSQST NVLGEAKKLN ESQAPK。ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQA PKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APKADAQQSKFYKDDQSAFYRILNMPNLNE EQRNGFIQSL KDDPSQSTNV LGEAKKLNESQAPKADAQQS KFYKDDQSAF YRILNMPNLNEEQRNGFIQS LKDDPSQSTNVLGEAKKLNE SQAPKADAQQ SKFYKDDQSA FYRILNMPNL NEEQRNGFIQSLKDDPSQST NVLGEAKKLN ESQAPK。

根据本发明的一个方面，提供了一种基因，该基因编码如所述重组Protein A蛋白的基因序列。According to one aspect of the present invention, there is provided a gene encoding the gene sequence of the recombinant Protein A protein.

根据本发明的另一个方面，如图1所示，提供了一种亲和层析介质的制备方法，该亲和层析介质的配基为所述重组Protein A蛋白，该亲和层析介质的制备方法包括以下步骤：According to another aspect of the present invention, as shown in FIG. 1, a method for preparing an affinity chromatography medium is provided, wherein the ligand of the affinity chromatography medium is the recombinant Protein A protein, and the affinity chromatography medium The preparation method comprises the following steps:

S1、合成重组Protein A蛋白的基因；S1. Gene for synthesizing recombinant Protein A protein;

S2、利用所述S1中合成的基因分别构建表达载体；S2, utilize the genes synthesized in the S1 to construct expression vectors respectively;

其中，所述S2中的表达载体为PET23a。Wherein, the expression vector in S2 is PET23a.

S3、培养并表达纯化得到序列对应的SEQ ID的重组Protein A溶液；S3, cultivate and express and purify the recombinant Protein A solution that obtains the SEQ ID corresponding to the sequence;

其中，所述S3中培养并表达纯化得到对应序列的重组Protein A溶液具体包括以下步骤：Wherein, the recombinant Protein A solution obtained by culturing and expressing and purifying the corresponding sequence in the S3 specifically includes the following steps:

S31、利用单个重组质粒转化到大肠杆菌，并通过LB液体培养基进行发酵培养并进行诱导表达；S31, transforming into Escherichia coli with a single recombinant plasmid, and fermenting and inducing expression in LB liquid medium;

S32、发酵后，收集菌体并采用热裂解的方式破坏细胞壁将表达产物释放出来并离心分离；S32, after fermentation, collect thalline and destroy the cell wall by means of thermal cracking to release the expression product and separate by centrifugation;

S33、将分离液体经过IgG亲和介质进行纯化，分离液上样经过亲和介质，用10mM的磷酸盐缓冲液清洗介质，并用pH 3.8的缓冲溶液收集目的蛋白，调节到中性环境，保存待用。S33. Purify the separation liquid through an IgG affinity medium, load the separation liquid into the affinity medium, wash the medium with 10 mM phosphate buffer, and collect the target protein with a pH 3.8 buffer solution, adjust to a neutral environment, and store it until use.

S4、利用所述重组Protein A制备得到亲和层析介质。S4, using the recombinant Protein A to prepare an affinity chromatography medium.

其中，所述S4中利用所述重组Protein A制备得到亲和层析介质具体包括以下步骤：Wherein, using the recombinant Protein A to prepare the affinity chromatography medium in the S4 specifically includes the following steps:

S41、活化：利用环氧氯丙烷活化高刚性琼脂糖；S41, activation: use epichlorohydrin to activate high rigidity agarose;

S42、交联：在活化后的琼脂糖中加入交联缓冲液，以及重组Protein A溶液，在34℃下恒温反应，完成活化后的琼脂糖与重组蛋白的交联；S42. Cross-linking: add cross-linking buffer and recombinant Protein A solution to the activated agarose, and react at a constant temperature at 34°C to complete the cross-linking of the activated agarose and the recombinant protein;

S43、活化基团封闭：在交联产物中加入封闭液，用氢氧化钠调整pH为8.6，26℃下恒温反应，完成对残留的环氧基团的封闭，减小影响。S43. Blocking of activated groups: adding blocking solution to the cross-linked product, adjusting the pH to 8.6 with sodium hydroxide, and reacting at a constant temperature at 26°C to complete the blocking of residual epoxy groups and reduce the impact.

