Mycobacterium tuberculosis antibody capture antigen as well as preparation method and kit thereof

Abstract

The invention relates to the technical field of biology. Specifically, the invention discloses a mycobacterium tuberculosis antibody capture antigen as well as a preparation method and a kit thereof. The mycobacterium tuberculosis antibody capture antigen comprises a recombinant MPB70 antigen with an amino acid sequence as shown in SEQ ID NO: 1, a recombinant MPB83 antigen with an amino acid sequence as shown in SEQ ID NO: 2 and a recombinant CE fusion antigen with an amino acid sequence as shown in SEQ ID NO: 3. By implementing the method, early recognition of tuberculosis can be realized, the recognition sensitivity is high, and the method is suitable for multiple species.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a mycobacterium tuberculosis antibody capture antigen and a preparation method and kit thereof. BACKGROUND

[0002] Tuberculosis is a chronic infectious disease of human and animals caused by Mycobacterium tuberculosis complex (MTBC), in which Mycobacterium bovis (M. bovis) is the main pathogen causing bovine tuberculosis. Mycobacterium tuberculosis complex Mycobacterium tuberculosis variant bovis Compared with M. tuberculosis, M. bovis can infect more animals, such as cattle, sheep, pigs, badgers and foxes, and can also be transmitted to humans through aerosols, infected milk and dairy products, especially children, the elderly and HIV-infected persons with low immunity are more susceptible to M. bovis infection. At present, the methods for laboratory diagnosis of animal tuberculosis mainly include pathogen detection (such as PCR) and immunological detection (such as intradermal allergic reaction test, gamma interferon release test, antibody detection, etc.). However, the intradermal allergic reaction test has the problems of insufficient specificity, time-consuming and laborious operation, and difficulty in carrying out the test in wild animals; the gamma interferon release test needs to establish a double-antibody sandwich ELISA method for detecting interferon levels in animal plasma with different animal monoclonal antibodies, has species specificity, and has high detection cost; the antibody indirect ELISA is simple and easy to operate, has a lagging diagnostic window period, and needs to use different animal IgG secondary antibodies or optimize the working concentration of SPG / SPA, and has limited universality. Therefore, it is an urgent need in the field of animal tuberculosis prevention and control to develop a new antibody detection method with high sensitivity, high specificity and wide applicability.

[0003] Traditional ELISA mainly detects IgG antibodies in serum against M. tuberculosis complex specific proteins, and cannot identify IgM and IgA appearing in the early stage of infection, and has a lagging detection window period; indirect method needs to rely on species-specific secondary antibodies (such as anti-cattle IgG-HRP) to detect objects with species specificity, and needs to develop antibody indirect ELISA detection kits for different species; when using SPA / SPA-HRP as secondary antibody, although the species restriction can be eliminated, the dilution of serum and enzyme-labeled antibody needs to be optimized for different animal serum, which limits its popularization and application in different species and actual scenes. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a mycobacterium tuberculosis antibody capture antigen, which can capture IgG antibodies, IgM antibodies and IgA antibodies at the same time, realize early identification of tuberculosis, and has high sensitivity and can be applied to multiple species.

[0005] ​The present application also aims to solve the technical problem of providing a DNA molecule that can encode the above-mentioned Mycobacterium tuberculosis antigen.

[0006] The present application also aims to solve the technical problem of providing a recombinant vector that can express the above-mentioned Mycobacterium tuberculosis antigen.

[0007] The present application also aims to solve the technical problem of providing a recombinant strain that can express the above-mentioned Mycobacterium tuberculosis antigen.

[0008] The present application also aims to solve the technical problem of providing a preparation method of the above-mentioned Mycobacterium tuberculosis antibody capture antigen.

[0009] The present application also aims to solve the technical problem of providing a kit for detecting tuberculosis, which can achieve early detection of tuberculosis of multiple species.

[0010] In order to solve the above technical problems, as a first aspect of the present application, the present application provides a Mycobacterium tuberculosis antibody capture antigen, which comprises a recombinant MPB70 antigen with an amino acid sequence as shown in SEQ ID NO: 1, a recombinant MPB83 antigen with an amino acid sequence as shown in SEQ ID NO: 2, and a recombinant CE fusion antigen with an amino acid sequence as shown in SEQ ID NO: 3. Through the combination of the above three kinds of recombinant antigens, the detection sensitivity can be greatly improved, the detection rate can be improved, and IgG antibodies, IgM antibodies and IgA antibodies can be captured at the same time, so as to realize early identification of tuberculosis; in addition, the capture antigen in the present application can also be applied to multiple species.

[0011] Specifically, the amino acid sequence (SEQ ID NO: 1) of the recombinant MPB70 antigen is as follows: LEVLFQGP GDLVGPGCAEYAAANPTGPASVQGMSQDPVAVAASNNPELTTLTAALSGQLNPQVNLVDTLNSGQYTVFAPTNAAFSKLPASTIDELKTNSSLLTSILTYHVVAGQTSPANVVGTRQTLQGASVTVTGQGNSLKVGNADVVCGGVSTANATVYMIDSVLMPPA. The underlined part is the HRV 3C cleavage site.

[0012] The amino acid sequence (SEQ ID NO: 2) of the recombinant MPB83 antigen is as follows: LEVLFQGPDTSPKPATSPAAPVTTAAMADPAADLIGRGCAQYAAQNPTGPGSVAGMAQDPVATAASNNPMLSTLTSALSGKLNPDVNLVDTLNGGEYTVFAPTNAAFDKLPAATIDQLKTDAKLLSSILTYHVIAGQASPSRIDGTHQTLQGADLTVIGARDDLMVNNAGLVCGGVHTANATVYMIDTVLMPPAQ. The underlined part is the HRV 3C enzyme cleavage site.

