CN103694355B - The recombinant antibodies of anti-human cardiac muscle troponin I and construction method thereof and application - Google Patents

Abstract

The present invention relates to a recombinant antibody against human cardiac troponin I, which comprises a light chain composed of the light chain variable region of the mouse antibody against human cardiac troponin I and the light chain constant region of human IgG1 and composed of A heavy chain composed of the heavy chain variable region of the murine antibody against human cardiac troponin I and the heavy chain constant region of human IgG1. Compared with the mouse-derived antibody, the recombinant antibody not only retains the specificity and affinity of the parent antibody's recognition antigen, but also avoids non-specific reaction with the anti-mouse antibody in human serum. Therefore, from the perspective of clinical diagnostic application , the above-mentioned recombinant antibody has more application value than the mouse-derived antibody. In addition, the present invention also relates to a method for constructing a recombinant antibody against human cardiac troponin I and the application of the recombinant antibody.

Description

Recombinant antibody against human cardiac troponin I and its construction method and application

technical field

The present invention relates to the field of antibody engineering, in particular to a recombinant antibody against human cardiac troponin I and its construction method and application.

Background technique

Before the 1980s, the World Health Organization (WHO) has always regarded myocardial enzyme activity as one of the diagnostic criteria for acute myocardial infarction (AMI). In the late 1980s, researchers discovered that the sensitivity and specificity of troponin (Tn) was higher than that of phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), lactate dehydrogenation enzymes and biomarkers such as aspartate aminotransferase. Cardiac troponin I (cTnI) exists only in the myocardium and is a marker of cardiomyocytes. Its abnormal changes can affect the diastolic and systolic functions of the heart, and can be used to diagnose myocardial necrosis and judge myocardial injury. One of the most specific markers is recognized as the main biochemical marker for the rapid diagnosis of AMI and acute coronary syndrome (acute coronary syndromes, ACS), as well as for assisting ACS risk stratification and reflecting its prognosis.

The cTnI content in normal blood is generally lower than 0.3 μg/L. When the integrity of the myocardial cell membrane is damaged due to ischemia or hypoxia, free cTnI can quickly penetrate the cell membrane and enter the blood stream. Therefore, the rapid, sensitive and accurate determination of cTnI in human blood and its changing trend in the early stage of the disease is of great importance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, and monitoring of myocardial injury caused by various factors. clinical significance. Clinical methods for detecting cTnI levels include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold, etc. Different methods have their own advantages and disadvantages, but they all require specific monoclonal antibodies against cTnI. Traditional clinical diagnosis uses monoclonal antibodies (mAbs) of mouse origin.

For a long time, mouse-derived monoclonal antibodies have been widely used in scientific research, clinical diagnosis and treatment, but because mouse-derived mAbs can cause human anti-mouse antibody (HAMA) reactions, their clinical application is limited. Therefore, it is very necessary to humanize murine antibodies. Since the 1980s, the development trend of monoclonal antibodies used for treatment has been from murine antibodies to humanized antibodies to fully human antibodies. Nearly half of the antibody drugs approved by the FDA are human. derivatized antibodies. In the field of clinical diagnosis, the mouse-derived antibodies are basically used now, but because people usually come into contact with items that rodents have touched, HAMA reactions may appear in the blood, and human anti-mouse antibodies exist. Nonspecific reactions can occur in the diagnosis, that is, false positives can occur.

Contents of the invention

Based on this, it is necessary to provide a recombinant antibody against human cardiac troponin I capable of significantly reducing the probability of false positives, its construction method and application.

A recombinant antibody against human cardiac troponin I, comprising a light chain composed of the light chain variable region of the mouse antibody against human cardiac troponin I and the light chain constant region of human IgG1 and an anti-human cardiac troponin I A heavy chain consisting of the heavy chain variable region of the murine antibody to calprotein I and the heavy chain constant region of human IgG1.

In one of the embodiments, the murine antibody against human cardiac troponin I is produced by hybridoma cells with deposit number CCTCC C2013185.

In one of the embodiments, the light chain variable region contains the amino acid sequence shown in SEQ ID NO.1.

In one of the embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.2.

A construction method of anti-human cardiac troponin I recombinant antibody, comprising the steps of:

Construct expression vectors containing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively, wherein the expression vectors containing the nucleotide sequences shown in SEQ ID NO.3 contain the light chain of human IgG1 The expression gene of the constant region, the expression vector containing the nucleotide sequence shown in SEQ ID NO.4 contains the expression gene of the heavy chain constant region of human IgG1;

Transfecting the expression vector into the same host cell;

The anti-human cardiac troponin I recombinant antibody is recovered from the host cell.

In one of the embodiments, the expression vector to be inserted into the nucleotide sequence shown in SEQ ID NO.3 is reserved with XbaI and PmlI double restriction sites; to be inserted into the nucleotide sequence shown in SEQ ID NO.4 The expression vector of the acid sequence has reserved NheI and HindIII double restriction sites.