此外，本发明中还包括采用上述优选实施例中的重组Protein A蛋白进行的以下实验分析，该实验包括实验组与对照组，对照组与实验组的实验分析相同，区别在于使用的蛋白不同，其中，实验组具体实验如下：In addition, the present invention also includes the following experimental analysis using the recombinant Protein A protein in the above preferred embodiment, the experiment includes an experimental group and a control group, and the experimental analysis of the control group and the experimental group is the same, the difference is that the proteins used are different, Among them, the specific experiments of the experimental group are as follows:

实施例一Example 1

使用下面描述的方法对表1的重组Protein A制备得到的亲和层析介质的能力和耐碱稳定性进行评价。结果见表1和图3。Affinity chromatography media prepared from recombinant Protein A of Table 1 were evaluated for their ability and alkali stability using the methods described below. The results are shown in Table 1 and Figure 3.

表1在柱(0.5M NaOH)中评价的具有重组Protein A的亲和层析介质Table 1 Affinity chromatography media with recombinant Protein A evaluated in column (0.5M NaOH)

Figure BDA0003060063290000141
Figure BDA0003060063290000141

(1)免疫球蛋白G(IgG)动态载量的测定(1) Determination of dynamic load of immunoglobulin G (IgG)

将2ml树脂装填到TRICORN TM 5 100柱中，用20mM磷酸缓冲液(pH 7.4)使柱平衡化后，将含有人多克隆IgG(5mg/mL)的20mM磷酸缓冲液(pH7)以线流速300cm/h流过，由吸光度监视器根据溶出液中的人多克隆IgG浓度突破(Breakthrough)10％时的人多克隆IgG吸附量和载体体积来求出动态结合容量(DBC)。2 ml of resin was loaded into a TRICORN™ 5 100 column, and after equilibrating the column with 20 mM phosphate buffer (pH 7.4), 20 mM phosphate buffer (pH 7) containing human polyclonal IgG (5 mg/mL) was added at a linear flow rate of 300 cm /h flow, and the dynamic binding capacity (DBC) was calculated from the absorbance monitor based on the amount of human polyclonal IgG adsorbed when the concentration of human polyclonal IgG in the eluate breaks through 10% and the volume of the carrier.

突破载量是使用AKTA Explorer 10系统在2.4分钟的停留时间确定的。使平衡缓冲液通过旁路色谱柱，直到获得稳定的基线。这是在自动归零之前完成的。将样品上样至色谱柱，直至获得100％的UV信号。然后，再次施加平衡缓冲液直到获得稳定的基线。The breakthrough load was determined using the AKTA Explorer 10 system at a dwell time of 2.4 minutes. Pass equilibration buffer through the bypass column until a stable baseline is obtained. This is done before auto-zeroing. The sample was loaded onto the column until a 100% UV signal was obtained. Then, equilibration buffer was applied again until a stable baseline was obtained.

将样品加载到色谱柱上，直到达到最大吸光度的85％的UV信号为止。Load the sample onto the column until a UV signal at 85% of the maximum absorbance is reached.

然后用平衡缓冲液洗涤色谱柱，直到流速为0.5ml/min时最大吸收度的20％的UV信号为止。在10个柱体积上以线性梯度洗脱蛋白质，起始pH为6.0，终止于pH 3.0，流速为0.5ml/min。The column was then washed with equilibration buffer until a UV signal of 20% of maximum absorbance was reached at a flow rate of 0.5 ml/min. The protein was eluted with a linear gradient over 10 column volumes starting at pH 6.0 and ending at pH 3.0 at a flow rate of 0.5 ml/min.

然后用0.5M NaOH以0.5ml/min的流速清洗色谱柱，并在用20％乙醇清洗之前用平衡缓冲液重新平衡。The column was then washed with 0.5M NaOH at a flow rate of 0.5ml/min and re-equilibrated with equilibration buffer before washing with 20% ethanol.