[0013] The amino acid sequence of the recombinant CE fusion antigen (SEQ ID NO: 3) is as follows: LEVLFQGP MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGFGGGGSGSGGGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA. The underlined part is the HRV 3C enzyme cleavage site.

[0014] Specifically, in the Mycobacterium tuberculosis antibody capture antigen, the weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen and the recombinant CE fusion antigen is (0.5~4):(0.5~4):(0.5~2), exemplarily 3:3:1, 3:2:2, 2:1.5:1.5, 1.5:3:3, 1:1:3, but not limited thereto. Preferably, it is (1~3):(1~3):(1~1.5), more preferably (1~3):(1~3):(1~1.2).

[0015] Preferably, in some embodiments, when the Mycobacterium tuberculosis antibody capture antigen is used as a detection antigen, the weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen and the recombinant CE fusion antigen is (2~4):(2~4):(0.8~1.5), preferably (2.5~3.5):(2.5~3.5):(0.8~1.5).

[0016] When the Mycobacterium tuberculosis antibody capture antigen is used as a coating antigen, the weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen and the recombinant CE fusion antigen is (0.5~3):(0.5~3):(0.5~2), preferably (0.5~1.5):(0.5~1.5):(0.5~1.5). When the Mycobacterium tuberculosis antibody capture antigen is used as a coating antigen, the weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen and the recombinant CE fusion antigen is (0.5~3):(0.5~3):(0.5~2), preferably (0.5~1.5):(0.5~1.5):(0.5~1.5).

[0017] Preferably, in some embodiments, when the Mycobacterium tuberculosis antibody capture antigen is used as a detection antigen, the weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen, and the recombinant CE fusion antigen is 3:3:1, and based on this ratio, the detection specificity, accuracy, and sensitivity are higher.

[0018] When the Mycobacterium tuberculosis antibody capture antigen is used as a coating antigen, the weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen, and the recombinant CE fusion antigen is 1:1:1, and based on this ratio, the detection specificity and sensitivity are higher.

[0019] As a second aspect of the present application, the present application provides a DNA molecule comprising a first DNA molecule for encoding the recombinant MPB70 antigen, a second DNA molecule encoding the recombinant MPB83 antigen, and a third DNA molecule encoding the recombinant CE fusion antigen.

[0020] Preferably, in some embodiments, the nucleotide sequence of the first DNA molecule is as shown in SEQ ID NO: 4, the nucleotide sequence of the second DNA molecule is as shown in SEQ ID NO: 5, and the nucleotide sequence of the third DNA molecule is as shown in SEQ ID NO: 6.

[0021] More specifically, the nucleotide sequence of the first DNA molecule (SEQ ID NO: 4) is shown as follows: CTGGAAGTTCTGTTCCAGGGGCCCGGCGATCTGGTGGGCCCGGGCTGCGCGGAATACGCGGCAGCCAATCCCACTGGGCCGGCCTCGGTGCAGGGAATGTCGCAGGACCCGGTCGCGGTGGCGGCCTCGAACAATCCGGAGTTGACAACGCTGACGGCTGCACTGTCGGGCCAGCTCAATCCGCAAGTAAACCTGGTGGACACCCTCAACAGCGGTCAGTACACGGTGTTCGCACCGACCAACGCGGCATTTAGCAAGCTGCCGGCATCCACGATCGACGAGCTCAAGACCAATTCGTCACTGCTGACCAGCATCCTGACCTACCACGTAGTGGCCGGCCAAACCAGCCCGGCCAACGTCGTCGGCACCCGTCAGACCCTCCAGGGCGCCAGCGTGACGGTGACCGGTCAGGGTAACAGCCTCAAGGTCGGTAACGCCGACGTCGTCTGTGGTGGGGTGTCTACCGCCAACGCGACGGTGTACATGATTGACAGCGTGCTAATGCCTCCGGCGTGA. The underlined portion is the HRV 3C enzyme cleavage site.

[0022] The nucleotide sequence of the second DNA molecule (SEQ ID NO: 5) is shown below: CTGGAAGTTCTGTTCCAGGGGCCCGACACCAGCCCGAAACCGGCGACCAGCCCGGCGGCGCCCGTTACCACGGCGGCAATGGCTGACCCCGCAGCGGACCTGATTGGTCGTGGGTGCGCGCAATACGCGGCGCAAAATCCCACCGGTCCCGGATCGGTGGCCGGAATGGCGCAAGACCCGGTCGCTACCGCGGCTTCCAACAACCCGATGCTCAGTACCCTGACCTCGGCTCTGTCGGGCAAGCTGAACCCGGATGTGAATCTGGTCGACACCCTCAACGGCGGCGAGTACACCGTTTTCGCCCCCACCAACGCCGCATTCGACAAGCTGCCGGCGGCCACTATCGATCAACTCAAGACTGACGCCAAGCTGCTCAGCAGCATCCTGACCTACCACGTGATAGCCGGCCAGGCGAGTCCGAGCAGGATCGACGGCACCCATCAGACCCTGCAAGGTGCCGACCTGACGGTGATAGGCGCCCGCGACGACCTCATGGTCAACAACGCCGGTTTGGTATGTGGCGGAGTTCACACCGCCAACGCGACGGTGTACATGATCGATACGGTGCTGATGCCCCCGGCACAGTGA. The underlined portion is the HRV 3C enzyme cleavage site.

[0023] The nucleotide sequence of the third DNA molecule (SEQ ID NO: 6) is shown as follows: CTGGAAGTTCTGTTCCAGGGGCCC ATGGCAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCGACGGCAGGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTC GGTGGCGGAGGATCCGGTTCTGGTGGCGGAATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGCAACGTCACTGGGATGTTCGCATGA. Wherein, the straight line underlined part is HRV 3C enzyme cutting point, and the wavy underlined part is linker.