In one of the embodiments, the nucleotide sequence shown as SEQ ID NO.3 is prepared by the following steps:

RNA was extracted from hybridoma cells with the preservation number CCTCC C2013185, and RT-PCR was performed with a reverse transcription kit. The RT-PCR amplification product was inactivated at 70°C as a template, and SEQ ID NO.5 and SEQ ID NO. Perform PCR with the primer sequence shown in 6, recover the PCR product, insert the PCR product into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transform it into DH5α competent cells for expression;

Recovering the product expressed by DH5α competent cells, using the primer sequences shown in SEQ ID NO.7 and SEQ ID NO.8 to perform PCR to obtain the nucleotide sequence shown in SEQ ID NO.3;

The nucleotide sequence shown as SEQ ID NO.4 is prepared by the following steps:

RNA was extracted from hybridoma cells with the preservation number CCTCC C2013185, and RT-PCR was performed with a reverse transcription kit. The RT-PCR amplification product was inactivated at 70°C as a template, and SEQ ID NO.9 and SEQ ID NO. Perform PCR with the primer sequence shown in 10, recover the PCR product, insert the PCR product into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transform it into DH5α competent cells for expression;

The product expressed by DH5α competent cells was recovered, and PCR was performed using the primer sequences shown in SEQ ID NO.11 and SEQ ID NO.12 to obtain the nucleotide sequence shown in SEQ ID NO.4.

A DNA encoding the amino acid sequence shown in SEQ ID NO.1.

A DNA encoding the amino acid sequence shown in SEQ ID NO.2.

A eukaryotic cell expression vector comprising the DNA encoding the amino acid sequence shown in SEQ ID NO.1 and/or the DNA encoding the amino acid sequence shown in SEQ ID NO.2.

A host cell transfected with the eukaryotic expression vector described above.

Application of the above-mentioned recombinant antibody in the preparation of detection reagents or detection instruments for detecting cardiac troponin I.

A test paper for detecting cardiac troponin I, comprising the above-mentioned recombinant antibody.

In one of the embodiments, the detection test paper includes a supporting sheet, a sample pad, a gold standard pad, a nitrocellulose membrane, an absorption pad, a detection line and a quality control line, the sample pad, the gold standard pad, the The nitrocellulose membrane and the absorbent pad are sequentially arranged on the support sheet from one end to the other end of the support sheet, the sample pad partially overlaps with the gold standard pad, the gold standard pad overlaps with the nitric acid The cellulose membrane is partially overlapped, the nitrocellulose membrane is partially overlapped with the absorbent pad, the detection line and the quality control line are arranged on the nitrocellulose membrane, and the detection line is arranged near the One end of the gold label pad, the quality control line is located near the end of the absorbent pad, the gold label pad is coated with the recombinant antibody wrapped with colloidal gold particles to form a colloidal gold-labeled recombinant antibody, the detection line It is an affinity-purified rabbit polyclonal antibody against cardiac troponin I, and the quality control line is a goat anti-mouse IgG antibody.

A detection kit, comprising the above-mentioned cardiac troponin I detection test paper.

Compared with the mouse-derived antibody, the above-mentioned recombinant antibody not only retains the specificity and affinity of the parent antibody's recognition antigen, but also avoids non-specific reaction with the anti-mouse antibody in human serum. Therefore, from the perspective of clinical diagnostic application , the above-mentioned recombinant antibody has more application value than the mouse-derived antibody.

Description of drawings

Fig. 1 is the front schematic view of the detection test paper of an embodiment;

Fig. 2 is the longitudinal section schematic diagram of detection test paper in Fig. 1;

Figure 3 is a schematic diagram of the detection kit;

Figure 4 is the electrophoresis pattern of gene fragments containing _VH and _VL amplified by using the cDNA obtained after reverse transcription of RNA extracted from hybridoma cells as a template, using heavy chain and light chain degenerate primers, and the mKF5/mKR primers of _VL A band of about 420bp is amplified by the pair of mHF2/mHR primers for V and _H , and M is DL2000DNAMarker;

Figure 5 is a recombinant antibody eukaryotic expression vector, a is pFP-IgCK, b is pFP-IgCH;

Figure 6 is the V _L (395bp) and V _H (405bp) gene fragments amplified with V _L and V _H specific primers, M is DL2000DNAMarker;

Figure 7 is the breakthrough peak and absorption peak when the recombinant antibody is purified with proteinG affinity chromatography column;

Figure 8 is the SDS-PAGE analysis of purified recombinant antibody, a is 4 μg of mouse antibody, b is 4 μg of recombinant antibody, and M is Protein Marker.

detailed description

The recombinant antibody against human cardiac troponin I of the present invention and its construction method and application will be further described in detail below in conjunction with the accompanying drawings and specific examples.