(2)0.5M NaOH稳定性的测定(2) Determination of the stability of 0.5M NaOH

在用0.5M NaOH清洗色谱柱之前和之后测定IgG结合能力。具体操作过程为：将载体填充柱设定为AKTA，将0.5M氢氧化钠20ml流入柱内。将柱从装置卸除，密封后在室温下放置一定时间(1小时)后，测定线流速300cm/h的人多克隆IgG的结合容量，总共进行25个循环，共计24小时。将0.5M氢氧化钠处理之前的人多克隆IgG结合量设为100％时的结合容量，求出0.5M氢氧化钠处理之后的结合容量维持率。IgG binding capacity was determined before and after column washing with 0.5M NaOH. The specific operation process is as follows: the carrier packed column is set to AKTA, and 20 ml of 0.5M sodium hydroxide is flowed into the column. The column was removed from the apparatus, sealed and left at room temperature for a certain period of time (1 hour) to measure the binding capacity of human polyclonal IgG at a linear flow rate of 300 cm/h. A total of 25 cycles were performed for a total of 24 hours. Taking the binding capacity of human polyclonal IgG before 0.5M sodium hydroxide treatment as the binding capacity when 100%, the binding capacity retention ratio after 0.5M sodium hydroxide treatment was determined.

实施例二Embodiment 2

用表2中的重组Protein A制备得到的亲和层析介质重复实施例一，但用1.0MNaOH代替0.5M NaOH清洗色谱柱。结果见表2和图4。Example 1 was repeated with the affinity chromatography medium prepared from recombinant Protein A in Table 2, but the column was washed with 1.0M NaOH instead of 0.5M NaOH. The results are shown in Table 2 and Figure 4.

表2在柱(0.5M NaOH)中评价的具有重组Protein A的亲和层析介质Table 2 Affinity chromatography media with recombinant Protein A evaluated in column (0.5M NaOH)

Figure BDA0003060063290000151
Figure BDA0003060063290000151

Figure BDA0003060063290000161
Figure BDA0003060063290000161

根据本发明的另一个方面，提供了一种亲和层析介质，该层析介质采用上述亲和层析介质的制备方法制备得到。According to another aspect of the present invention, an affinity chromatography medium is provided, and the chromatography medium is prepared by the above-mentioned preparation method of an affinity chromatography medium.

根据本发明的又一个方面，提供了一种重组ProteinA蛋白在制备亲和层析介质中的应用。According to another aspect of the present invention, there is provided the use of a recombinant ProteinA protein in the preparation of an affinity chromatography medium.

根据本发明的又一个方面，提供了一种亲和层析介质在抗体分离纯化的应用。According to yet another aspect of the present invention, an application of an affinity chromatography medium in the separation and purification of antibodies is provided.

综上所述，借助于本发明的上述技术方案，本发明通过对氨基酸序列重新设计，大大提高Protein A在碱液中的化学稳定性，从而提供一种对抗体具有特异性结合作用的Protein A，其在碱性条件下具有良好的化学稳定性，且重组Protein A亲和层析介质结合载量显著提高，可耐受0.5-1.0M NaOH在位清洗，IgG结合载量60-90mg/ml。To sum up, with the help of the above technical solutions of the present invention, the present invention greatly improves the chemical stability of Protein A in lye by redesigning the amino acid sequence, thereby providing a Protein A that has a specific binding effect on antibodies. , it has good chemical stability under alkaline conditions, and the binding capacity of recombinant Protein A affinity chromatography medium is significantly increased, it can withstand 0.5-1.0M NaOH in-situ cleaning, and the binding capacity of IgG is 60-90mg/ml .

此外，本发明还提供了一种利用该突变结构域的Protein A为配基的亲和层析介质制备方法，与以结构域Z及其寡聚体为配基的亲和层析介质相比，本发明制备的亲和层析介质的抗体结合能力更为优异。In addition, the present invention also provides a method for preparing an affinity chromatography medium using Protein A of the mutant domain as a ligand, compared with an affinity chromatography medium using the domain Z and its oligomers as ligands , the antibody binding ability of the affinity chromatography medium prepared by the present invention is more excellent.

