CN118085108B

CN118085108B - A Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and its preparation method and application

Info

Publication number: CN118085108B
Application number: CN202410150601.4A
Authority: CN
Inventors: 章金勇; 廖静雯; 张晓丽; 敬海明; 陈致富; 苟强; 袁月; 邹全明
Original assignee: Shenzhen Kangtai Biological Products Co ltd; Third Military Medical University TMMU
Current assignee: Shenzhen Kangtai Biological Products Co ltd
Priority date: 2024-02-02
Filing date: 2024-02-02
Publication date: 2025-09-30
Anticipated expiration: 2044-02-02
Also published as: CN118085108A

Abstract

The invention discloses a klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, and the amino acid sequence of the antigen protein is shown as SEQ ID NO. 1. Wherein mHla is nontoxic mutant of staphylococcus aureus alpha hemolysin, which can be used as carrier protein of epitope to form heptamer, epiVac contains three immunodominant epitopes of klebsiella pneumoniae, and is formed by linking linker. The invention also discloses a preparation method and application of the antigen protein. The antigen protein prepared by the method can effectively stimulate organisms to generate high-efficiency humoral response and cellular immune response, can provide obvious protection effect on killing dose of Klebsiella pneumoniae infection, and can be used as candidate antigens of Klebsiella pneumoniae vaccine.

Description

Klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and preparation method and application thereof

Technical Field

The invention belongs to the field of biotechnology pharmacy, and relates to a klebsiella pneumoniae vaccine candidate antigen and a preparation method thereof, and application of the antigen protein in preparation of klebsiella pneumoniae recombinant protein vaccine.

Background

In 2017, the world health organization has first released a list of 12 key pathogens that are serious hazards to human health, with enterobacteriaceae bacteria, represented by klebsiella pneumoniae (Klebsiella pneumoniae, KP), being listed as a very important class (Tacconelli E, et al LANCET INFECT dis 2018). KP is a gram-negative bacterium and is one of the most common opportunistic pathogens in clinic. KP can be planted in the intestinal tract, nasopharynx, armpit and other parts, wherein the digestive tract is the most main planting part. The colonic setting rate in western countries is 5% -35%, while asia countries are generally higher, and individual countries (such as malaysia) are even more than 85% (Zhang Xin, et al, tuberculosis and respiratory journal 2020). KP can cause infection at various parts of the whole body, is common in the elderly, malnutrition, chronic diseases and patients with whole body failure, and can cause systemic or local infection such as pneumonia, urinary tract infection, meningitis, septicemia and the like (Lee CR, et al Front CELL INFECT Microbiol. 2017). According to different virulence and pathogenic characteristics, KP is divided into two types at present, one type is classical Klebsiella pneumoniae (cKP), which mainly causes hospital acquired infection, such as pneumonia, urinary tract infection, septicemia and the like, has high drug resistance rate, and is common to people with basic diseases or low immunity. The other group is klebsiella pneumoniae (hvKP) which is a high virulence klebsiella pneumoniae and mainly causes infection of healthy people without basic diseases in communities, most commonly liver abscess, 66% of which are caused by KP infection (Russo TA, et al Clin Microbiol Rev.2019).

In recent years, the drug resistance situation of KP against various common antibiotics is more and more severe, and the drug resistance monitoring result of CHINET Chinese bacteria in 2022 shows that the separation rate of KP is the second place of gram negative bacteria, which is inferior to that of Escherichia coli, and reaches 13.99% in 2022. The drug resistance rate of the anti-aging agent to ampicillin is up to 88.58%, and the drug resistance rate of the anti-aging agent to piperacillin is over 50%. Particularly, with the wide clinical application of carbapenem medicines, the detection rate of the carbapenem-resistant klebsiella pneumoniae (CRKP) is increased year by year, the drug resistance rate of KP to imipenem and meropenem in China is respectively increased from about 3% in 2005 to 22.6% and 24.2% in 2022, and the modes are very severe (https:// www.chinets.com/Document). Moreover, KP infection has extremely high mortality rate, and is counted to be up to 45% -72% of patients with community pneumonia caused by 22-32% of KP patients need ICU treatment, and further, KP accounts for 5-20% of cases of sepsis caused by gram negative bacteria infection, and the mortality rate is up to 27.4% -37% (Paczosa MK, et al Microbiol Mol Biol Rev.2016). More importantly, research shows that the KP infection mortality rate sensitive to carbapenem antibacterial drugs is 20% -30%, and CRKP infection mortality rate is remarkably increased by 40% -70% (Iredell J, et al BMJ 2016), and CRKP is called "super bacterial king" due to super drug resistance and pathogenicity. Considering that the KP drug resistance situation is very severe, and especially the wide popularity of CRKP makes antibiotic treatment difficult, the development of new effective control means is urgent, and vaccine development is one of the most promising strategies.