[0024] As a third aspect of the present application, the present application provides a recombinant vector comprising the above-mentioned first DNA molecule, second DNA molecule or third DNA molecule. Specifically, the recombinant vector can comprise any one, any two or any three of the three. Specifically, the recombinant vector adopts a pET32a plasmid, but is not limited thereto.

[0025] Preferably, in some embodiments, in order to facilitate purification, HRV 3C protease and sites and 6xHis tags are connected to the N- and C-termini of the DNA molecule.

[0026] As a fourth aspect of the present application, the present application provides a recombinant strain obtained by transforming the above-mentioned recombinant vector into a host bacterium. Wherein, the host bacterium can be E. coli, but is not limited thereto. Preferably, in some embodiments, the host bacterium is BL21 (DE3).

[0027] As a fifth aspect of the present application, the present application provides a kit for detecting tuberculosis, comprising the above-mentioned Mycobacterium tuberculosis antibody capture antigen. Accordingly, the kit also comprises sample diluent, washing solution, color developing solution and termination solution commonly used in the art, but is not limited thereto. Based on the above-mentioned capture antigen, the kit, on the one hand, improves the sensitivity of antibody recognition and the detection rate of clinical samples, and improves the sensitivity of detection; on the other hand, realizes the simultaneous capture of IgM, IgA and IgG antibodies, which makes the kit of the present application effectively recognize multiple types of antibodies in the early stage of infection without relying on species-specific secondary antibodies, improves the detection rate, and is suitable for multiple species of serum samples.

[0028] Specifically, in the kit, the Mycobacterium tuberculosis antibody capture antigen of the present application can be used as a coating antigen and also as a detection antigen; it can also be used as both a coating antigen and a detection antigen, i.e. forming a double-antigen sandwich structure, based on which the sensitivity of recognizing multiple antibodies in the early infection stage can be further improved, and the detection rate can be improved.

[0029] When the Mycobacterium tuberculosis antibody capture antigen of the present application is used as a coating antigen, its coating concentration is 1.5 μg / mL to 9 μg / mL, exemplarily 1.5 μg / mL, 3 μg / mL, 6 μg / mL or 9 μg / mL, but not limited thereto. Preferably, the coating concentration is 1.5 μg / mL to 3 μg / mL or 3 μg / mL to 4.5 μg / mL, more preferably 3.0 μg / mL.

[0030] When the Mycobacterium tuberculosis antibody capture antigen of the present application is used as a detection antigen, it is labeled with N-hydroxysuccinimide-biotin, which can reduce the detection limit and is particularly suitable for the diagnosis of individuals infected with low antibody levels. Specifically, the protein concentration of the detection antigen is 5 to 10 mg / mL, exemplarily 5.5 mg / mL, 6.5 mg / mL, 8 mg / mL or 9 mg / mL, but not limited thereto. Preferably, it is 6 mg / mL to 10 mg / mL or 6 mg / mL to 8 mg / mL, more preferably 7 mg / mL. The dilution of the detection antigen is 1:4000 to 1:8000, exemplarily 1:4000, 1:5000 or 1:6000, but not limited thereto. Preferably, it is 1:5000 to 1:7000, more preferably 1:6000 to 1:7000, further preferably 1:6000.

[0031] Preferably, in some embodiments, when the Mycobacterium tuberculosis antibody capture antigen of the present application is used as a detection antigen, a streptavidin-horseradish peroxidase (SA-HRP) amplification system is also added to the kit, and the combination of the two can greatly improve the detection sensitivity, effectively overcoming the problem of weak signal caused by low antibody titer in the early stage, thereby realizing early diagnosis. Specifically, the dilution of the streptavidin-horseradish peroxidase (SA-HRP) amplification system is 1:3000 to 1:15000, exemplarily 1:3000, 1:5000, 1:8000, 1:10000 or 1:15000, but not limited thereto. Preferably, it is 1:3000 to 1:10000, more preferably 1:5000 to 1:8000, further preferably 1:5000.

[0032] As a fifth aspect of the present application, the present application also provides a detection method based on the above-mentioned kit, which is suitable for non-diagnostic purposes, for detecting the concentration of Mycobacterium antibody in a sample to be tested, which specifically comprises: (1) The sample to be tested is added to the coated plate and incubated; the coated plate is coated with the above-mentioned Mycobacterium tuberculosis antibody capture antigen; wherein the incubation temperature is 20℃~40℃ and the incubation time is 20min~60min; (2) Add the detection antigen and bind for a first preset time; wherein, the detection antigen is the above-mentioned Mycobacterium tuberculosis antibody capture antigen labeled with N-hydroxysuccinimide-biotin; wherein, the binding temperature is 20℃~40℃ and the binding time is 20min~60min; (3) After washing, add the streptavidin-horseradish peroxidase amplification system and bind for the second preset time; wherein the binding temperature is 20℃~40℃ and the binding time is 5min~30min; (4) After washing, add color developing solution, develop color, and measure; wherein, the color developing temperature is 20℃~40℃, and the color developing time is 5min~30min.

