Antibody for resisting mycobacterium tuberculosis MPT64 protein or antigen binding fragment thereof and application of antibody or antigen binding fragment

Abstract

The invention relates to an antibody for resisting mycobacterium tuberculosis MPT64 protein or an antigen binding fragment thereof in the technical field of immunological detection, the antibody can specifically bind to mycobacterium tuberculosis MPT64 protein, the antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 4. The antibody or the antigen binding fragment thereof is used as a key recognition molecule in an immunodetection system, is used for immunological detection of MPT64 protein, and provides a basis for construction of stable and reliable mycobacterium tuberculosis related immunodetection methods and detection reagents.

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology, specifically to an antibody against Mycobacterium tuberculosis MPT64 protein or its antigen-binding fragment and its applications. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease primarily caused by Mycobacterium tuberculosis (MTB). It can affect multiple organs throughout the body, with pulmonary tuberculosis being the most common. Currently, TB is the single leading cause of death worldwide, seriously threatening human health. my country is one of the countries with a high burden of TB globally; therefore, developing rapid, accurate, and convenient diagnostic techniques is of great significance for the prevention, control, and treatment of TB.

[0003] Diagnostic techniques for Mycobacterium tuberculosis mainly include various methods such as the tuberculin skin test, bacteriological detection, molecular biology techniques, and immunological detection. While the tuberculin skin test (TST) is simple and rapid, its specificity is insufficient, making it difficult to differentiate between Mycobacterium tuberculosis infection, BCG vaccination response, and non-tuberculous mycobacterial (NTM) infection. In bacteriological detection, acid-fast staining can only identify the genus Mycobacteria but cannot distinguish specific species, while fluorescent staining has limitations such as a high false-positive rate. Traditional culture methods, considered the "gold standard" for diagnosis, suffer from long culture cycles and low positive rates, while instrumental culture methods (such as the BACTEC MGIT 960) face challenges such as expensive equipment and radioactive contamination. Molecular biology techniques such as GeneXpert, TB-LAMP, and whole-genome sequencing have good diagnostic value, but their widespread application is limited by high equipment costs and specialized technical requirements. The interferon-gamma release assay (IGRA) recommended by the World Health Organization, while highly specific and unaffected by BCG vaccination, has high testing costs due to the need for specialized equipment. Since one of the earliest biomarkers produced after infection with Mycobacterium tuberculosis is the tuberculosis-specific antigen, antigen detection can serve as direct evidence of tuberculosis infection. Immunological detection methods based on antigen-antibody specific reactions have become an important auxiliary means for the early diagnosis of tuberculosis due to their high specificity and sensitivity, as well as their simplicity, speed, and efficiency. In recent years, research on immunological detection of Mycobacterium tuberculosis-specific antigens and their corresponding antibodies has received increasing attention from scholars both domestically and internationally.

[0004] Among Mycobacterium tuberculosis-associated antigens, MPT64 is one of the immunogenic proteins secreted by the Mycobacterium tuberculosis complex. It exhibits good antigen specificity and has received widespread attention in Mycobacterium tuberculosis research and immunological detection. Detection strategies based on the MPT64 antigen typically rely on the antibody's specific recognition ability of the target antigen, and their detection effectiveness largely depends on the specificity, stability, and batch-to-batch consistency of the antibody used.

[0005] In the existing technology, antibodies against MPT64 still vary in terms of source, preparation method and immunological properties. Some antibodies may have problems such as non-specific binding or insufficient reaction consistency in practical applications, which may affect the reliability and reproducibility of immunoassay results.

[0006] Therefore, obtaining highly specific monoclonal antibodies against the MPT64 protein and systematically identifying their immunological characteristics is of great significance for constructing a stable and reliable MPT64 antigen immunoassay system. Summary of the Invention

[0007] The present invention aims to provide an antibody or antigen-binding fragment thereof against Mycobacterium tuberculosis MPT64 protein, which can be used as a key recognition molecule in an immunoassay system for the immunological detection of MPT64 protein, thus providing a foundation for constructing stable and reliable immunoassay methods and reagents related to Mycobacterium tuberculosis.