In one embodiment, the recombinant antibody against human cardiac troponin I comprises the light chain variable region (L chain V region, V _L ) of the mouse antibody (mAb) against human cardiac troponin I (hcTnI) and The light chain composed of the light chain constant region of human IgG1 (L chain C region, _CL , such as the k chain C region (C _k ), etc.) and the heavy chain variable region of the mouse antibody against human cardiac troponin I (H chain V region, V _H ) and heavy chain constant region ( _H chain C region, CH ) of human IgG1.

Among them, the above-mentioned mouse-derived antibody against human cardiac troponin I is produced by a hybridoma cell with the preservation number CCTCC C2013185, and the hybridoma cell is named CTNI-C4, which was deposited in Wuhan City, Hubei Province on November 13, 2013. Wuchang Luojia Mountain Collection Center of Wuhan University (namely the Chinese Type Culture Collection Center). The above light chain variable region contains the amino acid sequence shown in SEQ ID NO.1, and its corresponding DNA sequence is SEQ ID NO.3 in the sequence listing; the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.2 The corresponding DNA sequence is SEQ ID NO.4 in the sequence listing.

The sequence of SEQ ID NO.3 is: GAT GTTTTGATGA CCCAAACTCC ACTCTCCCTG CCTGTCAGTC TTGGAGATCA GCCTCCATC TCTTGC TGGTAC CTGCAGAAAC CAGGCCAGTC TCCAAAACTC CTGATCTAC GGGGTCCCAG ACAGGTTCAG TGGCAGTGGA TCAGGGACAG ATTTCACACT CAAGATCAGC AGAGTGGAGG CTGAGGATCT GGGAGTTTAT TACTGC TTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG . The sequence of SEQ ID NO.4 is: CAG GTCCAACTGC AGCAGCCTGG GTCTGAACTG GTGAGGCCTG GGGCTTCAGT GAAGCTGTCC TGCAAGGCTT CTGGCTACAC CTTCACC TGGGTGAA GCAGAGGCCT GGACAAGGCC TTGAATGGAT TGGT AAGGC CACATTGACT GTAGACAAAT CGTCCAGCGC AGCCTACATG CACCTCAACA GCCTGACATC TGAGGACTCT GCGGTCTATT ACTGTGCACA A TGGGGTCA AGGAACCTCA GTCACTGTCT CTGCA . Among them, the sequence marked with an underline is the signal peptide (Signal Peptide, SP) sequence of the antibody, the sequence marked with a solid underline is the framework region (framework Region, FR) sequence, and the sequence in italics is the complementarity determining region (complementary determining region, CDR) sequence , the hypervariable region.

This embodiment also provides a method for constructing an anti-human cardiac troponin I recombinant antibody, comprising the following steps:

Construct expression vectors containing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively, wherein the expression vectors containing the nucleotide sequences shown in SEQ ID NO.3 contain the light chain of human IgG1 The expression gene of the constant region, the expression vector containing the nucleotide sequence shown in SEQ ID NO.4 contains the expression gene of the heavy chain constant region of human IgG1;

Transfect the expression vector into the same host cell;

The anti-human cardiac troponin I recombinant antibody was recovered from the host cells.

Wherein, the expression vector to be inserted into the nucleotide sequence shown in SEQ ID NO.3 has reserved XbaI and PmlI double restriction sites; the expression vector to be inserted into the nucleotide sequence shown in SEQ ID NO.4 NheI and HindIII double enzyme cutting sites are reserved.

The nucleotide sequence shown as SEQ ID NO.3 is prepared by the following steps:

RNA was extracted from hybridoma cells with the preservation number CCTCC C2013185, and RT-PCR was performed with a reverse transcription kit. The RT-PCR amplification product was inactivated at 70°C as a template, and SEQ ID NO.5 and SEQ ID NO. Perform PCR with the primer sequence shown in 6, recover the PCR product, insert the PCR product into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transform it into DH5α competent cells for expression;

Recovered DH5α Competent Cells Expressing

The product was subjected to PCR using the primer sequences shown in SEQ ID NO.7 and SEQ ID NO.8 to obtain the nucleotide sequence shown in SEQ ID NO.3.

The nucleotide sequence shown as SEQ ID NO.4 is prepared by the following steps:

RNA was extracted from hybridoma cells with the preservation number CCTCC C2013185, and RT-PCR was performed with a reverse transcription kit. The RT-PCR amplification product was inactivated at 70°C as a template, and SEQ ID NO.9 and SEQ ID NO. Perform PCR with the primer sequence shown in 10, recover the PCR product, insert the PCR product into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transform it into DH5α competent cells for expression;

The product expressed by DH5α competent cells was recovered, and PCR was performed using the primer sequences shown in SEQ ID NO.11 and SEQ ID NO.12 to obtain the nucleotide sequence shown in SEQ ID NO.4.

