A recombinant protein antigen and its use in the preparation of a kit for detecting bovine gamma interferon

Abstract

The application provides a recombinant protein antigen and application thereof in preparation of a kit for detecting bovine gamma interferon, and belongs to the field of biomedical technology. The LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein are obtained through modification and design of CFP10, ESAT6 and Rv3615c; and the recombinant protein antigen is obtained by mixing the three fusion proteins. The recombinant protein antigen obtained by the application is compared with avian and bovine PPD, and it is found that the recombinant protein antigen obtained by the application has higher bovine mycobacterium specificity and sensitivity; and the recombinant protein culture tube prepared by using the culture composition has the advantages of ready-to-use blood sampling anticoagulation, simultaneous incubation and long-distance transportation of plasma after centrifugation.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical technology, and particularly relates to a recombinant protein antigen and application thereof in preparing a bovine gamma interferon kit. BACKGROUND

[0002] Bovine tuberculosis is mainly caused by Mycobacterium tuberculosis complex (MTBC) member M. bovis infection, and is a zoonosis. M. bovis is most likely to infect cattle, and can also infect dogs, cats and other poultry. Bovine tuberculosis can not only be transmitted to humans by inhaling bacteria-containing aerosols or consuming milk that has not been pasteurized, but also can affect animal production performance, food supply stability and public health safety. The World Organization for Animal Health (WOAH) has listed it as a disease that must be reported and a disease that must be inspected in international trade.

[0003] In recent years, the gamma interferon in vitro release test developed abroad has significantly improved the sensitivity of M. bovis infection detection. The principle is that sensitized peripheral blood lymphocytes are activated by specific T cell antigens (such as PPD) during in vitro culture, thereby expressing and secreting gamma interferon, and the release level of interferon in the supernatant is detected by corresponding technical means, so as to determine whether it is infected.

[0004] The gamma interferon release test simulates the immune response of M. bovis infection in the body in vitro, and to some extent reflects the immune status of the host in the early stage of M. bovis infection. Therefore, this method can be used for early detection of bovine tuberculosis, especially when M. bovis is in a latent infection state, the body has no obvious humoral immune response and pathological changes, and this method has more advantages.

[0005] At present, the T cell antigen mainly applied in bovine tuberculosis gamma interferon release test is still avian or bovine PPD. Although the patent CN109776661B "a cocktail stimulating antigen for bovine tuberculosis gamma-interferon ELISA detection", which is different from the avian or bovine PPD, is also a T cell antigen, the mass ratio of ESAT6 antigen, CFP10 antigen, FixB antigen and Rv3615c antigen is 2:2:1:1. However, since lymphocytes cannot directly recognize soluble antigens outside the cells, the soluble antigens must be first taken up by antigen presenting cells, digested into short peptides, and then form MHC peptide complexes on the surface of the antigen presenting cells, so that the MHC peptide complexes can be recognized and combined by the TCR on the surface of the lymphocytes to activate the downstream cellular immune response. Although the aPPD, bPPD and the cocktail antigen obtained by mixing the patent CN109776661B can be taken up by antigen presenting cells in an endocytosis manner, the utilization rate of the endocytosis form of the antigen and the efficiency of the short peptides obtained by downstream digestion are insufficient, resulting in insufficient MHC peptide complexes to cause the downstream lymphocytes to produce a more extensive cellular immune response intensity, which restricts the immunogenicity application of the specific T cell antigen. Therefore, it is urgent to obtain an antigen that can widely cause the downstream lymphocytes to produce an immune response intensity. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a recombinant protein antigen and its application in preparing a bovine gamma interferon detection kit. The present application obtains LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein by modification and design of CFP10, ESAT6 and Rv3615c, and obtains a recombinant protein antigen by mixing the three fusion proteins. The recombinant protein antigen obtained by the present application can cause a more extensive cellular immune response intensity downstream, thereby achieving the purpose of detecting bovine gamma interferon.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a recombinant protein antigen, which is composed of LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein with a mass ratio of 1:0.5-1.5:0.5-1.5; the amino acid sequence of the LFnCFP10 fusion protein is shown in SEQ ID NO. 1; the amino acid sequence of the LFnESAT6 fusion protein is shown in SEQ ID NO. 2; and the amino acid sequence of the LFnRv3615c fusion protein is shown in SEQ ID NO. 3.

[0009] Preferably, the purity of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein is all ≥ 80%.