Since the advent of KP vaccine-related studies in the 70 s of the 20 th century, many different types of vaccine studies have emerged, early studies being based on inactivated, attenuated and bacterin-split vaccines, which are complex in composition, difficult to control quality, and may have residual toxicity, failing to enter clinical studies based on safety considerations (Ahmad TA, et al vaccine, 2012). Later, research into ribosome vaccines was started, but since it is an intracellular component, immunoprotection was limited, and intensive studies have not been performed. And secondly, polysaccharide vaccine, wherein the antigen adopted by the polysaccharide vaccine mainly comprises capsular polysaccharide and LPS, but as the capsular polysaccharide (K-antigen) of klebsiella pneumoniae has more than 80 serotypes, the LPS (O-antigen) has 12 serotypes, the K-antigen and the O-antigen have great variation among different serotypes, and although research shows that the vaccine shows good immunogenicity and safety in human body experiments, the passive immunity also has better protection effect, but the cross protection capability on other serotypes is limited, and the application of the vaccine is objectively limited (Jenney AW, et al J Clin microbiol 2006). Recent studies have focused mainly on recombinant protein vaccine studies, where various secreted proteins and outer membrane proteins exhibit a protective effect against KP infection, such as outer membrane proteins (OmpA, ompK36, fepA, ompK17, ompW), colicin I receptor proteins, adhesin MrkD proteins, pilin, cell surface iron regulatory proteins, toxoids, etc. (Zhang BZ, et al Front immunol 2021), are currently the most promising type of vaccine.

Alpha hemolysin (Hla) is a member of the perforin family secreted by staphylococcus aureus, hla can self-assemble into heptameric structures on host cell membranes, leading to cell lysis and death, and a Hla mutant (mHla) lacking two β fragments forming a pore-like structure retains the ability to assemble into heptameric structures, but loses its biological activity (Zou JT, et al Front immunol 2021). mHla can be used as a universal carrier protein to improve the immunogenicity of protein antigens. First, the formation of oligomers increases the size of the antigenic protein. Second, fusion with mHla facilitates the exposure of hidden epitopes on antigen proteins (Zou JT, et al PLoS pathway 2021).

Disclosure of Invention

Aiming at the high infection rate, high pathogenicity and high drug resistance of the klebsiella pneumoniae, the invention provides a klebsiella pneumoniae vaccine fusion protein antigen mHla-EpiVac which can be applied to the preparation of a klebsiella pneumoniae recombinant subunit vaccine.

The klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac provided by the invention has an amino acid sequence shown in SEQ ID NO. 1. Preferably, the fusion antigen contains three immunodominant epitopes of klebsiella pneumoniae origin.

The amino acid sequences of the three immunodominant epitopes are FepA to 423, shown as SEQ ID NO. 2, ompA148 to 165, shown as SEQ ID NO. 3, and OmpW144 to 163, shown as SEQ ID NO. 4.

The invention also provides a preparation method of the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac, which mainly comprises the following steps:

1) mHla-EpiVac synthesis and subcloning;

2) mHla-EpiVac;

3) mHla-EpiVac protein antigen is prepared;

4) mHla-EpiVac protein purification.

Specifically, step 1) comprises ligating mHla to the FepA396-423 sequence shown in SEQ ID NO. 2 with a flexible chain, then ligating the OmpW144-163 sequence shown in SEQ ID NO. 4 with a flexible chain, and then ligating the OmpA148-165 sequence shown in SEQ ID NO. 3 with a flexible chain to form fusion antigen mHla-EpiVac.