[0033] Implementing this invention has the following beneficial effects: The Mycobacterium tuberculosis antibody capture antigen of this invention includes recombinant MPB70 antigen, recombinant MPB83 antigen, and recombinant CE fusion antigen. The combination of these three recombinant antigens significantly improves detection sensitivity and increases the detection rate. Furthermore, it can simultaneously capture IgG, IgM, and IgA antibodies, enabling early identification of tuberculosis. In addition, the capture antigens of this invention are applicable to multiple species. Therefore, the kit based on these capture antibodies can effectively identify multiple types of antibodies in the early infection stage without relying on species-specific secondary antibodies, improving the detection rate and being applicable to serum samples from multiple species. Furthermore, the kit of this invention also incorporates a streptavidin-biotin amplification system, which further enhances detection sensitivity and effectively overcomes the weak signal problem caused by low antibody titers in the early stages. In summary, this invention, based on a dual-antigen sandwich structure, multi-antibody subtype recognition capability, and signal amplification strategy, constructs a kit with high sensitivity, multi-species adaptability, and early diagnostic capability, possessing significant clinical and preventative value. Attached Figure Description

[0034] Figure 1 This is an SDS-PAGE image of the antigen purified by enzyme digestion in Example 2 of this invention; Figure 2 This is an SDS-PAGE image of the antigen after multi-stage purification in Example 2 of this invention; Figure 3 This is a Western blot verification image of the antigen-labeled biotin in Example 3 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below.

[0036] Example 1 Construction of recombinant plasmid of capture antigen MPB70 / MPB83 / CE protein According to the reference sequence of Mycobacterium bovis MPB70 / MPB83 / CF-10 / ESTA-6 protein with NCBI accession number (AF2122 / 97), the signal peptide and transmembrane region were removed, and the enzyme cutting site of HRV 3C protease was added at the N-terminal, which was used for subsequent removal of the tag on the vector. Then, the homologous recombination primers were designed to amplify the target fragment (the specific primer sequences are shown in Table 1), which was connected to the PET32a (+) vector between the BamHI and XhoI double enzyme cutting sites. The correct recombinant plasmid with the inserted sequence was sequenced and named as PET32a-MPB70, PET32a-MPB83 and PET32a-CE, respectively, which were transformed into BL21 (DE3) competent cells for antigen expression.

[0037] Table 1 Amplification primer table

[0038] Note: The straight line underlined part is the vector homologous arm sequence, the wavy line underlined part is the primer sequence, and the remaining part is the enzyme cutting site sequence.

[0039] Example 2 Expression and purification of capture antigen (1) Affinity chromatography: the expression strain was transferred in LB medium, and when OD600 was about 0.6, 0.5 mM IPTG was added for induction at 16°C overnight. After centrifugal harvesting of the bacterial cells, PB buffer (20 mM Na2HPO4 / NaH2PO4+150 mM NaCl, pH 7.5) was used as the lysis solution, and the bacterial cells were broken by ultrasonic and centrifuged. The supernatant was used for purification. The supernatant was added to the Ni-NTA affinity column, which was first washed with PB buffer containing 20 mM imidazole to remove non-specific binding proteins, and then eluted with PB buffer containing 500 mM imidazole to elute the target His-tag protein. The eluate was dialyzed to remove salt, and HRV 3C protease was added for overnight incubation at 4°C to remove the Trx, S-tag, His and other tags. The uncut fusion protein and free tag were removed again by Ni-NTA column to obtain the antigen protein without any tag (as shown in the figure). Figure 1

[0040] (2) Ion exchange chromatography: the protein after removing the tag was loaded onto the anion exchange column, and the binding liquid was PB buffer without sodium chloride. The unbound impurity proteins were eluted with the binding buffer, and then eluted in a linear elution mode (0→100% PB buffer containing 1 M NaCl, 15 CV). Finally, the target protein was collected at 20% gradient (i.e. about 200 mM NaCl), and PB buffer was used for dialysis. ​

[0041] (3) Endotoxin removal: Since animals can be infected with gram-negative bacteria such as E. coli, antibodies against LPS can be produced, in order to eliminate the reaction between the antibodies against LPS in the serum and the LPS carried by the antigen, the endotoxin in the recombinantly expressed capture antigen was removed by phase separation. Triton X-114 was added to the protein solution at a final concentration of 0.1% at 4°C, mixed and shaken, incubated at 37°C for 30 min, centrifuged at 12000g for 10 min, the upper aqueous phase was recovered, and after repeating twice, the protein in the upper aqueous phase was recovered, and after verification by SDS-PAGE (as shown in FIG. 3), the protein was aliquoted and stored at -80°C for later use. Figure 2

[0042] Example 3 Detection of antigen labeling construction and screening Using MPB83 as a test case, four methods of C-terminal tandem SA monomers, C-terminal tandem AVI tags and enzymatic biotinylation, HRP direct chemical labeling, and NHS-Biotin chemical labeling were used to construct and label the antigen for detection. After comparing the detection efficiency of clinical serum, the best labeling method was selected for the next test.

[0043] 1. Biotin labeling The purified MPB83 protein described above was adjusted to a final concentration of 1.0 mg / mL, and an appropriate amount of N-hydroxysuccinimide-biotin (NHS-Biotin) was weighed, added to the protein solution at a protein to NHS-Biotin molar ratio of 1:20, gently inverted to mix, and then reacted overnight at 4°C in the dark; after the reaction was completed, the reaction was terminated with Tris-HCl buffer (pH 7.5) (final concentration 5 mM) and incubated for 10 min; then the free biotin and small molecule impurities were removed by exchanging the solution with a PD-10 desalting column or an ultrafiltration concentration tube (10 kDa cutoff), and the biotinylated antigen MPB83-Biotin was obtained. The biotinylated antigen was aliquoted and stored at -80°C for later use.