[0010] The application provides a preparation method of the recombinant protein antigen, and comprises the following steps:

[0011] (1) fusing the Mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c with LFn respectively to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein;

[0012] (2) mixing the obtained LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein according to a mass ratio of 1:0.5-1.5:0.5-1.5 to obtain a recombinant protein antigen.

[0013] The application provides a culture composition comprising the recombinant protein antigen.

[0014] Preferably, the concentration of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein is all 2-3 mg / mL.

[0015] Preferably, the culture composition further comprises 0.5%-1.5% of sodium heparin, 4%-6% of trehalose and 0.5%-1.5% of glycine.

[0016] The application provides a recombinant protein culture tube comprising the culture composition.

[0017] The application further provides a kit for detecting bovine gamma interferon, which comprises the recombinant protein antigen, the recombinant protein antigen obtained according to the preparation method, the culture composition or the recombinant protein culture tube.

[0018] The application further provides application of the recombinant protein antigen, the recombinant protein antigen obtained according to the preparation method, the culture composition or the recombinant protein culture tube in preparing a kit for detecting bovine gamma interferon.

[0019] Compared with the prior art, the application has the following beneficial effects: after LFn is fused with protein antigens, the protein antigens carried by LFn can be taken into the cytoplasm in a membrane-penetrating manner, and meanwhile, LFn participates in MHC-I and MHC-II pathways, thereby inducing CD4 + and CD8 +T cell immune response. Unlike the way of antigen presenting cells through endocytosis of soluble antigen, LFn carries protein antigen across the cell membrane into the cytoplasm of antigen presenting cells, greatly improves the utilization rate of protein antigen, and degrades the protein antigen into short peptides under the hydrolysis of proteasome. In this process, the processing result of the dominant epitope by the antigen presenting cell is autonomous, and finally forms a high output MHC peptide complex, which can cause the characteristics of a wider downstream cellular immune response intensity, and the bovine Mycobacterium antigens CFP10, ESAT6 and Rv3615c are fused with LFn by genetic engineering technology, LFn molecule is located at the N terminal of the fusion protein, and specific antigen CFP10, ESAT6 or Rv3615c is located at the C terminal, to form LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein. And the fusion protein is purified through fermentation and series column chromatography by using E. coli expression system, to obtain LFnCFP10 protein stock solution, LFnESAT6 protein stock solution and LFnRv3615c protein stock solution, and a recombinant protein antigen is obtained by mixing the three kinds of fusion proteins, and the recombinant protein antigen obtained by the application can cause a wider downstream cellular immune response intensity, and thus achieves the purpose of detecting bovine gamma interferon. It is found that the recombinant protein antigen obtained by the application has higher Mycobacterium bovis specificity and sensitivity compared with avian and bovine PPD; and the recombinant protein culture tube prepared by using the culture composition of the application has the advantages of ready-to-use blood sampling anticoagulation, simultaneous incubation and long-distance transportation of plasma after centrifugation. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Plasmid construction map;

[0021] Figure 2 LFnCFP10 plasmid agarose gel map, wherein M is KB Ladder, 1 is LFnCFP10 plasmid, and 2 is LFnCFP10 plasmid after Mlu1 and Xho1 enzyme digestion;

[0022] Figure 3 LFnESAT6 plasmid agarose gel map, wherein M is KB Ladder, 1 is LFnESAT6 plasmid, and 2 is LFnESAT6 plasmid after Smal and Xho1 enzyme digestion;

[0023] Figure 4 LFnRv3615c plasmid agarose gel map, wherein M is KB Ladder, 1 is LFnRv3615c plasmid, and 2 is LFnRv3615c plasmid after Nco1 and Xho1 enzyme digestion;

[0024] Figure 5Figure 3 is a SDS-PAGE analysis diagram of three fusion protein antigens, wherein M is Marker; 1 is LFnCFP10, 2 is LFnESAT6, and 3 is LFnRv3615c. DETAILED DESCRIPTION

[0025] The present application provides a recombinant protein antigen, which is composed of LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein with a mass ratio of 1:0.5-1.5:0.5-1.5; the amino acid sequence of the LFnCFP10 fusion protein is shown as SEQ ID NO. 1; the amino acid sequence of the LFnESAT6 fusion protein is shown as SEQ ID NO. 2; and the amino acid sequence of the LFnRv3615c fusion protein is shown as SEQ ID NO. 3, specifically as follows:

[0026] SEQ ID NO. 1: MVLSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLF TNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSGKTQVLKNDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQAQWRGAAGTAAQAAVVRFQEAANKQKAELDEISTNWRGNAIR;

[0027] SEQ ID NO. 2: MVLSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLF TNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSNVTSIMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMQQKWDFA;

[0028] SEQ ID NO. 3: MVLSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSGVLSAMTENLTVQPERLGVLASHHDNAAVDASSG VEAAAGLGESVAITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFSLRAFT.