Step 1) further comprises the construction of a recombinant expression plasmid using the ampicillin-resistant prokaryotic expression plasmid pGEX-6 p-1.

Step 3) comprises culturing the induced protein obtained in step 2) in an amplified manner.

Step 4) comprises collecting genetically engineered bacteria expressing mHla-EpiVac, disrupting at high pressure, centrifuging, cation exchanging, and purifying mHla-EpiVac by hydrophobic chromatography.

The klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac can be used for preparing an immune preparation for preventing or treating klebsiella pneumoniae.

The invention also provides a klebsiella pneumoniae injection immune preparation, which contains the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac and any one or any combination of an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, an MF59, a complete Freund adjuvant, an incomplete Freund adjuvant and a mycobacteria BCG vaccine adjuvant.

As above, the present invention links mHla and FepA396-423 using flexible LINKERGGGGS strands, then links OmpW144-163 using flexible LINKER GGGGS, and finally links OmpA148-165 using flexible LINKER GSGGSG by fusion expression of 3 epitopes and mHla. The fusion protein is named mHla-EpiVac, and the amino acid sequence of the fusion protein is SEQ ID NO. 1.

The invention preferably adopts a prokaryotic expression plasmid pGEX-6p-1 to construct a recombinant expression plasmid, and the vector has ampicillin resistance and can be used for screening positive recombinants. To facilitate subsequent industrialization, no additional tag sequences were introduced at both ends of the fusion protein.

The invention provides a purification method of klebsiella pneumoniae recombinant protein antigen mHla-EpiVac. The main technical scheme is that the method comprises the steps of collecting genetically engineered bacteria expressing mHla-EpiVac, breaking bacteria according to high pressure, centrifuging, exchanging cations, and purifying mHla-EpiVac by hydrophobic chromatography. The method has the advantages of simple process, high purity of the obtained target protein, easy amplification, good repeatability and good recovery rate.

The recombinant protein is cloned and expressed by adopting a genetic engineering technology, is convenient for separation and purification, can be directly matched with an adjuvant (such as an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, an MF59, a complete Freund adjuvant, an incomplete Freund adjuvant, a mycobacteria BCG vaccine adjuvant and the like) for use, and is suitable for injection immunization.

The genetic engineering recombinant mHla-EpiVac protein has the following advantages:

1) The recombinant mHla-EpiVac antigen protein contains three immunodominant epitopes of three antigens of klebsiella pneumoniae, and can simultaneously generate specific immune responses aiming at the three proteins;

2) The recombinant mHla-EpiVac antigen protein can be expressed in a prokaryotic expression system, namely escherichia coli, and has low cost and high yield;

3) When pGEX-6p-1 vector is selected, mHla-EpiVac recombinant protein is expressed in soluble form;

4) mHla-EpiVac has mild purification conditions, simple steps, no need of adding denaturant, easy amplification, good repeatability and good recovery rate.

5) The recombinant mHla-EpiVac protein can induce animals to generate specific antibodies, and subunit vaccines prepared by utilizing the recombinant mHla-EpiVac protein can be immunized by a subcutaneous (intramuscular) injection way, so that organisms can be stimulated to generate high-titer IgG antibodies, and the antibodies have good immunogenicity.

6) The recombinant mHla-EpiVac protein can induce specific humoral immunity and also can induce cellular immunity.

The klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac has an amino acid sequence shown in SEQ ID NO. 1. Wherein mHla is a nontoxic mutant of staphylococcus aureus alpha hemolysin, carrier protein （ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN）, serving as an epitope can form a heptamer, epiVac contains three immunodominant epitopes (KDNASNTQALSGGEIPGYDSTGR, NEDFNDTGKAAGLSDLSLKD, ADSKGNYASTGVSRSEHD) of klebsiella pneumoniae and is formed by connecting the epitopes (GGGGS, GGGGS, GSGGSG). The invention also discloses a preparation method and application of the antigen protein. The antigen protein prepared by the method can effectively stimulate organisms to generate higher humoral immune response and cellular response, can provide obvious protection effect on killing dose of Klebsiella pneumoniae infection, and can be used as candidate antigens of Klebsiella pneumoniae vaccine.

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Drawings

The above and other features, properties and advantages of the present invention will become more apparent from the following description in conjunction with the accompanying drawings and embodiments.