In this embodiment, the light chain and heavy chain variable region genes of the anti-hcTnI antibody were cloned from the self-constructed and cultured anti-hcTnI mouse monoclonal antibody hybridoma cell line (CCTCC C2013185) using a set of designed primers. The sequences of the sequenced light and heavy chain variable regions were analyzed in the IMGT antibody database, and the results showed that the VH gene (heavy chain variable region gene) had a high homology with the mouse VH gene in the database, and It belongs to the mouse VH1 gene family; the VL gene (light chain variable region gene) has high homology with the mouse VL gene in the database, and belongs to the mouse Vk2 gene family.

In addition, this embodiment also provides an expression vector comprising DNA encoding the sequence of SEQ ID NO.3 and/or SEQ ID NO.4, such as pFP-IgCH and pFP-IgCK or other eukaryotic expression vectors commonly used in genetic engineering , and the host cells transfected with the DNA expression vector, such as Freestyle CHO-S cells or other expression cells commonly used in genetic engineering.

The above-mentioned recombinant antibody can be widely used in the field of preparing detection reagents or detection instruments for detecting cardiac troponin I. For example, a detection kit for cardiac troponin I, which includes a casing, detection test paper and other detection reagents.

As shown in FIGS. 1 and 2 , the detection test paper 100 of this embodiment includes a support sheet 110 , a sample pad 120 , a gold standard pad 130 , a nitrocellulose membrane 140 , an absorbent pad 150 , a detection line 160 and a quality control line 170 . The sample pad 120 , the gold standard pad 130 , the nitrocellulose membrane 140 and the absorbent pad 150 are sequentially arranged on the support sheet 110 from one end to the other end of the support sheet 110 . The sample pad 120 partially overlaps the gold standard pad 130 , the gold standard pad 130 partially overlaps the nitrocellulose membrane 140 , and the nitrocellulose membrane 140 partially overlaps the absorbent pad 150 . The detection line 160 and the quality control line 170 are set on the nitrocellulose membrane, and the detection line 160 is set at the end close to the gold standard pad 130 , and the quality control line 170 is set at the end close to the absorbent pad 150 . The support sheet 110 is made of non-absorbent material. The sample pad 120 is used for sample application. The colloidal gold-labeled recombinant antibody is evenly coated on the gold label pad 130 by encapsulating the colloidal gold particles with the recombinant antibody. The detection line 160 is the affinity-purified rabbit polyclonal antibody against cardiac troponin I, and the quality control line 170 is the goat anti-mouse IgG antibody.

As shown in FIG. 3 , the test strip 100 can be placed in the casing 200 of the test kit. The casing 200 is provided with a sample injection hole 210 and an observation window 220 . The sample injection hole 210 corresponds to the position of the sample pad 120 . The detection line 160 and the quality control line 170 are exposed in the observation window 220 for convenient observation.

Other detection reagents can be prepared directly in the laboratory as needed.

The above detection kit utilizes the double antibody sandwich method to detect cardiac troponin I in the material to be tested. During detection, all cTnI in the sample first binds to the recombinant antibody labeled with gold-labeled anti-cardiac troponin I. Due to capillary action, the reaction complex moves forward along the nitrocellulose membrane 140. If there is cTnI in the sample, it reaches the detection line. At 160, when encountering the rabbit polyclonal antibody against cardiac troponin I coated on the nitrocellulose membrane 140, a rabbit polyclonal antibody-cardiac troponin I-gold-labeled recombinant antibody complex will be formed, thereby enriching in On the detection line 160, a red precipitation line is formed; the gold-labeled recombinant antibody that is not bound to cTnI passes through the detection line 160, is captured by the goat anti-mouse IgG antibody, and is enriched on the quality control line 170, forming a red precipitation line. When there is a red precipitation line on the detection line 160 and the quality control line 170 at the same time, it is judged as a positive result. If the sample does not contain cTnI, when the reaction complex reaches the detection line 160, it will not form a rabbit polyclonal antibody-cTnI-gold-labeled recombinant antibody complex when it encounters the capture polyclonal antibody. The reaction complex passes through the detection line 160 and is only enriched in A red precipitation line is formed on the quality control line 170, and it is judged as a negative result at this time.

In addition, in other embodiments, the structure of the detection kit is not limited to the above description. In addition to being used in the above-mentioned colloidal gold-labeled monoclonal antibody detection kit, the above-mentioned recombinant antibody can also be used in other cardiac troponin I detection kits or equipment. Those skilled in the art can understand that the recombinant antibody of this embodiment is directly or indirectly bound to other signaling groups (such as magnetic microspheres, horseradish peroxidase, etc.), or the recombinant antibody of this embodiment is used as a coating antibody (such as ELISA), can be used in other forms of cardiac troponin I detection reagents or equipment.