以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

SEQUENCE LISTINGSEQUENCE LISTING

<110> 博格隆（浙江）生物技术有限公司<110> Boglong (Zhejiang) Biotechnology Co., Ltd.

<120> 一种重组 Protein A 蛋白及亲和层析介质的制备方法<120> A kind of preparation method of recombinant Protein A protein and affinity chromatography medium

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<170> PatentIn version 3.3<170> PatentIn version 3.3

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Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 7<210> 7

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 7<400> 7

Ala Tyr Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu AsnAla Tyr Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 8<210> 8

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 8<400> 8

Ala Tyr Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu AsnAla Tyr Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Phe Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Phe Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 9<210> 9

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 9<400> 9

Ala Ile Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu ThrAla Ile Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Thr

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 10<210> 10

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 10<400> 10

Ala Gln Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu AsnAla Gln Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 11<210> 11

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 11<400> 11

Ala Asp Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu SerAla Asp Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Ser

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Phe Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Phe Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 12<210> 12

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 12<400> 12

Ala Ile Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu AsnAla Ile Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 13<210> 13

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 13<400> 13

Ala Gln Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu ArgAla Gln Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu Arg

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 14<210> 14

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 14<400> 14

Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu AsnAla Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 15<210> 15

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 15<400> 15

Ala Gln Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu GluAla Gln Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu Glu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ala Glu Ala Asp Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ala Glu Ala Asp Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 16<210> 16

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 16<400> 16

Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu AsnAla Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 17<210> 17

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 17<400> 17

Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu LeuAla Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 18<210> 18

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 18<400> 18

Ala Ile Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu IleAla Ile Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu Ile

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ala Glu Ala Phe Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ala Glu Ala Phe Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 19<210> 19

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 19<400> 19

Ala Asp Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu ThrAla Asp Gln Asn Ala Phe Tyr Phe Val Leu Asn Met Pro Asn Leu Thr

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Gln Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Gln Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 20<210> 20

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 20<400> 20

Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu LeuAla Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Ile Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Ile Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 21<210> 21

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 21<400> 21

Ala Phe Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu ArgAla Phe Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Arg

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 22<210> 22

<211> 51<211> 51

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 22<400> 22

Ala Tyr Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu LeuAla Tyr Gln Asn Ala Phe Tyr Asp Val Leu Asn Met Pro Asn Leu Leu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ala Glu Ala Phe Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ala Glu Ala Phe Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro LysAla Pro Lys

50 50

<210> 23<210> 23

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 23<400> 23

Ala Asp Ala Gln Gln Thr Lys Phe Asn Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Thr Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 24<210> 24

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 24<400> 24

Ala Asp Ala Gln Gln Ser Lys Phe Asn Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 25<210> 25

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 25<400> 25

Ala Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 26<210> 26

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 26<400> 26

Ala Asp Ala Gln Gln Arg Lys Phe Asn Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Arg Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 27<210> 27

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 27<400> 27

Ala Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 28<210> 28

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 28<400> 28

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 29<210> 29

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 29<400> 29

Ala Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 30<210> 30

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 30<400> 30

Ala Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 31<210> 31

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 31<400> 31

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 32<210> 32

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 32<400> 32

Ala Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala PheAla Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 33<210> 33

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 33<400> 33

Ala Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Leu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn GlyTyr Leu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 34<210> 34

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 34<400> 34

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 35<210> 35

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 35<400> 35

Ala Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala PheAla Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Asp Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 36<210> 36

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 36<400> 36

Ala Asp Ala Gln Gln Arg Lys Phe Asn Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Arg Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Gly Ile Leu Asn Met Pro Asn Leu Ile Glu Gly Gln Arg Asn GlyTyr Gly Ile Leu Asn Met Pro Asn Leu Ile Glu Gly Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn His Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn His Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 37<210> 37

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 37<400> 37

Ala Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Leu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn AlaTyr Leu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn Ala

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 38<210> 38

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 38<400> 38

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn SerTyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Ser

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 39<210> 39

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 39<400> 39

Ala Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala PheAla Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Ala Glu Ala Lys Lys Leu Asn Asp Ser Gln Ala Pro LysAla Glu Ala Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 40<210> 40