FIG. 1 shows the results of double digestion assay of recombinant plasmid pGEX-6p-1-mHla-EpiVac

Lane M, molecular weight standard (Marker) of nucleic acid (DNA) with sizes of 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100 bp from top to bottom, lane 1, identification of recombinant expression plasmid pGEX-6p-1-mHla-EpiVac after double digestion with Nde1 and Xho1, about 5900 bp and about 1020 bp of fragments isolated after digestion, lane 2, plasmid pGEX-6p-1-mHla-EpiVac.

FIG. 2 shows the identification result of mHla-EpiVac protein induced expression

Lane M, protein molecular weight standard (Marker), size 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa, 10 kDa, respectively, from top to bottom, lane 1, broken supernatant, lane 2, broken pellet, lane 3, bound effluent, lane 4, bound filler, lane 5, cleaved protein, lane 6, cleaved filler. The target protein is about 37kDa in size and is expressed in both supernatant and precipitate, but the protein has a large number of impurities and requires further purification.

FIG. 3 SDS-PAGE result of purified mHla-EpiVac protein

Lane M shows the molecular weight standard (Marker) of 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, 15 kDa and 10 kDa from top to bottom, and lane 1 shows mHla-EpiVac protein after cation exchange chromatography with better purity.

FIG. 4 specific IgG antibody titres to EpiVac after three immunizations

After three immunizations, specific IgG antibody titers against EpiVac were obtained, mHla-EpiVac immunization effectively increased antibody production, while EpiVac immunization produced little corresponding antibody.

FIGS. 5A to 5C of FIG. 5 spleen cell ELISPot after three immunizations

The specific cell number of IL-4 and IFN-gamma is measured, spleen cells of mice 7 days after three immunizations are stimulated again, and the result obtained by photographing and statistics after 40 hours of stimulation is proved to be effective cell immunity induction by experiments mHla-EpiVac.

FIG. 6A to FIG. 6C of FIG. 6, protective Effect on mice after immunization mHla-EpiVac

Mice were given a lethal dose of YBQ x 10 ⁶ CFU lung infection 7 days after three immunizations, and then observed for ten days for survival, body weight, and clinically scored. Experiments show that the protection rate of mHla-EpiVac alone can reach 50%, the protection rate of the aluminum adjuvant can reach 80%, the immunization effect of EpiVac is equal to that of PBS, the body weight of the immunized mHla-EpiVac mice is small, the recovery is rapid, and the clinical grading state is good.

FIG. 7 uptake of mHla-EpiVac by mice BMDCs

Immature mice BMDCs were incubated with mHla-EpiVac and EpiVac for 10h, respectively, and experiments demonstrated that immature mice BMDCs had more uptake of mHla-EpiVac and less uptake of EpiVac.

Detailed Description

The invention is further described below with reference to the drawings and examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The strains and various reagents used in the invention are as follows:

Plasmid pGEX-6p-1, E.coli strain BL21 were stored as the unit of the inventors, restriction enzymes Nde I and Xho I, protein Marker were the products of company TakaRa, plasmid extraction kit and gel recovery kit were the products of company Omega, U.S.A., and cation exchange chromatography column (HITRAPSP HP ml) and hydrophobic chromatography column (HITRAP PHENYL HP ml) were the products of company GE HEALTHCARE, U.S.A.

The inventors have studied and found 5 outer membrane proteins OMPs, ompW, fepA, ompK, ompA, ompK36, respectively, from KP outer membrane proteins by reverse vaccinology. The structure of the membrane is screened to obtain 22 loop structures and sequences thereof, serum of 700721 standard strain infection recovery period is used for screening by ELISA method, and three high immunogenicity epitopes of FepA396-423, ompA148-165 and OmpW144-163 are obtained.