[0029] In the present application, the mass ratio of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein is preferably 1:0.7-1.2:0.7-1.2, and further preferably 1:1:1, the nucleotide sequence of the LFnCFP10 fusion protein is shown in SEQ ID NO. 4, specifically: ATGGTGCTGAGCCGCGATATTCTGAGCAAAATTAACCAGCCGTATCAGAAATTTCTGGATGTGCTGAACACCATTAAAAACGCGAGCGATAGCGATGGCCAGGATCTGCTGTTTACCAACCAGCTGAAAGAACATCCGACCGATTTTAGCGTGGAATTTCTGGAACAGAACAGCAACGAAGTGCAGGAAGTGTTTGCGAAAGCGTTTGCGTATTATATTGAACCGCAGCATCGCGATGTGCTGCAGCTGTATGCGCCGGAAGCGTTTAACTATATGGATAAATTTAACGAACAGGAAATTAACCTGAGCGGCAAAACCCAGGTGCTGAAAAACGATGCGGCGACCCTGGCGCAGGAAGCGGGCAACTTTGAACGCATTAGCGGCGATCTGAAAACCCAGATTGATCAGGTGGAAAGCACCGCGGGCAGCCTGCAGGCGCAGTGGCGCGGCGCGGCGGGCACCGCGGCGCAGGCGGCGGTGGTGCGCTTTCAGGAAGCGGCGAACAAACAGAAAGCGGAACTGGATGAAATTAGCACCAACTGGCGCGGCAACGCGATTCGC; the nucleotide sequence of the LFnESAT6 fusion protein is shown in SEQ ID NO. 5, specifically:5, specifically: ATGGTGCTGAGCCGCGATATTCTGAGCAAAATTAACCAGCCGTATCAGAAATTTCTGGATGTGCTGAACACCATTAAAAACGCGAGCGATAGCGATGGCCAGGATCTGCTGTTTACCAACCAGCTGAAAGAACATCCGACCGATTTTAGCGTGGAATTTCTGGAACAGAACAGCAACGAAGTGCAGGAAGTGTTTGCGAAAGCGTTTGCGTATTATATTGAACCGCAGCATCGCGATGTGCTGCAGCTGTATGCGCCGGAAGCGTTTAACTATATGGATAAATTTAACGAACAGGAAATTAACCTGAGCAACGTGACCAGCATTATGACCGAACAGCAGTGGAACTTTGCGGGCATTGAAGCGGCGGCGAGCGCGATTCAGGGCAACGTGACCAGCATTCATAGCCTGCTGGATGAAGGCAAACAGAGCCTGACCAAACTGGCGGCGGCGTGGGGCGGCAGCGGCAGCGAAGCGTATCAGGGCGTGCAGCAGAAATGGGATGCGACCGCGACCGAACTGAACAACGCTGCAGAACCTGGCGCGCACCATTAGCGAAGCGGGCCAGGCGATGGCGAGCACCGAAGGCAACGTGACCGGCATGCAGCAGAAATGGGATTTTGCG; the nucleotide sequence of the LFnRv3615c fusion protein is shown as SEQ ID NO.6, specifically: ATGGTGCTGAGCCGCGATATTCTGAGCAAAATTAACCAGCCGTATCAGAAATTTCTGGATGTGCTGAACACCATTAAAAACGCGAGCGATAGCGATGGCCAGGATCTGCTGTTTACCAACCAGCTGAAAGAACATCCGACCGATTTTAGCGTGGAATTTCTGGAACAGAACAGCAACGAAGTGCAGGAAGTGTTTGCGAAAGCGTTTGCGTATTATATTGAACCGCAGCATCGCGATGTGCTGCAGCTGTATGCGCCGGAAGCGTTTAACTATATGGATAAATTTAACGAACAGGAAATTAACCTGAGCGGCGTGCTGAGCGCGATGACCGAAAACCTGACCGTGCAGCCGGAACGCCTGGGCGTGCTGGCGAGCCATCATGATAACGCGGCGGTGGATGCGAGCAGCGGCGTGGAAGCGGCGGCGGGCCTGGGCGAAAGCGTGGCGATTACCCATGGCCCGTATTGCAGCCAGTTTAACGATACCCTGAACGTGTATCTGACCGCGCATAACGCGCTGGGCAGCAGCCTGCATACCGCGGGCGTGGATCTGGCGAAAAGCCTGCGCATTGCGGCGAAAATTTATAGCGAAGCGGATGAAGCGTGGCGCAAAGCGATTGATGGCCTGTTTAGCCTGCGCGCGTTTACC; the purities of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein are all ≥80%.