[0008] In a first aspect, to achieve the above objectives, the present invention provides an antibody against Mycobacterium tuberculosis MPT64 protein or an antigen-binding fragment thereof, wherein the antibody is capable of specifically binding to Mycobacterium tuberculosis MPT64 protein, and the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0009] Preferably, as an improvement, the antibody is a monoclonal antibody.

[0010] Preferably, as an improvement, the heavy chain subclass of the antibody is IgG1, and the light chain is kappa type.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect, the nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 1; and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 3.

[0012] Thirdly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0013] Preferably, as an improvement, the vector is a pET28a(+) expression vector.

[0014] Fourthly, the present invention provides a host cell comprising the recombinant expression vector described in the third aspect.

[0015] Fifthly, the present invention provides a cell line capable of producing the antibody.

[0016] In a sixth aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment in the preparation of reagents or kits for detecting Mycobacterium tuberculosis MPT64 protein.

[0017] The beneficial effects of this invention are: This invention relates to an antibody or its antigen-binding fragment targeting the MPT64 protein of Mycobacterium tuberculosis. It specifically recognizes the MPT64 protein, and the monoclonal antibody based on it exhibits good specificity and no significant cross-reactivity with non-target proteins. This antibody or its antigen-binding fragment can serve as a key recognition molecule in an immunoassay system for the immunological detection of the MPT64 protein, providing a foundation for constructing stable and reliable immunoassay methods and reagents related to Mycobacterium tuberculosis. Attached Figure Description

[0018] Figure 1 The image shows the SDS-PAGE results of imidazole eluted samples collected at different times in Example 1; where M is the protein molecular weight standard (Marker), and 1-4 are eluted samples with a concentration of 250 mM imidazole collected at different times. Figure 2 This is a graph showing the results of serum titer detection in mice after immunization in Example 2; Figure 3 This is a graph showing the results of ascites titer detection in mice after injection of MPT64-38 cell line in Example 3; Figure 4 This is the result of SDS-PAGE analysis of the purified MPT64-38 monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker) and 1 is the purified MPT64-38 monoclonal antibody. Figure 5 This is a graph showing the results of the MPT64-38 monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0019] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0020] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0021] Example 1: Obtaining recombinant MPT64 protein from Mycobacterium tuberculosis In this embodiment, recombinant MPT64 protein was prepared. The specific preparation method includes the following steps: 1. Construction of recombinant expression vectors Reference Mycobacterium tuberculosis (MTB) strain H37Rv mpt64 The gene sequence (GenBank accession number: NC_000962.3) was obtained based on its coding region information in the genome. mpt64 The nucleic acid coding sequence of the gene; Anshengda Biotechnology Co., Ltd. was commissioned to use molecular cloning technology to synthesize the gene. mpt64 The gene's nucleic acid coding sequence was ligated into the expression vector pET28a(+) with a 10×His tag to construct the recombinant expression vector pET28a(+)- mpt64 (10×His). Restriction endonuclease digestion analysis and sequencing verification showed that... mpt64 The gene was correctly ligated into the pET28a(+) vector, the sequence was correct, the reading frame was complete, and a recombinant plasmid for subsequent expression of MPT64 protein was successfully obtained.

[0022] 2. Expression and purification of MPT64 protein The recombinant expression vector pET28a(+)- was correctly identified. mpt64 After amplification and culture of (10×His)-positive BL21(DE3) clones, plasmids were extracted, transformed using standard methods, and single colonies were picked and cultured in LB medium containing kanamycin resistance to achieve an A concentration. 600When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16℃. The bacterial cells were collected by centrifugation at 4℃ and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and then sonicated in an ice bath with Binding buffer at 30 W. The sonication was repeated for 2 s with a 2 s interval until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4℃ and 12000 rpm for 20 min for purification.