Step of ELISA screening for strongly immunogenic loop:

1) Diluting the purified 22 loop proteins to 5 ug/mL by using coating liquid;

2) Coating, namely adding recombinant protein diluent into an ELISA plate, washing 3 times with a washing solution after the dilution is 100 mu L/hole at 4 ℃ overnight, wrapping the diluted recombinant protein diluent with a preservative film after the diluted recombinant protein diluent is air-dried, and placing the recombinant protein diluent in a refrigerator at 4 ℃ for standby;

3) Sealing, namely adding 200 mu L/hole of sealing liquid into an ELISA plate, placing the ELISA plate into a 37 ℃ incubator for 2 hours, and washing for 3 times;

4) Serum from the third day after infection of the klebsiella pneumoniae 700721 strain is diluted 1:200;

5) Taking a sealed ELISA plate, sequentially adding diluted serum and 100 mu L/hole, placing the diluted serum and the hole in a 37 ℃ incubator 1h, washing for 3 times, and air-drying;

6) Diluting the goat anti-mouse IgG antibody preservation solution added with HRP label by 1:10000 to prepare an antibody working solution;

7) Adding diluted antibody working solution, 100 mu L/hole, placing in a 37 ℃ incubator 40 min, washing for three times, and air drying;

8) Adding 100 mu L/hole of a substrate color development solution (TMB), and reacting for 5min at room temperature in a dark place;

9) Stop solution (2M H ₂SO₄) was added and immediately placed on an microplate reader to determine the OD at a wavelength of 450nm, screening for higher OD450 loop.

The 3 epitopes are fused and expressed as fusion antigen EpiVac, then mHla and EpiVac are fused to form antigen protein mHla-EpiVac, the antigen and aluminum adjuvant are combined to prepare vaccine immunized mice for three times, the toxicity attack protection rate of the high virulence strain YBQ screened in the early stage of a laboratory can reach 80%, the mHla-EpiVac can induce high-efficiency humoral immune response after the titer is measured, and ELISPot experiments prove that the protein can induce high-efficiency cellular immunity and can be used as effective vaccine candidate antigens.

Example 1 Synthesis and subcloning of genes

1. The synthesis of the amino acid sequence encoding mHla-EpiVac (SEQ ID NO: 1) and the ligation of the sequence to pGEX-6p-1 were synthesized by the company Wohan Jin Kairui bioengineering Co., ltd.

2. Transformation of recombinant plasmid 1 tube E.coli BL21 competent cells (Shanghai Biotechnology Co., ltd.) were taken from a-80℃refrigerator and 4. Mu.l of the synthetic pGEX-6p-1-mHla-EpiVac plasmid was added. Ice bath 30min, hot impact at 42 ℃ for 90s in metal bath, and rapid ice bath 2min. 600 μl of LB blank medium was added, mixed well and placed in a 37℃shaker at 220rpm for 1h. Mu.l of the bacterial liquid was applied to an ampicillin-resistant LB plate. Plates were placed in an incubator at 37℃for 14h. Well-separated colonies on the transformation plates were picked and inoculated in ampicillin-resistant LB medium and shake-cultured overnight at 37 ℃.

3. Double enzyme cutting identification

The plasmid of the positive clone was extracted by a rapid plasmid miniprep kit (Tiangen Biotechnology Co., ltd.) according to the procedure of the specification by shaking overnight at 37 ℃. Digestion was performed using Nde1 (Takara Co.) and Xhol (Takara Co.) in a 37℃water bath for half an hour. The system is as follows:

Reagent(s)	Volume (ul)
		plasmid DNA o	3
NdeI	0.5
		XhoI	0.5
10×buffer(H)	1
		ddH₂O	5
total volume	10

A1.0% agarose gel was prepared by casting, 0.5ug/ml of EB (Shanghai Jun Cheng Biotechnology Co., ltd.) was added to each of the above cleavage reaction systems, 1. Mu.l of 6X Loading buffer was added, and after electrophoresis at 20: 20 min on gel 80V, the result of cleavage was observed by a UV scanner. As a result, the plasmid of the positive clone was found to be cut into 2 fragments, the large fragment of about 5900 bp being part of the expression vector pGEX-6p-1 and the small fragment of about 1020 bp being the inserted DNA fragment encoding mHla-EpiVac (FIG. 1).