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 40<400> 40

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Ile Glu Glu Gln Arg Asn SerTyr Arg Ile Leu Asn Met Pro Asn Leu Ile Glu Glu Gln Arg Asn Ser

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 41<210> 41

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 41<400> 41

Ala Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala PheAla Asp Ala Gln Gln Glu Lys Phe Asn Lys Asp Phe Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr His Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn GlyTyr His Ile Leu Asn Met Pro Asn Leu Leu Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Ala Glu Ala Lys Lys Leu Asn Asp Ser Gln Ala Pro LysAla Glu Ala Lys Lys Leu Asn Asp Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 42<210> 42

<211> 61<211> 61

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 42<400> 42

Ala Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala PheAla Asp Ala Gln Gln Thr Lys Phe Ser Lys Asp Gln Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Leu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn AlaTyr Leu Ile Leu Asn Met Pro Asn Leu Glu Glu Glu Gln Arg Asn Ala

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55 60 50 55 60

<210> 43<210> 43

<211> 204<211> 204

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 43<400> 43

Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu AsnAla Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro Asn Leu Asn

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met ProAla Pro Lys Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn Met Pro

50 55 60 50 55 60

Asn Leu Asn Ala Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys AspAsn Leu Asn Ala Asp Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp

65 70 75 8065 70 75 80

Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu AsnAsp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn

85 90 95 85 90 95

Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Gln Val LeuAsp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu

100 105 110 100 105 110

Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Ser Phe Ile Gln SerAsn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Ser Phe Ile Gln Ser

115 120 125 115 120 125

Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala AspLeu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Asp

130 135 140 130 135 140

Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe TyrLys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr

145 150 155 160145 150 155 160

Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Ser PheGln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Ser Phe

165 170 175 165 170 175

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu SerIle Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser

180 185 190 180 185 190

Glu Ala Asp Lys Leu Asn Asp Ser Gln Ala Pro LysGlu Ala Asp Lys Leu Asn Asp Ser Gln Ala Pro Lys

195 200 195 200

<210> 44<210> 44

<211> 204<211> 204

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 44<400> 44

Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu LeuAla Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met ProAla Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro

50 55 60 50 55 60

Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys AspAsn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp

65 70 75 8065 70 75 80

Asp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu AsnAsp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn

85 90 95 85 90 95

Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val LeuAsp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu

100 105 110 100 105 110

Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln SerAsn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser

115 120 125 115 120 125

Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala IleLeu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala Ile

130 135 140 130 135 140

Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe TyrLys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr

145 150 155 160145 150 155 160

Tyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly PheTyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe

165 170 175 165 170 175

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu AlaIle Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ala

180 185 190 180 185 190

Glu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro LysGlu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro Lys

195 200 195 200

<210> 45<210> 45

<211> 204<211> 204

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 45<400> 45

Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu LeuAla Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Ile Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Ile Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met ProAla Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro

50 55 60 50 55 60

Asn Leu Leu Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys AspAsn Leu Leu Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp

65 70 75 8065 70 75 80

Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Ile Lys Leu AsnAsp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Ile Lys Leu Asn

85 90 95 85 90 95

Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val LeuAsp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu

100 105 110 100 105 110

Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Ala Phe Ile Gln SerAsn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser

115 120 125 115 120 125

Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala IleLeu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Ile

130 135 140 130 135 140

Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe TyrLys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr

145 150 155 160145 150 155 160

Tyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Ala PheTyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Ala Phe

165 170 175 165 170 175

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu SerIle Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser

180 185 190 180 185 190

Glu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro LysGlu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro Lys

195 200 195 200

<210> 46<210> 46

<211> 204<211> 204

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 46<400> 46

Ala Phe Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu ArgAla Phe Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Arg

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro Lys Ala Phe Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met ProAla Pro Lys Ala Phe Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro

50 55 60 50 55 60

Asn Leu Arg Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys AspAsn Leu Arg Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp

65 70 75 8065 70 75 80

Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu AsnAsp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Asp Lys Leu Asn