[0023] The target protein (MPT64 recombinant protein) was purified by nickel-column affinity chromatography: A 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The lysate containing the MPT64 recombinant protein was added to the column, and the permeate was collected and loaded again. Unbound proteins were washed away with binding buffer, followed by 20 mL of washing buffer. Proteins were then eluted with 250 mM imidazole elution buffer. The purity of the eluted sample was determined by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown: MPT64 recombinant protein was obtained by affinity chromatography. The concentration of purified MPT64 recombinant protein was determined by BCA method.

[0024] Example 2: Establishment of MPT64 recombinant protein monoclonal antibody cell line This embodiment prepared a cell line for MPT64 recombinant protein monoclonal antibody. The specific preparation method includes the following steps: 1. Mouse immunization The MPT64 recombinant protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of the immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, and administered subcutaneously at five sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg of the immunogen, following the same immunization method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing; if the antibody titer was greater than 1:10000, a pulse immunization of 100 μg of the immunogen was administered intraperitoneally three days before cell fusion.

[0025] 2. Mouse serum titer detection (1) Antigen coating: The MPT64 recombinant protein obtained in Example 1 was adjusted to 1 μg / mL in the coating buffer. 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C.

[0026] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0027] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37℃ for 1 h.

[0028] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0029] (5) Add primary antibody: Use blocking solution to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking solution as a blank control. Incubate at 37℃ for 1 h, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

[0030] (6) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking buffer, 100 μL / well, incubate at 37℃ for 1 h, discard the liquid in the well, and pat dry.

[0031] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution to each well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2 M sulfuric acid to terminate the reaction. Measure the A value at 450 nm using a microplate reader. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well (P / N) is ≥2.1 is taken as the serum titer. Mice with a dilution greater than 1:10000 are prepared for the next fusion step. The results are as follows: Figure 2 As shown, the reaction between recombinant MPT64 protein and immune serum was identified by indirect ELISA, with a mouse serum titer of 1:64000.

[0032] 3. Cell fusion Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible. The mixture was allowed to stand for 1 min. 20 mL of preheated serum-free DMEM culture medium at 37°C was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a density of 200 μL per well and incubated.

[0033] 4. Screening of positive hybridoma cells Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0034] 5. Subcloning of positive hybridoma cells Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 100 μL of the diluted cells in 96-well plates, labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was measured using an indirect ELISA method. The positive rate reached 100%. The culture was expanded, and the cell line was preserved and numbered as MPT64-38.

[0035] Example 3: Preparation and Identification of MPT64 Protein Monoclonal Antibody In this embodiment, a monoclonal antibody against the MPT64 protein was prepared and its subclass was identified. The specific steps are as follows: 1. Preparation and titer determination of ascites fluid from MPT64 protein monoclonal antibody-conjugated ascites fluid. 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of a suspension of the MPT64-38 cell line preserved in Example 2 (5 × 10⁻⁶ cells). 5 (Each mouse was injected with the cell suspension, and the day of injection was counted as day 1). On day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, and the supernatant was collected and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA. A negative control group was set up, using ascites fluid obtained from injection of SP2 / 0 myeloma cells as a negative control. The results are as follows: Figure 3 As shown: Ascites antibody titer 1:1280000.

[0036] 2. Purification of monoclonal antibodies Ascites fluid was collected, and MPT64-38 monoclonal antibody was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. The mixture was then incubated at 4°C for 1 h to allow for complete precipitation of contaminating proteins. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. SDS-PAGE was used to identify the purity of the monoclonal antibody. The results are shown below. Figure 4 As shown: MPT64-38 monoclonal antibody was obtained.

[0037] 3. Identification of Monoclonal Antibody Types and Subclasses The experimental procedure was performed using the mouse subtype identification kit instructions. The MPT64-38 monoclonal antibody was identified as IgG1, kappa light chain.