Example 2 identification of the expression form of the mHla-EpiVac antigen protein

1. MHla-EpiVac induced expression

Taking 100 mu L of pGEX-6p-1-mHla-EpiVac/BL21 bacterial liquid cultured overnight, adding into 10 mL Ampicillin resistant LB culture medium, culturing at 180 rpm and 37 ℃ for 5h until the OD600 is 0.6-0.8, adding IPTG to the final concentration of 200 mu M, placing in a shaking table for induced expression, and carrying out 16 ℃ induced expression for 14h. Taking out the bacterial liquid after induced expression, centrifuging 5min at 10000 rpm, discarding the supernatant, adding 1mL PBS, mixing uniformly, performing ultrasonic lysis for 3min, centrifuging at 12000rpm at 4 ℃ for 15min, and separating the supernatant and the precipitate.

2. SDS-PAGE electrophoresis

Pouring 10% separating gel into a gel plate, adding distilled water to flatten the gel, standing at room temperature for 30min to solidify, pouring distilled water on the upper layer to dry, pouring concentrated gel, immediately inserting comb, and standing at room temperature for 30min to solidify. And (5) taking 10 mu L of the treated samples respectively, and performing SDS-PAGE electrophoresis. The voltage is firstly 80v for 30min, then 180v is regulated, after electrophoresis for 1-2 h, the gel is taken out, put into transient blue staining solution for oscillation staining, put into primary water for oscillation decolorization, and then the result is observed under an imaging system, pGEX-6p-1-mHla-EpiVac/BL21 is expressed in a soluble form under the induction of 16 ℃ condition, and a large amount of protein is also present in the sediment (figure 2).

Example 3 preparation of mHla-EpiVac protein antigen

1. Obtaining protein by amplifying culture

Taking 100 mu L of pGEX-6p-1-mHla-EpiVac/BL21 bacterial liquid stored in a refrigerator at 4 ℃ for standby, adding the bacterial liquid into 10mL of LB culture medium containing AMPICILLIN resistance for primary activation, culturing at 220rpm for 4-5h, adding 10mL of the primary activated bacterial liquid into 2000mL of LB culture medium containing AMPICILLIN resistance for secondary activation, adding 400 mu L of IPTG (with the final concentration of 200 mu M) when the bacterial liquid is cultured at 37 ℃ for 4-5h until the OD600 is 1.0, placing the bacterial liquid in a 16 ℃ shaking table for 14h after induction, centrifuging at 10000rpm for 15min, collecting bacterial bodies, adding 50mL of PBS (same as in example 2) for re-suspending bacterial bodies, performing ultrasonic lysis on the bacterial liquid for 3min (200V), and collecting supernatant for subsequent purification.

2. MHla-EpiVac protein purification

After washing Glutathione Sepharose mL of the affinity filler with 20mM PBS, pH7.5 for 3 times, the prepared supernatant was mixed with the affinity filler and combined at room temperature for 1 hour. Unbound supernatant was removed by air gravity column, the affinity packing was washed 3 times with PBS, and 1mg of PreScission protease was added in appropriate amount for excision of GST tag, and digested at room temperature for 2h. After the cleavage, the supernatant was collected for ion exchange chromatography.

Taking a cation exchange chromatographic column (HITRAPSP HP ml), adopting a PBS (same as in example 2) balance chromatographic system and an SP HP chromatographic column, loading the supernatant, and carrying out linear gradient elution by using a wash buffer (50 mM PB, pH 6.25, 1M NaCl), wherein the elution flow rate is set to be 5ml/min, the elution gradient is from 0 to 100% of the wash buffer, and the elution volume is 50ml. The eluted target protein is collected and stored at 4 ℃ for standby. The result of electrophoresis is shown in FIG. 3.

Example 4 immunization and antibody detection of animals

The vaccine was prepared by diluting mHla-EpiVac antigen with PBS and adding Al (OH) ₃ at a concentration of 1mg/mL, immunizing BALB/C mice with 5-gauge semi-needle by double-sided thigh intramuscular injection at 0, 14 and 21 days, each with 100 μl of antigen, immunizing blank with PBS of the same volume at 30 μl, collecting tail vein blood of BALB/C mice at 7 days after the last immunization, and detecting antigen-specific IgG response level after immunization of mice by ELISA.