[0030] The present application provides a preparation method of the recombinant protein antigen, comprising the following steps:

[0031] (1) fusing the Mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c with LFn respectively to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein;

[0032] (2) mixing the obtained LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein according to a mass ratio of 1:0.5-1.5:0.5-1.5 to obtain the recombinant protein antigen.

[0033] In the present application, the mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c are fused with LFn respectively to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein; the mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c are fused with LFn respectively by genetic engineering technology in the present application, LFn molecules are located at the N terminal of the fusion protein, and specific antigens CFP10, ESAT6 or Rv3615c are located at the C terminal to form fusion proteins LFnCFP10, LFnESAT6 and LFnRv3615c, the fusion proteins are purified by fermentation and series column chromatography by using E. coli expression system to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein. LFn is a new type of recombinant T cell antigen delivery system, and the specific antigens in the LFn fusion protein prepared by using LFn can be effectively delivered to the cytoplasm of antigen presenting cells to participate in the MHC processing and presentation pathway (i.e. the internal presentation pathway of the antigen) so as to activate specific T cell response.

[0034] In the present application, the obtained LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein are mixed according to the mass ratio of 1:0.5-1.5:0.5-1.5 to obtain recombinant protein antigens; the mass ratio of the LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein is preferably 1:0.7-1.2:0.7-1.2, and further preferably 1:1:1.

[0035] The present application provides a culture composition comprising the recombinant protein antigens.

[0036] In the present application, the concentration of the LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein is 2-3 mg / mL, preferably 2.3-2.8 mg / mL, and further preferably 2.5 mg / mL.

[0037] In the present application, the culture composition further comprises 0.5%-1.5% sodium heparin, 4%-6% trehalose and 0.5%-1.5% glycine, the concentration of the sodium heparin is preferably 0.7%-1.2%, and further preferably 1%, the concentration of the trehalose is preferably 4.5%-5.5%, and further preferably 5%, and the concentration of the glycine is preferably 0.7%-1.2%, and further preferably 1%.

[0038] The present application provides a recombinant protein culture tube comprising the culture composition.

[0039] In the present application, the preparation method of the recombinant protein culture tube is as follows: mixing the recombinant protein antigen with 0.5-1.5% heparin sodium, 4-6% trehalose, and 0.5-1.5% glycine, and vacuum drying to obtain the recombinant protein culture tube. The mass ratio of the recombinant protein antigen to 0.5-1.5% heparin sodium, 4-6% trehalose, and 0.5-1.5% glycine is 4-6:1-3:0.4-0.8:1, preferably 4.5-5.5:1.5-2.5:0.5-0.7:1, and further preferably 5:2:0.6:1.

[0040] The present application also provides a kit for detecting bovine gamma interferon, which comprises the recombinant protein antigen, the recombinant protein antigen obtained according to the preparation method, the culture composition, or the recombinant protein culture tube.

[0041] The present application also provides the use of the recombinant protein antigen, the recombinant protein antigen obtained according to the preparation method, the culture composition, or the recombinant protein culture tube in the preparation of a kit for detecting bovine gamma interferon.

[0042] The technical solutions provided by the present application will be described in detail below with reference to the embodiments, but they should not be understood as limiting the scope of the present application.