[0038] Meanwhile, GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) was commissioned to sequence the monoclonal antibody produced by the MPT64-38 cell line preserved in Example 2 (i.e., the MPT64-38 monoclonal antibody purified in step 2 of Example 3). The results are as follows: The nucleotide sequence of the heavy chain variable region of the MPT64-38 monoclonal antibody is: 5'-GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAAACCTTTCAGTCTCTGTCTCACCTGCTACGCTCACTGGCTACGCCATCATCAGTGGTTATTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCT ACGACGGTAGAAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAGGTTGAATTCTGTGACTTCTGAGGACACAGCTACATATTACTGTACAAGTGGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA-3' (SEQ ID NO: 1), Its corresponding amino acid sequence is: DVQLQESGPGLVKPSQSLSLTCSVTGYAIISGYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDTSKNQFFLRLNSVTSEDTATYYCTSGAWFAYWGQGTLVTVSA (SEQ ID NO: 2); The nucleotide sequence of the light chain variable region is: 5'-AGTATTGTCATGACCCAGACTCCCAAATTCCTGCTCGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGCCAGAGTGTGAGTAAAGATGTATCTTGGTACCAACAGAAGCCAGGGCAGTCTCCCAAAATGTTGATATATTATGCAT CCAATCCTTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA-3' (SEQ ID NO: 3), The corresponding amino acid sequence is: SIVMTQTPKFLLVSAGDRVTITCKASQSVSKDVSWYQQKPGQSPKMLIYYASNPYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK (SEQ ID NO: 4).

[0039] 4. Specificity detection of monoclonal antibodies The antigens used were Mycobacterium tuberculosis MPT64 protein (MPT64), Mycobacterium tuberculosis ESAT6 protein (ESAT6), Mycobacterium tuberculosis CFP10 protein (CFP10), human protein disulfide isomerase A6 (PDIA6), novel coronavirus N protein (N), novel coronavirus E protein (E), novel coronavirus M protein (M), swine foot-and-mouth disease virus 3ABC protein (3ABC), African swine fever virus I226R protein (I226R), porcine reproductive and respiratory syndrome virus Nsp protein (NSP), and bovine early pregnancy factor (PAG1). All the antigens used in the tests were obtained by constructing expression vectors in the laboratory and inducing prokaryotic expression.

[0040] The ELISA plate was coated under the same conditions, using the MPT64-38 monoclonal antibody purified in step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450 The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 5As shown, the monoclonal antibody produced by the MPT64-38 cell line preserved in Example 2 (i.e., the MPT64-38 monoclonal antibody purified in step 2 of Example 3) only reacts with the MPT64 protein (A 450 =2.33), and it does not cross-react with the other antigens mentioned above, indicating that it has good specificity and can be used for qualitative or quantitative detection of Mycobacterium tuberculosis MPT64 protein in the sample to be tested.

Claims

1. An antibody against Mycobacterium tuberculosis MPT64 protein or an antigen-binding fragment thereof, characterized in that: The antibody can specifically bind to the MPT64 protein of Mycobacterium tuberculosis. The antibody contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. The antibody against Mycobacterium tuberculosis MPT64 protein or its antigen-binding fragment according to claim 1, characterized in that: The antibody is a monoclonal antibody.

3. The antibody against Mycobacterium tuberculosis MPT64 protein or its antigen-binding fragment according to claim 2, characterized in that: The heavy chain subclass of the antibody is IgG1, and the light chain is kappa type.

4. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 3, characterized in that: The nucleic acid molecule contains a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 1; and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:

3.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. The recombinant expression vector for nucleic acid molecules according to claim 5, characterized in that: The vector is a pET28a(+) expression vector.

7. A host cell comprising the recombinant expression vector of claim 5 or 6.

8. A cell line, characterized in that: The cell line is capable of producing the antibody as described in any one of claims 1 to 3.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of reagents or kits for detecting Mycobacterium tuberculosis MPT64 protein.

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