1. Preparation of liquid

1) Preparing a coating liquid, namely weighing up 2.9g of Na ₂CO₃1.6g,NaHCO₃, dissolving in 1L of ddH ₂ O, and adjusting the pH to 9.6 by a PH meter;

2) Preparing a blocking solution, namely dissolving 1g of bovine serum albumin in 100mL of antibody diluent (1:100);

3) Preparing antibody diluent, namely dissolving phosphate in 1L ddH ₂ O, adding 500 mu L Tween 20, and regulating the pH to 7.4 by a PH meter;

4) Preparation of washing solution with antibody diluent

5) Color development liquid (TMB) is a product of Tiangen corporation;

6) Preparation of stop solution (2M H ₂SO₄) 22.2mL of concentrated sulfuric acid was poured into 177.8mL of ddH ₂ O.

2. ELISA detection mHla-EpiVac recombinant protein immune mouse generated antibody titer

1) Diluting the purified mHla-EpiVac, fepA396-423, ompA148-165 and OmpW144-163 proteins to 5 ug/mL by using a coating solution;

4) Serum was diluted at 1:1000, 1:2000, 1:4000, 1:8000, etc.;

9) Adding a stop solution (2M H ₂SO₄), immediately placing on an enzyme-labeled instrument, and measuring an OD value at a wavelength of 450 nm;

10 Results were judged positive for a _{Sample of}∕A_{Negative of} value ∈ 2.1 (negative control is 1:1000 dilution of serum before mouse immunization).

As a result, the geometrical average titer of the EpiVac specific IgG antibody produced by the mHla-EpiVac protein antigen immunized mice is 1:4096000, the geometrical average titer of the EpiVac specific IgG antibody produced by the combination of mHla-EpiVac and aluminum adjuvant is 1:8192000, the geometrical average titer of the specific IgG antibody against FepA396-423, ompA148-165 and OmpW144-16 is 1:8192000, 1:1024000, 1:2048000 respectively, and the positive rate of the antibody reaches 100% on the 7 th day after the last immunization (figure 4), which shows that the mHla-EpiVac recombinant protein constructed by the invention has good immunogenicity.

Example 5 animal immunization and challenge protection experiments

The preparation method comprises the steps of diluting mHla-EpiVac antigen with PBS, adding Al (OH) ₃ with the concentration of 1mg/mL to prepare a vaccine, injecting BALB/C mice by intramuscular injection of 5-gauge semi-needles on the thighs at days 0, 14 and 21, wherein the injection amount of each mouse is 100 mu L, the antigen content is 30 mu L, a blank group is immunized with PBS with the same volume, the mice are subjected to virus attack by adopting klebsiella pneumoniae clinical strain YBQ with the dosage of 8X 10 ⁶ CFU on the 7 th day after final immunization in a tracheal instillation mode, the survival state of the mice is observed, the observation period is 10 days, the death number of the mice is recorded every day, and the survival rate of the mice is calculated after the observation period is ended. The results of three continuous animal experiments show that compared with a control group, the survival rate of mHla-EpiVac immunized mice is obviously improved, the protection rate is about 50%, and the survival rate can reach 80% after the mice are combined with an aluminum adjuvant (shown in fig. 6A to 6C), so that the mice can be used as candidate antigens for vaccine research and development.

EXAMPLE 6 ELISpot

Spleens of mice 7 days after three immunizations were taken, homogenized under sterile conditions, red-split using erythrocyte lysate, adjusted to a cell concentration of 1×10 ⁷, and subsequently added to ELISpot plates (MabTech).

1) Each group of treatments:

(1) Activation of the pre-coated plate, which is to add 200uL of sterile PBS to wash each well, leave it for two minutes, then repeat 4 times, then add 200uL of 1640 complete medium to each well, leave it for 30 minutes at room temperature, and leave it out.

(2) Cell suspension was added by adding the cell suspension at the adjusted concentration to each experimental well, 100. Mu.L/well.

(3) Stimulus was added at 100. Mu.L/well as follows:

Positive control wells were added with positive stimulant working fluid.

Negative control wells, adding medium (or medium for resuspension of cells);

Experimental wells mHla-EpiVac and EpiVac (formulated in serum-free medium or RPMI 1640) were added.

2) Color development

(1) The plate was emptied to remove cells and washed 5 times with 200 μl/well of PBS.

(2) The detection antibody (R4-6A 2-biotin) was diluted to 1. Mu.g/ml in PBS containing 0.5% fetal calf serum (PBS-0.5% FCS). 100 μl/well was added and incubated for 2 hours at room temperature

(3) 200 Μl/well of PBS was washed 5 times.