Compared with the mouse-derived antibody, the above-mentioned recombinant antibody not only retains the specificity and affinity of the parent antibody's recognition antigen, but also avoids non-specific reaction with the anti-mouse antibody in human serum. Therefore, from the perspective of clinical diagnostic application , the above-mentioned recombinant antibody has more application value than the mouse-derived antibody.

The following is the specific embodiment part:

In this example, the Freestyle CHO-S cells, the transfection reagent FreeStyle ^TM MAX Reagent and the cell culture medium were all purchased from Life Technologies. Prime Star DNA polymerase was purchased from Takara Company. Trizol RNA extraction kit was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. Restriction enzymes were purchased from NEB Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen. Human recombinant cardiac troponin I antigen was produced by Shenzhen Faipeng Biological Co., Ltd., product number AG-CTNI-HP0005.

1. Primer design and synthesis

Amplify V _L gene upstream primer:

mkF1: 5'>ATGGAGACAGACACACTCCTGCTAT<3' (SEQ ID NO. 13);

mkF2: 5'>ATGGATTTTCAAGTGCAGATTTTCAG<3' (SEQ ID NO. 14);

mkF3: 5'>ATGGAGWCACAKWCTCAGGTCTTTRTA<3' (SEQ ID NO. 15);

mkF4: 5'>ATGKCCCCWRCTCAGYTYCTKGT<3' (SEQ ID NO. 16);

mkF5: 5'>ATGAAGTTGCCTGTTAGGCTGTTG<3' (SEQ ID NO. 5).

Amplify _VL gene downstream primers:

mkR: 5'>GGATACAGTTGGTGCAGCATCAGCCCGTTT<3' (SEQ ID NO. 6).

Amplify V _H gene upstream primer:

mHF1: 5'>SAGGTGMAGCTKCASSARTCWGG<3' (SEQ ID NO. 17);

mHF2: 5'>ATGGRATGSAGCTGKGTMATSCTCT<3' (SEQ ID NO.9);

mHF3: 5'>ATGRACTTCGGGYTGAGCTKGGTTTT<3' (SEQ ID NO. 18);

mHF4: 5'>ATGGCTGTCTTGGGGCTGCTCTTCT<3' (SEQ ID NO. 19).

Downstream primers for amplifying the _VH gene:

mHR: 5'>TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA<3' (SEQ ID NO. 10).

2. Antibody variable region gene cloning and sequencing

Extract medium RNA from hybridoma cTnI-C4, use a reverse transcription kit to perform RT-PCR, use the amplified product as a template after enzyme inactivation at 70°C, use the above-mentioned synthesized primers to perform PCR, and amplify 5 tubes of V _L gene , 4 tubes of _VH gene amplification, wherein the mKF5/mKR primer pair of _VL amplified the target band of about 420bp, and the mHF2/mHR primer pair of _VH amplified the target band of about 420bp, as shown in Figure 4 Show. Purified and recovered by agarose gel electrophoresis, the product was subjected to A reaction with rTaq DNA polymerase, then inserted into the pMD-18T vector, transformed into DH5α competent cells, and cloned V _H and V _L genes after colonies were grown. Two clones were sent to Invitrogen for sequencing.

3. Sequence analysis of cTnI-C4 antibody variable region gene

The gene sequence obtained by the above sequencing was analyzed in the IMGT antibody database, and the VNTI11.5 software was used to analyze and confirm that the genes amplified by the primers for the heavy chain and the light chain were correct, and the genes amplified by mKF5/mKR were correct. In the 420bp V _L gene fragment, the V _L gene sequence is 339bp, which belongs to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; in the 423bp V _H gene fragment amplified by the mHF2/mHR primer pair, the V _H gene sequence It is 348bp, belongs to the VH1 gene family, and has a 57bp leader peptide sequence in front of it.

4. Construction of recombinant antibody expression plasmid

The pFP-IgCK and pFP-IgCH vectors are recombinant antibody eukaryotic expression vectors. The former has been inserted into the constant region gene of the human IgG k chain, and XbaI and PmlI restriction sites have been reserved; the latter has been inserted into the constant region of the human IgG1 heavy chain. Region gene, and reserved NheI and HindIII restriction sites, the plasmid map is shown in Figure 5.

According to the above pMD-18T antibody variable region gene sequencing results, design the _VL and _VH gene-specific primers of the cTnI-C4 antibody, with restriction sites and protective bases at both ends. The primers are as follows:

C4-VHF: 5'> GGGGCTAGCATGGAATGGAGCTGTGTCATC <3' (SEQ ID NO. 7);

C4-VHR: 5'>CCC AAGCTT GCTGCAGAGACAGTGACTGAGG<3' (SEQ ID NO. 8);

C4-VKF: 5'>CTAG TCTAGA ATGAAGTTGCCTGTTAGG<3' (SEQ ID NO. 11);

C4-VKR: 5'>CCGTTTCAGTCCAGCTTGG<3' (SEQ ID NO. 12).