[0044] 2. HRP-labeled antigen ​The purified recombinant MPB83 protein was adjusted to a concentration of 2 mg / mL, and dialyzed using 50 mM carbonate buffer (pH 9.6) to remove amine buffer substances (such as Tris, glycine, etc.) in the sample that can affect the coupling reaction. 2 mg of HRP (horseradish peroxidase) dry powder was weighed and dissolved in 100 μL of ultrapure water; 20 mg of NaIO4 was weighed and dissolved in 1 mL of water, 100 μL of the HRP solution was mixed with 100 μL of the NaIO4 solution, and the oxidation reaction was carried out at 4°C in the dark for 30 min to generate aldehyde groups in the oxidized HRP; then 2 μL of ethylene glycol was added and incubated at room temperature in the dark for 30 min to terminate the reaction; the oxidized HRP solution was mixed with a protein solution of the same concentration, and incubated at room temperature with gentle shaking for 2 h, and 0.4 mg of NaBH4 was added and incubated at 4°C for 2 h to terminate the reaction; after the reaction, PBS was used for dialysis overnight, and 50% glycerol was added to the dialysis solution as a stabilizer. The final labeled antigen MPB83-HRP was stored at -80°C for later use.

[0045] 3. Streptavidin and detection antigen expressed in tandem The tubercle MPB83 antigen gene was designed to be C-terminally fused with the streptavidin (SA) gene through a flexible linker peptide (GGGGS), and the expression vector pET32a-MPB83-SA was constructed. The above-mentioned plasmid was transformed into E. coli BL21 (DE3), and after induction with 0.5 mM IPTG at 16°C for 24 h, a soluble fusion protein was obtained. The fusion protein was directly used as a detection antigen (MPB83-SA) after His affinity chromatography and enzyme digestion purification. The HRP-labeled Biotin (HRP-Biotin) was used as an enzyme-labeled color system, and specifically combined with the SA part in the fusion protein to complete the detection signal transduction.

[0046] 4. Addition of AVI tag biotinylation to the detection antigen The MPB83 protein recombinant tuberculosis antigen with a C-terminal AVI tag (Avidin recognition peptide, GLNDIFEAQKIEWHE) was designed, and the AVI tag was connected to the C-terminus through molecular cloning technology to construct the expression vector pET32a-MPB83-AVI. The above-mentioned plasmid was transformed into E. coli BL21 (DE3), and after induction with 0.5 mM IPTG at 16°C for 24 h, a soluble fusion protein was obtained. The expression product was subjected to Ni-NTA affinity purification and enzyme digestion purification, and then subjected to in vitro reaction using BirA biotin ligase. The reaction system contained BirA enzyme, ATP (10 mM), Mg 2+(10 mM), D-biotin (50 μM) and target protein, and the reaction was carried out at 30 °C for 1 h; after the reaction, free biotin and enzyme were removed by gel filtration to obtain purified single-site labeled antigen MPB83-AVI.

[0047] 5. Clinical serum validation test antigen To systematically evaluate the detection efficiency of the four labeling methods, five bovine tuberculosis positive sera and three healthy bovine negative sera were selected for parallel detection by double antigen sandwich ELISA, and the results are shown in Table 2 (OD 450nm ) : Table 2. Clinical serum reactivity validation results of detection antigens

[0048] Comprehensive comparison of the performance of the four labeling methods, MPB83 labeled by NHS-Biotin multi-site chemical labeling, combined with SA-HRP amplification system, showed the highest detection signal in sandwich ELISA. AVI tag method can achieve site-specific biotinylation of the C-terminal of the antigen, but due to only a single labeling site, the signal strength is significantly lower than that of multi-site chemical labeling; the strategy of fusing the antigen with streptavidin (SA) monomers for expression also has limited amplification capacity and unsatisfactory detection sensitivity due to the single binding site. Direct HRP labeling may result in lower signal due to the large molecular weight of HRP (about 44 kDa) affecting the conformation of the antigen or the exposure of the antibody epitope. In summary, in terms of sensitivity, amplification efficiency and feasibility, NHS-Biotin multi-site labeling combined with SA-HRP system is the optimal solution for tuberculosis antibody detection, especially for the diagnosis of individuals with low antibody levels. Using the same labeling method, MPB70 and CE were labeled to prepare MPB70-Biotin and CE-Biotin, and WB verification was performed, and the results are shown in Figure 3 .

[0049] Example 4. Screening of coated antigen combinations The purified Mycobacterium bovis antigens MPB70, MPB83 and CE proteins were diluted with carbonate buffer (0.05 mol / L, pH = 9.6) to a protein concentration of 2 μg / mL, and the antigens were coated on the enzyme-labeled plate (100 μL / well) individually, in any two combinations, and in three combinations, respectively, at 4 °C overnight. The next day, the plate was washed once with PBST, and 2% BSA in PBS was added as blocking solution (200 μL / well), and the plate was blocked at room temperature for 2 h.

[0050] Three parts of diagnosed tuberculosis positive bovine serum and three parts of negative serum were diluted by 1:10, and then incubated with biotin-labeled MPB70-Biotin, MPB83-Biotin and CE-Biotin protein (weight ratio of 3:3:1). Then, streptavidin-horseradish peroxidase (SA-HRP) of the same concentration was added uniformly to complete the color reaction and measure the OD value at 450 nm wavelength.

[0051] As shown in Table 3, the use of mixed coating of the three antigens produced higher light absorption values in all positive sera, indicating that compared with single antigen and any two antigens mixed, the three mixed antigens can more comprehensively recognize tuberculosis-specific antibodies in the serum, improve the positive detection rate of detection, for example, serum No. 1, when coated with MPB83 or CE alone, the antibody level is negative, but after mixed coating of the three antigens, the antibody detection is positive. Further, when the three antigens are mixed at a weight ratio of 1:1:1, the positive / negative value is relatively highest, indicating that the use of antigens mixed at this weight ratio can improve the detection sensitivity. In addition, the detection of negative serum (Table 3, Nos. 4-6) shows that when MPB70+CE and MPB83+CE are used (i.e., after introducing CE protein), the OD value of the negative sample is relatively high. When the three are combined at a specific weight ratio, the OD value of the negative sample is lower than that of MPB70+CE and MPB83+CE. By using the combination of the three, the detection sensitivity, specificity and accuracy can be effectively optimized.