[0043] Embodiments

[0044] 1. Construction, fermentation and purification of LFnESAT6, LFnCFP10 and LFnRv3615c The amino acid sequences of CFP10 (Accession: NP_218391), ESAT6 (Accession: YP_178023) and Rv3615c (Accession: NP_218132) were queried from NCBI, and bovine Mycobacterium antigens CFP10, ESAT6 and Rv3615c were respectively fused with LFn through genetic engineering technology, with LFn molecules located at the N-terminus of the fusion protein and specific antigens CFP10, ESAT6 or Rv3615c located at the C-terminus, to form fusion proteins LFnCFP10, LFnESAT6 and LFnRv3615c. The fusion proteins were subjected to fermentation and series column chromatography purification by using an E. coli expression system to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein, which are three recombinant protein antigens, and their amino acid sequences are shown as follows:

[0045] The amino acid sequence of the LFnCFP10 fusion protein is: MVLSRDILSKINQPYQKFLDVL NTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSGKTQVLKNDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQAQWRGAAGTAAQAAVVRFQEAANKQKAELDEISTNWRGNAIR;

[0046] The amino acid sequence of the LFnESAT6 fusion protein is: MVLSRDILSKINQPYQKFLDVL NTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSNVTSIMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMQQKWDFA;

[0047] The amino acid sequence of the LFnRv3615c fusion protein is: MVLSRDILSKINQPYQKFLDVL NTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSGVLSAMTENLTVQPERLGV LASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFSLRAFT.

[0048] The amino acid sequences of the three recombinant protein antigens are translated into nucleotide sequences and codon-optimized for the E. coli expression system, NcoI and XhoI enzyme cutting sites are added at the 5' end and 3' end of the nucleotide sequence, respectively, and the nucleotide sequence is constructed in the pET-28b(+) expression vector (C-terminal containing His tag) Figure 1). The three constructed recombinant protein antigens were transformed into the expression strain E. coli Rosetta (DE3) pLysS, respectively, to obtain the expression engineering bacteria of LFnCFP10, LFnESAT6 and LFnRv3615c, respectively. The construction of the expression vector and the expression strain was entrusted to Nanjing Kingsriver Biotech Co., Ltd., and the vector and the strain were provided by Kingsriver Biotech Co., Ltd.

[0049] The three expression engineering bacteria of the fusion protein antigens were inoculated into 2 mL LB medium containing kanamycin (35 μg / mL) at 37°C until OD600nm=1.0, and then inoculated into 400 mL LB medium containing the corresponding antibiotic at 37°C until OD600nm=0.6 or so, and then 1 mmol / L IPTG was added, and the bacteria were induced for 1.5 h, centrifuged and harvested. The bacteria were resuspended with purified water and then added with loading buffer, and a part was subjected to 12% SDS-PAGE analysis, and another part was subjected to SDS-PAGE separation and then transferred to a nitrocellulose membrane, and Western blot analysis was performed with mouse anti-His-tag monoclonal antibody, and the results are shown in Figures 2 to 5 . Figures 2 to 5 The results show that the three proteins can be effectively expressed.

[0050] The three engineering bacteria of the fusion protein antigen were respectively inoculated into 100 mL LB medium containing 35 μg / mL kanamycin, and cultured at 30±2°C and 215±10 rpm for 12-16 h. Then, 10 mL seed liquid was inoculated into 1000 mL fermentation medium containing 2% peptone, 1% yeast powder and 0.5% glucose, and 1 mL kanamycin with a concentration of 35 mg / mL was added. The fermentation was carried out at 37±2°C and 200 rpm for about 3 h, then 2 mL 1M IPTG was added for induction, and the culture was continued for 3 h. The bacteria were collected by centrifugation at 9000 rpm for 20 min. The bacteria were thawed and resuspended in 5 times the wet weight of resuspension buffer (40 mM sodium phosphate, 0.1 M sodium chloride, pH 7.0) at 37°C, and then cycled 3 times in a homogenizer at 827 bar (1200 psi). Then, the bacteria were centrifuged at 2-8°C for 60 min, and the precipitate (inclusion body) was collected. The inclusion body was resuspended in 4 times the weight of washing buffer (40 mM sodium phosphate, 0.1 M sodium chloride, 0.5 M urea, pH 7.0), and then centrifuged to collect the precipitate. The washing process was repeated once. The inclusion body was dissolved in 2 times the weight of dissolution buffer (2% N-Lauroyl Sarcosine, 8 M urea, 500 mM sodium chloride, 10 mM imidazole, 100 mM triethanolamine, pH 8.0) by stirring, and then centrifuged at 20°C for 30 min. The supernatant was filtered and then purified by one-step affinity chromatography using Chelating Sepharose FastFlow medium. The eluted target protein was changed into the final buffer (25 mM sodium phosphate, 140 mM sodium chloride, pH 8.0) using G-25 Medium Sephadex medium, and then renatured. Finally, the fusion protein antigen stock solution was obtained by sterilizing filtration using a 0.22 μm filter, and stored in a refrigerator at ≤-70°C.