The underlined part is the restriction site, the sites at both ends of the V _H are NheI/HindIII, the 5' segment of the V _L gene is XbaI, and the 3' segment is PmlI. This enzyme is a flat-end enzyme, so there is no restriction on the primer. Dots, the fragments amplified by Prime Star DNA Polymerase are blunt-ended.

The 396bp _VL gene fragment and the 405bp _VH gene fragment amplified by PCR are shown in FIG. 6 . The V _L gene was digested with XbaI, pFP-IgCK was digested with XbaI/PmlI, the _VH gene and pFP-IgCH were digested with NheI/HindIII, the fragment and vector were purified and recovered, and the _VL gene was connected to pFP-IgCK In the vector, the V _H gene is connected to the pFP-IgCH vector to obtain the recombinant expression plasmids of the heavy chain and the light chain respectively.

5. The recombinant antibody expression plasmid was transfected into CHO cells, and the product was detected

The day before transfection, inoculate 5×10 ⁵ /mL cells in a 6-well culture plate, use Freestyle CHO Expression Medium containing 8mM glutamine, and culture in a 37°C, 8% CO ₂ incubator with circular shaking at 150rpm for 16-22h , the cell density at the time of transfection was 1×10 ⁶ /mL, transfection of 3mL cells required 3.75 μg of plasmids (1.875 μg of heavy chain and light chain expression plasmids), and 3.75 μL of transfection reagent FreeStyle ^TM MAX Reagent, as recommended by Life Technologies The transfection method is used for transfection. After 72 hours of transfection, samples can be taken to detect the expressed recombinant antibody.

6. Detection of recombinant antibodies expressed in eukaryotes

Dilute human recombinant cardiac troponin I antigen (manufactured by Shenzhen Faipeng Biological Co., Ltd., product number AG-CTNI-HP0005) with 0.06M pH9.6 carbonic acid buffer solution to make the final concentration 8 μg/mL. Add to a 96-well polystyrene plate, 0.1 mL per well, at 37°C for 2 hours or at 4°C overnight. The next day, block with 0.02M pH7.2PB containing 10% calf serum (NBS), 0.15mL/well, at 37°C for 2 hours for detection. On the sixth day after transfection, take 0.1 mL of the cell supernatant and put it in the above-mentioned 96-well detection plate, wash it with water six times at 37°C for 30 minutes, add 2000 times diluted horseradish peroxidase-labeled mouse anti-human IgG-18# ( Shenzhen Faipeng Biological Co., Ltd.), after washing at 37°C for 30 minutes, add 100 μL of 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH 5.0 citric acid to each well Phosphate buffer solution, 37°C for 15 minutes, add dilute sulfuric acid solution, 50 μL per well, measure the absorbance at 450 nm. The cell supernatant co-transfected with the empty vector pFP-IgCk and pFP-IgCH was used as a negative control, and the ratio of the measured value to the control value ≥ 2.0 was judged as positive. The results are shown in Table 1:

Table 1

The results showed that the active recombinant antibody was successfully expressed.

7. Purification of recombinant antibodies

Use the same method to amplify the expression of 500mL. After six days of expression, the cell culture medium was centrifuged at 12,000rpm for 20min. The supernatant was transferred to a clean bottle, and the proteinG affinity chromatography column was used for affinity purification. The purified breakthrough and absorption peaks are shown in Figure 7 shown. After purification, 15 mg of recombinant cTnI-C4 antibody was obtained. 4 μg of the purified antibody was subjected to reducing SDS-PAGE, and 4 μg of mouse cTnI-C4 antibody was used as a control. The electropherogram is shown in FIG. 8 . Two bands were shown after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain).

8. Recombinant cTnI-C4 antibody used in cardiac troponin I colloidal gold detection kit

The detection kit includes detection test paper and sample diluent. Among them, the sample diluent is 8% NaCl solution. Preparation method: 80gNaCl, add distilled water to make up to 1000mL.

The detection test paper is made through the following steps:

A. Preparation of Nitrocellulose Membrane

Preparation of coating buffer: 6% methanol, 0.01M pH7.2 PBS buffer as coating buffer, filtered through 0.22μm membrane, set at 4°C for later use, valid for one week. 1000mL 6% methanol 0.01M pH7.2PBS buffer formulation: NaCl8g, _KCl0.2g , _{Na2HPO4· 12H2O2.9g , KH2PO40.2g} , methanol60mL, distilled deionized water to 1000mL.

Preparation of nitrocellulose membrane: Dilute the anti-cardiac troponin I rabbit polyclonal antibody (manufactured by Shenzhen Faipeng Biological Co., Ltd., product number PAB-CTNI-AP0002) to 1-5 mg/mL with coating buffer, adjust Machine, marked as T line, which is the detection line, the T line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad; the goat anti-mouse IgG antibody (produced by Shenzhen Feipeng Biological Co., Ltd. , Cat. No. BA-PAB-MU0001) diluted to 1-5mg/mL, adjust the machine, draw the line C, which is the control line, and the line C is close to the absorbent pad, about 3mm away from the absorbent pad. The distance between the two lines is 5-8mm, uniform. Dry at 37°C and package for later use.