[0052] Table 3 Coating antigen clinical serum reactivity verification results

[0053] Example 5 Screening of detection antigen combination The purified Mycobacterium bovis antigens MPB70, MPB83 and CE protein were diluted with carbonate buffer (0.05 mol / L, pH=9.6) to a protein concentration of 3 μg / mL (weight ratio of MPB70, MPB83 and CE was 1:1:1), coated enzyme-labeled plate (100 μL / well), 4°C overnight. The next day, wash once with PBST, add 2% BSA-containing PBS as blocking solution (200 μL / well), and block at room temperature for 2 h.

[0054] Six bovine serum samples confirmed as positive for tuberculosis and four negative samples were diluted 1:10 and incubated separately with biotin-labeled MPB70-Biotin, MPB83-Biotin, and CE-Biotin proteins. They were also incubated with antigens prepared by mixing them in pairs (1:1 by weight) or by mixing them with different proportions of the three proteins (as specified in Table 4). Then, the same concentration of streptavidin-horseradish peroxidase (SA-HRP) was added to complete the colorimetric reaction, and the OD value was measured at 450 nm.

[0055] As shown in Table 4, the three biotinylated antigens produced higher absorbance values ​​in all positive sera when used in combination, indicating that compared to single antigens or any combination of two antigens, the three-antigen mixture can more comprehensively identify tuberculosis-specific antibodies in serum and improve the positive detection rate. Furthermore, the positive / negative value was highest when the three biotinylated antigens were mixed in a 3:3:1 weight ratio, indicating that this weight ratio improves detection sensitivity. In addition, the detection of negative sera (serial numbers 7-10 in Table 4) showed that the OD values ​​of negative samples were relatively high when MPB70-Biotin+CE-Biotin and MPB83-Biotin+CE-Biotin were used (i.e., after introducing CE-Biotin protein). When the three are combined in a specific weight ratio, the OD value of the negative sample is actually lower than that of MPB70-Biotin+CE-Biotin and MPB83-Biotin+CE-Biotin. This trend is consistent with the trend in Example 3, which also shows that by using the combination of the three, the detection sensitivity, specificity and accuracy can be effectively optimized.

[0056] Table 4 Results of clinical serum reactivity verification of biotinylated antigens

[0057] Example 6 Selection of antigen coating concentration Carbonate buffer (pH=9.6) was used as the coating buffer. MPB70, MPB83 and CE proteins were mixed at a weight ratio of 1:1:1 and coated with antigen-coated plates (100 μL / well) at total protein concentrations of 1.5 μg / mL, 3 μg / mL, 4.5 μg / mL, 6 μg / mL and 9 μg / mL. Positive and negative controls were detected according to the detection procedure in Example 8, and the P / N value (average OD of positive wells / average OD of negative wells) was calculated. The results are shown in Table 5. The P / N value was the highest when the antigen coating concentration was 3 μg / mL. Therefore, a concentration of 3 μg / mL was selected as the antigen coating amount of the kit.

[0058] Table 5 Results of antigen coating amount optimization

[0059] Example 7 Selection of working concentration of biotinylated antigen According to the optimized antigen coating amount of Example 6, using 2% BSA as diluent, mixing biotinylated antigens MPB70-Biotin, MPB83-Biotin and CE-Biotin in a weight ratio of 3:3:1 (total protein concentration of 7 mg / mL), respectively diluting 4K, 5K, 6K, 7K, 8K, and detecting positive control and negative control according to the detection method of Example 8, calculating P / N value (positive well OD average value / negative well OD average value), the results are shown in Table 6. As can be seen from the table: when the dilution ratio is 6K, the P / N value is the highest, so the dilution ratio of 6K is selected as the working concentration of biotinylated antigen of the kit.

[0060] Table 6 Optimization results of working concentration of biotinylated antigen

[0061] Example 8 Selection of working concentration of SA-HRP According to the optimized antigen coating amount of Example 6 and the optimized dilution ratio of biotinylated antigen of Example 7, using 2% BSA as diluent, SA-HRP was diluted at 3K, 5K, 8K, 10K, 15K, respectively, and positive control and negative control were detected according to the detection method of Example 9, and P / N value (positive well OD average value / negative well OD average value) was calculated, the results are shown in Table 7. As can be seen from the table, when the dilution ratio is 5K, the P / N value is the highest, so the dilution ratio of 5K is selected as the working concentration of SA-HRP of the kit.

[0062] Table 7 Optimization results of working concentration of SA-HRP

[0063] Example 9 Preparation and detection method of double antigen sandwich ELISA antibody detection kit 1. Preparation of kit (1) Mix the purified recombinant proteins (MPB70 / MPB83 / CE) in a weight ratio of 1:1:1 and dilute to 3 μg / mL with carbonate buffer (pH=9.6) for coating, 100 μL / well, 4°C coating overnight, wash once with PBST, add blocking solution (2% BSA in PBS) 200 μL / well, room temperature (about 25°C) blocking for 2h, discard the blocking solution and then fix with 2% M / V sucrose at room temperature for 2h, dry the coated plate after wiping (humidity not higher than 20%), vacuum seal in aluminum foil bag and store at 2~8°C for standby.

[0064] (2) Biotinylated antigen: Mix the NHS-biotin labeled antigens MPB70-Biotin, MPB83-Biotin and CE-Biotin in a weight ratio of 3:3:1 (total protein concentration is 7 mg / mL), dilute 60 times with enzyme-labeled protein protection solution to prepare 100x mother liquor A as the detection antigen, and store at 2-8°C.