85 90 95 85 90 95

Asp Ser Gln Ala Pro Lys Ala Phe Gln Asn Ala Phe Tyr Tyr Val LeuAsp Ser Gln Ala Pro Lys Ala Phe Gln Asn Ala Phe Tyr Tyr Val Leu

100 105 110 100 105 110

Asn Met Pro Asn Leu Arg Ala Asp Gln Arg Asn Ala Phe Ile Gln SerAsn Met Pro Asn Leu Arg Ala Asp Gln Arg Asn Ala Phe Ile Gln Ser

115 120 125 115 120 125

Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala AspLeu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser Glu Ala Asp

130 135 140 130 135 140

Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Phe Gln Asn Ala Phe TyrLys Leu Asn Asp Ser Gln Ala Pro Lys Ala Phe Gln Asn Ala Phe Tyr

145 150 155 160145 150 155 160

Tyr Val Leu Asn Met Pro Asn Leu Arg Ala Asp Gln Arg Asn Ala PheTyr Val Leu Asn Met Pro Asn Leu Arg Ala Asp Gln Arg Asn Ala Phe

165 170 175 165 170 175

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu SerIle Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ser

180 185 190 180 185 190

Glu Ala Asp Lys Leu Asn Asp Ser Gln Ala Pro LysGlu Ala Asp Lys Leu Asn Asp Ser Gln Ala Pro Lys

195 200 195 200

<210> 47<210> 47

<211> 306<211> 306

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 47<400> 47

Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu LeuAla Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu

1 5 10 151 5 10 15

Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro SerAla Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

20 25 30 20 25 30

Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser GlnGln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln

35 40 45 35 40 45

Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met ProAla Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro

50 55 60 50 55 60

Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys AspAsn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp

65 70 75 8065 70 75 80

Asp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu AsnAsp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn

85 90 95 85 90 95

Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val LeuAsp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr Tyr Val Leu

100 105 110 100 105 110

Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln SerAsn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser

115 120 125 115 120 125

Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala IleLeu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ala Glu Ala Ile

130 135 140 130 135 140

Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe TyrLys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn Ala Phe Tyr

145 150 155 160145 150 155 160

Tyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly PheTyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg Asn Gly Phe

165 170 175 165 170 175

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu AlaIle Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Ala

180 185 190 180 185 190

Glu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln AsnGlu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Gln Gln Asn

195 200 205 195 200 205

Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln ArgAla Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu Ala Asp Gln Arg

210 215 220 210 215 220

Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala AsnAsn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn

225 230 235 240225 230 235 240

Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro Lys AlaVal Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala

245 250 255 245 250 255

Gln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu AlaGln Gln Asn Ala Phe Tyr Tyr Val Leu Asn Met Pro Asn Leu Leu Ala

260 265 270 260 265 270

Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser GlnAsp Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln

275 280 285 275 280 285

Ser Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln AlaSer Ala Asn Val Leu Ala Glu Ala Ile Lys Leu Asn Asp Ser Gln Ala

290 295 300 290 295 300

Pro LysPro Lys

305305

<210> 48<210> 48

<211> 244<211> 244

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 48<400> 48

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp AlaGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala

50 55 60 50 55 60

Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Glu IleGln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Glu Ile

65 70 75 8065 70 75 80

Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile GlnLeu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln

85 90 95 85 90 95

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu AlaSer Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

100 105 110 100 105 110

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln SerLys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser

115 120 125 115 120 125

Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Glu Ile Leu Asn MetLys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met

130 135 140 130 135 140

Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu LysPro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys

145 150 155 160145 150 155 160

Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys LeuAsp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu

165 170 175 165 170 175

Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe TyrAsn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr

180 185 190 180 185 190

Lys Asp Asp Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn LeuLys Asp Asp Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu

195 200 205 195 200 205

Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp ProAsn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro

210 215 220 210 215 220

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu SerSer Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

225 230 235 240225 230 235 240

Gln Ala Pro LysGln Ala Pro Lys

<210> 49<210> 49

<211> 244<211> 244

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 49<400> 49

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp AlaGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala

50 55 60 50 55 60

Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg IleGln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile

65 70 75 8065 70 75 80

Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile GlnLeu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln

85 90 95 85 90 95

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu AlaSer Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