B. Preparation of colloidal gold and gold-labeled monoclonal antibodies

(1) Preparation of solution

①Preparation of chloroauric acid: Dissolve chloroauric acid in double-distilled deionized water to make a 1% solution, set it at 4°C for standby, and the validity period is four months. 1000mL1% chloroauric acid solution formula: 10g chloroauric acid: distilled deionized water to 1000mL.

②Preparation of trisodium citrate: Dissolve sodium citrate in double-distilled deionized water to make a 1% solution, filter through a 0.22 μm membrane, store at 4°C for later use, and have a validity period of up to 1000 mL.

③Preparation of 0.1M potassium carbonate: Prepare with double-distilled deionized water, filter through 0.22μm membrane, set aside at 4°C, valid for four months. 1000mL 0.1M potassium carbonate solution formula: 13.8g potassium carbonate; distilled deionized water to 1000mL.

④Preparation of 2% PEG-20000: Prepare with double distilled deionized water, filter with 0.22μm membrane, store at 4°C for later use, valid for four months. 1000mL 2% PEG-20000 solution formula: 20g PEG-20000; distilled deionized water to 1000mL.

⑤Preparation of labeling, washing and preservation solution: 2% bovine serum albumin (BSA), 0.05% sodium azide (NaN ₃ ), 0.01M pH7.2 PBS solution, 0.22μ membrane filtration, set at 4°C for later use, valid for four months . 1000mL marked washing preservation solution formula: 20g BSA, 0.5g NaN ₃ , 0.01M pH7.2 PBS solution to 1000mL.

(2) Preparation of colloidal gold:

Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water, boil in an electric furnace, add 2mL 1% trisodium citrate for every 100mL of 0.01% chloroauric acid, and continue to boil until the liquid turns bright red, then stop heating. Make up the lost water after cooling to room temperature. The appearance of the prepared colloidal gold should be pure, translucent, free of sediment and floating matter, and the validity period is one week.

(3) Preparation of colloidal gold-labeled monoclonal antibody:

Use 0.1M potassium carbonate to adjust the pH value of the colloidal gold to 8.2, add the anti-cardiac troponin I monoclonal antibody prepared in Example 1 at 8-10 μg antibody/mL colloidal gold, mix with a magnetic stirrer for 30 minutes, and stir Add BSA to a final concentration of 1% and let stand for 1 hour. Centrifuge at 13,000rpm at 4°C for 30min, discard the supernatant, wash the pellet twice with labeled washing and preservation solution, resuspend the pellet with one-tenth of the initial volume of colloidal gold in the labeled washing and preservation solution, store at 4°C for later use, and be valid for one week.

C. Preparation of gold standard pads

(1) Preparation of blocking solution:

2%BSA, 0.1%TritonX-100, 0.05%NaN ₃ , 0.01M pH7.2PBS solution, filtered through a 0.22μm membrane, stored at 4°C for later use, valid for four months. 1000mL blocking solution formula: 20g BSA, 0.5g NaN ₃ , 1mL TritonX-100, 0.01M pH7.2 PBS solution to 1000mL.

(2) Preparation of gold standard pad:

After soaking the gold standard pad in the blocking solution for 30 min, dry it at 37°C. Then spread the prepared gold-labeled antibody evenly on the gold label pad, spread 20 square centimeters per milliliter of solution, freeze-dry, package, and store at 4°C for later use.

D. Preparation of Test Strip Sample Pads

(1) Preparation of blocking solution:

2%BSA, 0.1%TrtionX-100, 0.05%NaN ₃ , 0.01M pH7.2PBS solution, filtered through a 0.22μm membrane, stored at 4°C for later use, valid for four months. 1000mL blocking solution formula: 20g BSA, 0.5g NaN ₃ , 1mL TritionX-100, 0.01M pH7.2 PBS solution to 1000mL.

(2) Preparation of sample pad:

Soak the sample pad in the blocking solution for 30 minutes, dry it at 37°C, package it, and store it at 4°C for later use.

E. Assembly of test strips

Set the absorbent pad (purchased from Millipore), nitrocellulose membrane, gold standard pad, and sample pad on a non-absorbent support sheet, and cut into strips with a width of 3 mm. Each pack of ten strips is added with a desiccant and vacuum-packed to obtain the detection test paper.