[0065] (3) SA-HRP: Dilute the commercial SA-HRP 50 times with enzyme-labeled protein protection solution to prepare 100x mother liquor B, and store at 2-8°C.

[0066] (4) Diluent: Dissolve 20 g of IgG-free bovine serum albumin in 1 L of PBST, add ProClin300 to a final concentration of 0.05%, completely dissolve, and then filter with a 0.22 μm filter membrane, and store at 2-8°C after dispensing.

[0067] (5) Positive control: Mix the expressed and purified recombinant protein with Freund's adjuvant, and then immunize the bovine by intramuscular injection (1 mg / bovine), and then perform booster immunization at the same dose and in the same way after 14 days. Collect blood 14 days after the third immunization, and separate the serum. Mix 2 mL of the serum of the immunized bovine with 50 μL of ProClin300, and then dilute with PBS to 100 mL. Filter with a 0.22 μm filter membrane, and then store at 4°C after dispensing.

[0068] (6) Negative control: Mix 10 mL of bovine serum with 50 μL of ProClin300, and then dilute with PBS to 100 mL. Filter with a 0.22 μm filter membrane, and then store at 4°C after dispensing.

[0069] (7) Color developing solution is a commercial single-component TMB color developing solution, and is stored at 2-8°C after dispensing. (8) Termination solution is 1M HCL solution.

[0070] 2. Detection steps (1) Sample preparation: Collect the blood sample in a collection tube (test tube or centrifuge tube) without anticoagulant, and then incline and stand at room temperature for natural coagulation for 30-60 min. After the blood coagulates, centrifuge at low speed for 10 min, and then the supernatant is the serum sample.

[0071] (2) Kit equilibration: Take the solutions in the kit (except 100x detection A solution and detection B solution) out from 2-8°C, and then restore to room temperature.

[0072] (3) Preparation of 1x washing solution: Before use, restore the 20x washing solution to room temperature (22-26°C) (crystals can be dissolved by heating in a 37°C water bath for 5-10 min), dilute 20 times with double distilled water, and then mix thoroughly.

[0073] (4) Add sample: Take the antigen-coated plate (according to the number of samples, it can be used in split), negative control, positive control and serum sample 100 μL per well, seal the plate, 37°C for 30 min. Take out the reaction plate and discard the reaction solution.

[0074] (5) Add detection antigen: dilute detection A liquid 100 times with diluent (freshly prepared, 0.1 mL of 100x detection A liquid is added to 9.9 mL of diluent), 100 μL per well, 37°C for 30 min. Take out the reaction plate and discard the reaction solution, add 300 μL of 1x washing solution per well, wash 5 times, and gently pat dry for the last time; dilute detection B liquid 100 times with diluent (freshly prepared, 0.1 mL of 100x detection B liquid is added to 9.9 mL of diluent), 100 μL per well, 37°C for 10 min. Take out the reaction plate and discard the reaction solution, add 300 μL of 1x washing solution per well, wash 5 times, and gently pat dry for the last time.

[0075] (6) Color development and termination Add substrate color developing liquid, 100 μL per well, 37°C for 10 min. According to the order of adding substrate color developing liquid, add 50 μL of termination liquid to each well in turn, mix gently, and measure OD 450nm value within 10 min using an enzyme label meter.

[0076] (7) Result determination: test condition: positive control OD 450nm value ≥ 0.35, negative control OD 450nm value ≤ 0.2, the test is valid; S / P = (test sample OD 450nm value - negative control OD 450nm average) / (positive control OD 450nm value - negative control OD4 50nm average), when S / P value < 0.3, no Mycobacterium tuberculosis antibody is detected in the test sample; S / P value ≥ 0.3, Mycobacterium tuberculosis antibody exists in the test sample.

[0077] Example 10 Detection of animal serum by TB double antigen sandwich ELISA antibody detection kit 1. Take 5 portions of SPF mouse serum, 5 portions of SPF rabbit serum, 5 portions of SPF guinea pig serum, 5 portions of healthy bovine serum and 5 portions of healthy deer serum, and detect them using the optimized kit, and the results are S / P < 0.3, all negative (Table 8), which shows that the kit has no non-specific reaction with various animal sera.

[0078] Table 8 Detection results of the kit on TB-negative animal serum

[0079] 2, Take tuberculosis positive serum rat serum 5, rabbit serum 5, guinea pig serum 5, cow serum 5, deer serum 5 with optimized reagent kit for detection, the results S / P>0.3, all are positive (Table 9), the results show that the kit can be used for the detection of tuberculosis antibody in various animals.

[0080] Table 9 Detection results of the kit on tuberculosis positive animal serum

[0081] Example 11 Clinical sample detection and comparison of the tuberculosis double antigen sandwich ELISA antibody detection kit 1, select 149 clinical bovine serum samples from a certain ranch, use the double antigen sandwich ELISA antibody detection kit of the application for detection, and compare with the currently marketed tuberculosis antibody detection kit (indirect ELISA method). Using the same sample, respectively according to the operation process of the respective kit instruction book. The results are shown in Table 10. As can be seen from the table: the kit detects 22 positive samples, the commercial kit detects 20 positive samples, and the two have 14 positive samples. Compared with the commercial kit, the double antigen sandwich ELISA kit of the application has higher detection rate, especially in the samples that are negative in some commercial kits but positive in the kit, the tuberculin test and interferon gamma release test (IFN-γ) are further confirmed, and 7 samples are detected as bovine tuberculosis infected individuals, indicating that the kit has higher sensitivity and stronger early infection detection ability.