100 105 110 100 105 110

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln SerLys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser

115 120 125 115 120 125

Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn MetLys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met

130 135 140 130 135 140

Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu LysPro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys

145 150 155 160145 150 155 160

Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys LeuAsp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu

165 170 175 165 170 175

Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe TyrAsn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr

180 185 190 180 185 190

Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn LeuLys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu

195 200 205 195 200 205

Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp ProAsn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro

210 215 220 210 215 220

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu SerSer Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

225 230 235 240225 230 235 240

Gln Ala Pro LysGln Ala Pro Lys

<210> 50<210> 50

<211> 244<211> 244

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 50<400> 50

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn SerTyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Ser

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp AlaGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala

50 55 60 50 55 60

Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg IleGln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile

65 70 75 8065 70 75 80

Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Ser Phe Ile GlnLeu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Ser Phe Ile Gln

85 90 95 85 90 95

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu AlaSer Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

100 105 110 100 105 110

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln SerLys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser

115 120 125 115 120 125

Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn MetLys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met

130 135 140 130 135 140

Pro Asn Leu Asn Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu LysPro Asn Leu Asn Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys

145 150 155 160145 150 155 160

Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys LeuAsp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu

165 170 175 165 170 175

Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe TyrAsn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr

180 185 190 180 185 190

Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn LeuLys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu

195 200 205 195 200 205

Asn Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp ProAsn Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro

210 215 220 210 215 220

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu SerSer Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

225 230 235 240225 230 235 240

Gln Ala Pro LysGln Ala Pro Lys

<210> 51<210> 51

<211> 244<211> 244

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 51<400> 51

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Ile Glu Glu Gln Arg Asn SerTyr Arg Ile Leu Asn Met Pro Asn Leu Ile Glu Glu Gln Arg Asn Ser

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp AlaGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala

50 55 60 50 55 60

Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg IleGln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile

65 70 75 8065 70 75 80

Leu Asn Met Pro Asn Leu Ile Glu Glu Gln Arg Asn Ser Phe Ile GlnLeu Asn Met Pro Asn Leu Ile Glu Glu Gln Arg Asn Ser Phe Ile Gln

85 90 95 85 90 95

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu AlaSer Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

100 105 110 100 105 110

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln SerLys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser

115 120 125 115 120 125

Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn MetLys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met

130 135 140 130 135 140

Pro Asn Leu Ile Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu LysPro Asn Leu Ile Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys

145 150 155 160145 150 155 160

Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys LeuAsp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu

165 170 175 165 170 175

Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe TyrAsn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr

180 185 190 180 185 190

Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn LeuLys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu

195 200 205 195 200 205

Ile Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp ProIle Glu Glu Gln Arg Asn Ser Phe Ile Gln Ser Leu Lys Asp Asp Pro

210 215 220 210 215 220

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu SerSer Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

225 230 235 240225 230 235 240

Gln Ala Pro LysGln Ala Pro Lys

<210> 52<210> 52

<211> 366<211> 366

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 52<400> 52

Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala PheAla Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe

1 5 10 151 5 10 15

Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn GlyTyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

20 25 30 20 25 30

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val LeuPhe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu

35 40 45 35 40 45

Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp AlaGly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala

50 55 60 50 55 60

Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg IleGln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile

65 70 75 8065 70 75 80

Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile GlnLeu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln

85 90 95 85 90 95

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu AlaSer Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

100 105 110 100 105 110

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln SerLys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser

115 120 125 115 120 125

Lys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn MetLys Phe Tyr Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met

130 135 140 130 135 140

Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu LysPro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys

145 150 155 160145 150 155 160

Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys LeuAsp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu

165 170 175 165 170 175

Asn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe TyrAsn Glu Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr

180 185 190 180 185 190

Lys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn LeuLys Asp Asp Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu

195 200 205 195 200 205

Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp ProAsn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro

210 215 220 210 215 220

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu SerSer Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

225 230 235 240225 230 235 240

Gln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp AspGln Ala Pro Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp

245 250 255 245 250 255

Gln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu GluGln Ser Ala Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu

260 265 270 260 265 270

Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln SerGln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser

275 280 285 275 280 285

Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala ProThr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro

290 295 300 290 295 300

Lys Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser AlaLys Ala Asp Ala Gln Gln Ser Lys Phe Tyr Lys Asp Asp Gln Ser Ala

305 310 315 320305 310 315 320

Phe Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg AsnPhe Tyr Arg Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn

325 330 335 325 330 335

Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn ValGly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val

340 345 350 340 345 350

Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro LysLeu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

355 360 365 355 360 365

<210> 53<210> 53

<211> 58<211> 58

<212> PRT<212> PRT

<213> Staphylococcus aureus<213> Staphylococcus aureus

<400> 53<400> 53

Val Asp Ala Lys Phe Asp Lys Glu Gln Gln Asn Ala Phe Tyr Glu IleVal Asp Ala Lys Phe Asp Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 151 5 10 15

Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile GlnLeu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30 20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu AlaSer Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45 35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro LysLys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55 50 55

Claims (5)

1.一种重组Protein A蛋白，其特征在于，所述的重组Protein A蛋白是由包含SEQIDNo 1指定的E结构域的突变体，所述突变体包含4个重复单元，其序列选自：1. a recombinant Protein A protein is characterized in that, described recombinant Protein A protein is by the mutant of the E structural domain that comprises SEQIDNo 1 designation, and described mutant comprises 4 repeating units, and its sequence is selected from: SEQ ID No 43、SEQ ID No44或SEQ ID No 45。SEQ ID No 43, SEQ ID No 44 or SEQ ID No 45. 2.根据权利要求1所述的一种重组Protein A蛋白，其特征在于，所述重组Protein A蛋白C端包含一个半胱氨酸残基的偶联基团。2 . The recombinant Protein A protein according to claim 1 , wherein the C-terminal of the recombinant Protein A protein comprises a coupling group of cysteine residues. 3 . 3.一种亲和层析介质的制备方法，其特征在于，该亲和层析介质的配基为权利要求1所述的重组Protein A蛋白，该亲和层析介质的制备方法包括以下步骤：3. A preparation method of an affinity chromatography medium, wherein the ligand of the affinity chromatography medium is the recombinant Protein A protein of claim 1, and the preparation method of the affinity chromatography medium comprises the following steps : S1、合成重组Protein A蛋白的基因；S1. Gene for synthesizing recombinant Protein A protein; S2、利用所述S1中合成的基因分别构建表达载体；S2, utilize the genes synthesized in the S1 to construct expression vectors respectively; S3、培养并表达纯化得到序列对应的SEQ ID的重组Protein A溶液；S3, cultivate and express and purify the recombinant Protein A solution that obtains the SEQ ID corresponding to the sequence; S4、利用所述重组Protein A制备得到亲和层析介质。S4, using the recombinant Protein A to prepare an affinity chromatography medium. 4.根据权利要求3所述的一种亲和层析介质的制备方法，其特征在于，所述S2中的表达载体为PET23a。4 . The method for preparing an affinity chromatography medium according to claim 3 , wherein the expression vector in the S2 is PET23a. 5 . 5.根据权利要求3所述的一种亲和层析介质的制备方法，其特征在于，所述S3中培养并表达纯化得到对应序列的重组Protein A溶液具体包括以下步骤：5. the preparation method of a kind of affinity chromatography medium according to claim 3, is characterized in that, in described S3, cultivate and express and purify the recombinant Protein A solution that obtains corresponding sequence specifically comprises the following steps: S31、利用单个重组质粒转化到大肠杆菌，并通过LB液体培养基进行发酵培养并进行诱导表达；S31, transforming into Escherichia coli with a single recombinant plasmid, and fermenting and inducing expression in LB liquid medium; S32、发酵后，收集菌体并采用热裂解的方式破坏细胞壁将表达产物释放出来并离心分离；S32, after fermentation, collect thalline and destroy the cell wall by means of thermal cracking to release the expression product and separate by centrifugation; S33、将分离液体经过IgG亲和介质进行纯化。S33, purifying the separation liquid through an IgG affinity medium.
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