[0051] 2. Manufacturing process of recombinant protein culture tube prepared from recombinant protein antigen

[0052] The LFnCFP10, LFnESAT6 and LFnRv3615c protein stock solutions were diluted to 2.5 mg / mL with buffer (containing 0.82% sodium chloride, 0.02% sodium dihydrogen phosphate dihydrate, 0.85% disodium hydrogen phosphate dodecahydrate), and then added to the culture tube containing a mixed solution of heparin sodium, trehalose and glycine, wherein the final concentration of heparin sodium was 1%, the final concentration of trehalose was 5%, and the final concentration of glycine was 1%. The mixed solution was filtered through a 0.22 μm filter or filter before being added to the culture tube for standby use.

[0053] 20 μL of the above 2.5 mg / mL LFnCFP10, LFnESAT6 and LFnRv3615c solutions were respectively dispensed into the above culture tubes.

[0054] Then the culture tube is placed in a vacuum drying machine for vacuum drying to obtain the recombinant protein culture tube, and the vacuum degree should be between 46000 Pa and 56000 Pa. The method for vacuum drying the protein refers to the patent ZL 201010294090.1.

[0055] The raw material of the culture tube used in the manufacturing process of the recombinant protein culture tube is a sterile siliconized blood collection tube containing a separation gel, and heparin sodium is added in the preparation process. The solution is dried by vacuum drying technology and has a vacuum degree of 1 mL. The recombinant protein culture tube can be directly used for blood collection of cattle, has the advantages of anticoagulation, simultaneous incubation and long-distance transportation of plasma after centrifugation, and the dosage of recombinant protein for stimulating heparin anticoagulant whole blood of cattle is 50 μg / mL of whole blood.

[0056] 3. The use method of the recombinant protein culture tube

[0057] Bovine purified tuberculin stimulating antigen (Bovine Tuberculin PPD3000, product number: 7600060, hereinafter referred to as bPPD) and avian purified tuberculin stimulating antigen (Avian Tuberculin PPD 2500, product number: 7600065, hereinafter referred to as aPPD) prepared by Prionics are purchased for comparison of the immunogenicity of the bovine Mycobacterium tuberculosis specific T cell antigen provided by the application.

[0058] At present, the marketable products of aPPD and bPPD can only be purchased in the form of solution or dry powder high-concentration reagent. When used, they need to be diluted in a biological safety cabinet and then added to a cell culture plate to mix with anticoagulant whole blood. The plasma after incubation needs to be additionally sucked into a proper container for storage and transportation. The recombinant protein culture tube provided by the application can be directly used for blood collection, anticoagulation, incubation, plasma separation and long-distance transportation.

[0059] The bovine tuberculosis gamma interferon enzyme-linked immunoassay kit, the negative control culture tube with PBS as the main component and the positive control culture tube with plant agglutinin as the main component are provided by Hainan Weiniayi Clinical Immunological Test Co., Ltd. The test process is as follows:

[0060] 1) Collect at least 5 mL of heparin (lithium) anticoagulant whole blood samples of cattle, mix 250 μL of anticoagulant whole blood and 25 μL of aPPD and bPPD in a cell culture plate according to the instructions of aPPD and bPPD; add 1 mL of anticoagulant whole blood to the recombinant protein culture tube (T), the negative control culture tube (N) and the positive control culture tube (P) and shake or directly use the culture tube to collect blood into the tube and shake.

[0061] 2) After 23±1 h of incubation at 37℃, the supernatant plasma is obtained, and aPPD, bPPD, N, P and T plasma are obtained.

[0062] 3) The plasma sample to be tested and the bovine tuberculosis gamma interferon enzyme-linked immunoassay kit should be equilibrated at room temperature (22±5℃) for more than 30 min.

[0063] 4) Add 50 μL of diluent and 50 μL of aPPD, bPPD, N, P and T plasma to the wells of the enzyme-labeled plate, replace the plasma with the diluent in the negative control wells, and add the same amount of recombinant bovine gamma interferon quality control as the plasma in the positive control wells, and incubate at room temperature (22±5℃) for 1 h.