(4) Streptavidin HRP (1:1000) was diluted in PBS-0.5% FCS and 100 μl/well was added. Incubate for 1 hour at room temperature.

(5) 200 Μl/well of PBS was washed 5 times.

(6) 100 Μl/well of the ready-to-use TMB matrix solution was added and developed until a visible spot appeared.

(7) The color development was stopped by extensive washing in deionized water. The rack is removed from the plastic tray and the underside of the membrane is then rinsed.

(8) ELISPOT plate spot counts and records various parameters of spots for statistical analysis

As a result, mHla-EpiVac immunization induced specific IL-4 and IFN-gamma cells, and further IL-4 and IFN-gamma secreting cells were produced by restimulation with mHla-EpiVac (FIGS. 5A-5C of FIG. 5), indicating that mHla-EpiVac immunization resulted in specific cellular immunity.

EXAMPLE 7 laser Cofocal

MHla-EpiVac and EpiVac were fluorescent according to the procedure of fluorescent dye AF488, fluorescent mHla-EpiVac and EpiVac were diluted at a concentration of 40. Mu.g/ml using 1640 medium containing 10% FBS, then incubated with 1X 10 ⁵ of immature BMDCs mice in confocal dishes for 10h in the dark, medium was discarded, washed 3 times with 37℃pre-warmed PBS, and fixed with 4% paraformaldehyde for 15 min. PBS was washed 3 times, 3% BSA and Triten-100 in PBS was added and allowed to pass through for 15 min, discarded, PBS was washed 3 times, 100nmol/l of phalloidin (absin) was used for staining for 10 min, discarded, PBS was washed 3 times, DAPI (Biyun) was used for staining for 5 min, discarded, PBS was washed 3 times, 100ul of PBS was added, and the mixture was observed on the machine.

As a result, mice immature BMDCs ingest mHla-EpiVac and EpiVac at the same time and concentration, more of mHla-EpiVac was ingested (FIG. 7), indicating that mHla-EpiVac promoted antigen uptake by immature BMDCs.

The above description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention, but the invention is modified or equivalent to be included in the scope of the appended claims without departing from the spirit and scope of the invention.

Claims

1. A klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac is characterized in that the amino acid sequence of the fusion antigen mHla-EpiVac is shown as SEQ ID NO. 1.

2. The fusion antigen mHla-EpiVac of claim 1, wherein the fusion antigen comprises three immunodominant epitopes of klebsiella pneumoniae origin.

3. The fusion antigen mHla-EpiVac of claim 2, wherein the amino acid sequences of the three immunodominant epitopes are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively.

4. The method for preparing the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of any of the preceding claims, comprising the steps of:

1) mHla-EpiVac synthesis and subcloning;

2) mHla-EpiVac;

3) mHla-EpiVac protein antigen is prepared;

4) mHla-EpiVac protein purification.

5. The method of claim 4, wherein step 1) comprises ligating mHla to the sequence shown in SEQ ID NO.2 with a flexible chain, then ligating the sequence shown in SEQ ID NO. 4 with a flexible chain, and then ligating the sequence shown in SEQ ID NO. 3 with a flexible chain to form fusion antigen mHla-EpiVac.

6. The method of claim 4 or 5, wherein step 1) comprises constructing a recombinant expression plasmid using ampicillin-resistant prokaryotic expression plasmid pGEX-6 p-1.

7. The method of claim 4 or 5, wherein step 4) comprises collecting genetically engineered bacteria expressing mHla-EpiVac, disrupting the bacteria at high pressure, centrifuging, cation exchange chromatography, and purifying mHla-EpiVac by a sequential combination of hydrophobic chromatography.

8. Use of the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of claim 1 in the preparation of an immune formulation for preventing or treating klebsiella pneumoniae infection.

9. A klebsiella pneumoniae injectable immune formulation comprising the klebsiella pneumoniae vaccine fusion antigen mHla-EpiVac of claim 1, and any one or any combination of an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, an MF59, a complete freund's adjuvant, an incomplete freund's adjuvant, and a mycobacterial bcg adjuvant.