[0082] Table 10 Detection results of commercial kit and the kit of the application on clinical bovine serum

[0083] 2, detection of other wild animal serum and milk samples In order to evaluate the adaptability of the kit to different animal species, the laboratory collected tuberculosis positive serum samples of wild animals and milk were detected. The operation process is consistent with that of bovine serum, and the kit does not need special adjustment; the detection results are shown in Table 11. The results show that the double antigen sandwich ELISA antibody detection kit of the application is suitable for tuberculosis antibody detection in various animals, has good species spectrum, and can provide an effective tool for wild animal tuberculosis epidemiological investigation.

[0084] Table 11 Detection results of the kit of the application on various animal serum

[0085] Example 12 Comparison of detection antigens reported in literature To further improve the detection sensitivity and specificity, the recombinant plasmid expressing Rv3616, Rv3872 was constructed according to the method of Example 1, and the protein was purified according to the method of Example 2, and after quantification, it was aliquoted and stored at -80°C for standby. According to the method of Example 3, the Rv3616 protein and Rv3872 protein were labeled with biotin, and were named Rv3616-Biotin and Rv3872-Biotin respectively, and after quantification, they were aliquoted and stored at -80°C for standby.

[0086] According to the method of Example 9, the recombinant protein was mixed and diluted to 3 μg / mL in the manner of combination 1 (MPB70 / MPB83 / CE / Rv3616) and combination 2 (MPB70 / MPB83 / CE / Rv3872) at a weight ratio of 1:1:1:1, and after drying, 10 dilutions of bovine tuberculosis positive serum and negative serum were added respectively for reaction, and then biotinylated antigen combination 1 (MPB70-Biotin / MPB83-Biotin / CE-Biotin / Rv3616-Biotin at a weight ratio of 3:3:1:1) and combination 2 (MPB70-Biotin / MPB83-Biotin / CE-Biotin / Rv3872-Biotin at a weight ratio of 3:3:1:1) were added respectively for detection. As shown in Table 12, after the introduction of Rv3616 or Rv3872, the OD value of the positive serum decreased, while the OD value of the negative serum increased, and the P / N value decreased significantly compared with the optimized value. The results show that in this detection system, the introduction of Rv3616 and Rv3872 cannot improve the detection sensitivity or specificity, and it is possible that these two antigens are not suitable for the double-antigen sandwich detection system.

[0087] Table 12 Comparison of detection antigens reported in the literature

[0088] The above is the preferred embodiment of the application. It should be noted that for those skilled in the art, without departing from the principles of the application, several improvements and refinements can be made, which are also considered within the scope of protection of the application.

Claims

1. A Mycobacterium tuberculosis antibody capture antigen, characterized in that, The recombinant MPB70 antigen comprising an amino acid sequence as shown in SEQ ID NO: 1, the recombinant MPB83 antigen comprising an amino acid sequence as shown in SEQ ID NO: 2, and the recombinant CE fusion antigen comprising an amino acid sequence as shown in SEQ ID NO:

3.

2. The M. tuberculosis antibody-captured antigen of claim 1, wherein the antibody is a monoclonal antibody. The weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen, and the recombinant CE fusion antigen is (0.5-4):(0.5-4):(0.5-2).

3. The method for capturing antigens using mycobacterial antibodies as described in claim 1 or 2, characterized in that, The weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen, and the recombinant CE fusion antigen is (2-4):(2-4):(0.8-1.5); or The weight ratio of the recombinant MPB70 antigen, the recombinant MPB83 antigen, and the recombinant CE fusion antigen is (0.5-3):(0.5-3):(0.5-2).

4. A DNA molecule encoding the antibody-captured antigen of Mycobacterium tuberculosis according to any one of claims 1 to 3, characterized in that, The first DNA molecule for encoding the recombinant MPB70 antigen, the second DNA molecule for encoding the recombinant MPB83 antigen, and the third DNA molecule for encoding the recombinant CE fusion antigen are included; The nucleotide sequence of the first DNA molecule is shown in SEQ ID NO: 4, the nucleotide sequence of the second DNA molecule is shown in SEQ ID NO: 5, and the nucleotide sequence of the third DNA molecule is shown in SEQ ID NO:

6.

5. A recombinant vector, characterized in that, The first DNA molecule, the second DNA molecule, and / or the third DNA molecule of claim 4 are included.

6. A recombinant bacterial strain, characterized in that, The recombinant strain of claim 5 is transformed into a host cell.

7. A method for preparing the antibody-captured antigen of Mycobacterium tuberculosis according to any one of claims 1 to 3, characterized by, The recombinant protein is expressed by the recombinant strain of claim 6. The recombinant protein is collected and purified. The Mycobacterium tuberculosis antibody capture antigen of any one of claims 1-3 is included. The Mycobacterium tuberculosis antibody capture antigen is used as a coating antigen, and the protein concentration of the coating antigen is 1.5 μg / mL-9 μg / mL; and / or The Mycobacterium tuberculosis antibody capture antigen is used as a detection antigen, and the N-hydroxysuccinimide-biotin labeling is used, the protein concentration of the detection antigen is 5-10 mg / mL, and the dilution ratio is 1:4000-1:8000; 8. A kit for detecting tuberculosis, characterized by comprising: The kit further comprises a streptavidin-horseradish peroxidase amplification system, and the dilution ratio is 1:3000-1:15000.

9. The kit for detecting tuberculosis according to claim 8, wherein The Mycobacterium tuberculosis antibody capture antigen is used as a coating antigen, and the total protein concentration of the coating antigen is 3.0 μg / mL; The Mycobacterium tuberculosis antibody capture antigen is used as a detection antigen, and the N-hydroxysuccinimide-biotin labeling is used, the protein concentration of the detection antigen is 7 mg / mL, and the dilution ratio is 1:6000; The kit further comprises a streptavidin-horseradish peroxidase amplification system, and the dilution ratio is 1:5000.

10. The kit for detecting tuberculosis according to claim 9, wherein ​ ​ ​

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