[0064] 5) After incubation, discard the liquid in the wells, add 200 μL of 1× washing solution to each well, then pat dry, and repeat 6 times.

[0065] 6) Add 100 μL of enzyme-labeled antibody to each well, incubate at room temperature (22±5℃) for 60 min, and repeat the washing of step 5).

[0066] 7) Add 100 μL of TMB color developing substrate to each well, incubate at room temperature (22±5℃) in the dark for 15 min, add 50 μL of stop solution to each well, and read and record the double wavelengths (450 nm, 650 nm) with an enzyme-labeled instrument within 5 min, and calculate OD450nm-OD650nm.

[0067] Experimental Example

[0068] 29 cows were selected for sampling and detection, and the corresponding plasma samples were prepared and the amount of bovine gamma interferon in the plasma was detected according to the use method of aPPD, bPPD and recombinant protein culture tubes, the bovine tuberculosis gamma interferon enzyme-linked immunoassay kit of Hainan Weiniye Clinical Immunological Inspection Co., Ltd., the negative control culture tube with PBS as the main component, and the positive control culture tube with plant agglutinin as the main component. The OD450nm-OD650nm of the samples is shown in Tables 1 and 2.

[0069] Table 1 Detection results of bovine gamma interferon in plasma samples of 29 cows after stimulation by different antigens (OD450nm-OD650nm)

[0070]

[0071]

[0072] Table 2 Comparison of immunogenicity of recombinant protein antigens provided by the application with aPPD and bPPD

[0073]

[0074] In the sampling detection of 29 cows, the immunogenicity of different types of antigens was analyzed, and the recombinant protein antigen provided by the application had significantly higher immunogenicity than aPPD and bPPD, and could induce stronger specific in vitro cell immune response, which was embodied in that higher bovine gamma interferon was detected in plasma.

[0075] In the sample grouping of aPPD <0.1 and bPPD <0.1, the mean and median of the novel T cell antigen provided by the application had no significant difference with aPPD and bPPD; but in the sample grouping of aPPD ≥0.1 and bPPD ≥0.1, the mean and median of the novel T cell antigen provided by the application had significant difference with aPPD and bPPD.

[0076] In summary, the bovine Mycobacterium specific recombinant protein antigen provided by the application has higher specificity and sensitivity than aPPD and bPPD as a novel T cell antigen; and the prepared recombinant protein culture tube has the advantages of ready-to-use blood sampling, simultaneous incubation and long-distance transportation of plasma after centrifugation.

[0077] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A recombinant protein antigen, characterized in that, The recombinant protein antigen is composed of LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein with a mass ratio of 1:0.5-1.5:0.5-1.5; the amino acid sequence of the LFnCFP10 fusion protein is shown as SEQ ID NO. 1; the amino acid sequence of the LFnESAT6 fusion protein is shown as SEQ ID NO. 2; and the amino acid sequence of the LFnRv3615c fusion protein is shown as SEQ ID NO.

3.

2. The recombinant protein antigen according to claim 1, characterized in that, The purity of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein is all ≥80%.

3. The method of producing a recombinant protein antigen according to claim 1 or 2, characterized in that, The method comprises the following steps: (1) fusing Mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c with LFn respectively to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein; (2) mixing the obtained LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein according to a mass ratio of 1:0.5-1.5:0.5-1.5 to obtain a recombinant protein antigen.

4. A culture composition, characterized by, The culture composition comprises the recombinant protein antigen of claim 1 or 2.

5. The culture composition of claim 4, wherein, The concentration of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein is all 2-3 mg / mL.

6. The culture composition of claim 4, wherein The culture composition further comprises 0.5%-1.5% sodium heparin, 4%-6% trehalose and 0.5%-1.5% glycine.

7. A recombinant protein culture tube, comprising: The recombinant protein culture tube comprises the culture composition of claim 6.

8. A kit for detecting bovine gamma interferon, characterized by, The kit comprises the recombinant protein antigen of claim 1 or 2, the recombinant protein antigen obtained by the preparation method of claim 3, the culture composition of any one of claims 4-6 or the recombinant protein culture tube of claim 7.

9. Use of the recombinant protein antigen of claim 1 or 2, the recombinant protein antigen obtained by the preparation method of claim 3, the culture composition of any one of claims 4-6 or the recombinant protein culture tube of claim 7 in the preparation of a bovine gamma interferon detection kit.

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