The positive samples and negative samples detected by Roche Cardiac Troponin I Diagnostic Kit (Troponin I STAT) were used as the test samples of this kit, of which 205 cases were detected positive samples of cardiac Troponin I, and 800 cases were detected negative samples, The detection kit based on the mouse cTnI-C4 antibody was used as a control, and the detection results are shown in Table 2. The results showed that this kit detected 201 positive samples, with a relative sensitivity of 98.05%, which was consistent with the detection results of the control mouse cTnI-C4 antibody; 788 of 800 negative samples were detected, of which 12 were false positives, which were relatively specific The relative specificity was 98.5%, while 771 murine cTnI-C4 antibodies were detected, 29 of which were false positives, and the relative specificity was 96.38%. Therefore, the recombinant cTnI-C4 antibody used in the diagnosis of cardiac troponin I not only maintained The affinity of the mouse antibody reduces the false positive rate and improves the specificity of diagnosis, which is better than the cardiac troponin I diagnostic kit based on the mouse antibody.

Table 2 Detection results of cardiac troponin I detection kit based on mouse antibody and recombinant antibody

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (6)

1. A recombinant antibody against human cardiac troponin I, characterized in that it comprises a light chain consisting of the light chain variable region of the murine antibody against human cardiac troponin I and the light chain constant region of human IgG1 and a heavy chain consisting of the heavy chain variable region of a murine antibody against human cardiac troponin I and the heavy chain constant region of human IgG1; Wherein, the murine antibody against human cardiac troponin I is produced by a hybridoma cell with a deposit number of CCTCC C2013185; The light chain variable region contains the amino acid sequence shown in SEQ ID NO.1; The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO.2. 2. A method for constructing a recombinant antibody against human cardiac troponin I, characterized in that, comprising the steps of: Construct expression vectors containing the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively, wherein the expression vectors containing the nucleotide sequences shown in SEQ ID NO.3 contain the light chain of human IgG1 The expression gene of the constant region, the expression vector containing the nucleotide sequence shown in SEQ ID NO.4 contains the expression gene of the heavy chain constant region of human IgG1; Transfecting the expression vector into the same host cell; recovering the anti-human cardiac troponin I recombinant antibody from the host cell; Wherein, the expression vector to be inserted into the nucleotide sequence shown in SEQ ID NO.3 has reserved XbaI and PmlI double restriction sites; the expression vector to be inserted into the nucleotide sequence shown in SEQ ID NO.4 NheI and HindIII double restriction sites are reserved; The nucleotide sequence shown as SEQ ID NO.3 is prepared by the following steps: RNA was extracted from hybridoma cells with the preservation number CCTCC C2013185, and RT-PCR was performed with a reverse transcription kit. The RT-PCR amplification product was inactivated at 70°C as a template, and SEQ ID NO.5 and SEQ ID NO. Perform PCR with the primer sequence shown in 6, recover the PCR product, insert the PCR product into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transform it into DH5α competent cells for expression; Recovering the product expressed by DH5α competent cells, using the primer sequences shown in SEQ ID NO.7 and SEQ ID NO.8 to perform PCR to obtain the nucleotide sequence shown in SEQ ID NO.3; The nucleotide sequence shown as SEQ ID NO.4 is prepared by the following steps: RNA was extracted from hybridoma cells with the preservation number CCTCC C2013185, and RT-PCR was performed with a reverse transcription kit. The RT-PCR amplification product was inactivated at 70°C as a template, and SEQ ID NO.9 and SEQ ID NO. Perform PCR with the primer sequence shown in 10, recover the PCR product, insert the PCR product into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transform it into DH5α competent cells for expression; The product expressed by DH5α competent cells was recovered, and PCR was performed using the primer sequences shown in SEQ ID NO.11 and SEQ ID NO.12 to obtain the nucleotide sequence shown in SEQ ID NO.4. 3. the recombinant antibody as claimed in claim 1 is in the application of preparation detection cardiac troponin I detection reagent. 4. A detection test paper for cardiac troponin I, characterized in that it comprises the recombinant antibody as claimed in claim 1. 5. the detection test paper of cardiac troponin I as claimed in claim 4, is characterized in that, comprises support sheet, sample pad, gold standard pad, nitrocellulose membrane, absorption pad, detection line and quality control line, described The sample pad, the gold standard pad, the nitrocellulose membrane and the absorbent pad are sequentially arranged on the support sheet from one end to the other end of the support sheet, and the sample pad and the gold standard pad part overlapping, the gold standard pad partially overlaps the nitrocellulose membrane, the nitrocellulose membrane partially overlaps the absorbent pad, and the detection line and the quality control line are arranged on the nitrocellulose membrane , and the detection line is set at one end close to the gold label pad, the quality control line is set at one end close to the absorption pad, and the gold label pad is coated with the recombinant antibody-coated colloidal gold particles. Colloidal gold-labeled recombinant antibody, the detection line is affinity-purified anti-cardiac troponin I rabbit polyclonal antibody, and the quality control line is goat anti-mouse IgG antibody. 6. A detection kit, comprising the detection test paper of cardiac troponin I as claimed in claim 4 or 5.