Recombinant protein antigen and application thereof in preparation of kit for detecting bovine gamma interferon

Abstract

The invention provides a recombinant protein antigen and application thereof in preparation of a kit for detecting bovine gamma interferon, and belongs to the technical field of biomedicine. According to the present invention, the CFP10, the ESAT6 and the Rv3615c are modified and designed to obtain the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein, and the three fusion proteins are mixed to obtain the recombinant protein antigen; compared with poultry type and bovine type PPD, the recombinant protein antigen obtained by the invention is found to have higher specificity and sensitivity of mycobacterium bovis; the recombinant protein culture tube prepared from the culture composition has the advantages that blood sampling and anticoagulation can be achieved immediately, and meanwhile long-distance plasma transportation is achieved after incubation and centrifugation.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a recombinant protein antigen and an application thereof in preparing a kit for detecting bovine gamma interferon. Background Art

[0002] Bovine tuberculosis is mainly caused by infection with Mycobacterium bovis (M. bovis), a member of the Mycobacterium tuberculosis complex (MTBC). It is a zoonotic infectious disease. Mycobacterium bovis is most likely to infect cattle, but it can also infect other poultry such as dogs and cats. Bovine tuberculosis can not only be transmitted to humans through inhalation of bacterial aerosols or consumption of unpasteurized milk, but also affect animal production performance, stable food supply 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 for international trade.

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

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

[0005] At present, the T cell antigen mainly used in the gamma interferon release test for bovine tuberculosis is still avian or bovine PPD. Although the patent CN109776661B "A cocktail stimulation antigen for bovine tuberculosis gamma interferon ELISA detection" is a T cell antigen that is different from avian or bovine PPD by mixing ESAT6 antigen, CFP10 antigen, FixB antigen and Rv3615c antigen in a mass ratio of 2:2:1:1 to prepare a bovine Mycobacterium specific cocktail antigen that is different from avian or bovine PPD. However, since lymphocytes cannot directly recognize extracellular soluble antigens, soluble antigens must first be taken up by antigen presenting cells, digested into short peptides, and then form MHC peptide complexes on the surface of antigen presenting cells. Only then can the MHC peptide complexes be recognized by the TCR on the surface of lymphocytes and bind to activate downstream cellular immune responses. Although the cocktail antigen obtained by mixing aPPD, bPPD and patent CN109776661B can be taken up by antigen presenting cells in the form of endocytosis, the utilization rate of the antigen in the form of endocytosis and the efficiency of short peptides obtained by downstream digestion are insufficient, resulting in the MHC peptide complex being insufficient to induce a wider range of cellular immune response intensity in downstream lymphocytes, which restricts its application as a specific T cell antigen with immunogenicity. Therefore, it is urgent to obtain an antigen that can widely induce the immune response intensity of downstream lymphocytes. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a recombinant protein antigen and its use in the preparation of a kit for detecting bovine interferon-gamma. The present invention obtains LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein by transforming and designing CFP10, ESAT6 and Rv3615c, and obtains a recombinant protein antigen by mixing the three fusion proteins. The recombinant protein antigen obtained by the present invention can induce a wider cellular immune response intensity downstream, thereby achieving the purpose of detecting bovine interferon-gamma.

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

[0008] The present invention provides a recombinant protein antigen, which consists of a LFnCFP10 fusion protein, a LFnESAT6 fusion protein and a LFnRv3615c fusion protein in 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 are all ≥80%.

[0010] The present invention provides a method for preparing the recombinant protein antigen, comprising the following steps:

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

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

[0013] The present invention provides a culture composition, which comprises the recombinant protein antigen.

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

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

[0016] The invention provides a recombinant protein culture tube, which comprises the culture composition.

[0017] The present invention also provides a kit for detecting bovine interferon-gamma, 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 present invention 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 interferon gamma.

[0019] Compared with the prior art, the present invention has the following beneficial effects: after the fusion of LFn and protein antigen, the protein antigen carried can be brought into the cytoplasm in a transmembrane manner, and at the same time participate in the MHC-I and MHC-II pathways to induce CD4 + and CD8 +T cell immune response. Unlike antigen presenting cells that endocytose soluble antigens, LFn carries protein antigens across the cell membrane and enters the cytoplasm of antigen presenting cells, greatly improving the utilization rate of protein antigens, and degrading protein antigens into short peptides under the hydrolysis of proteasomes. In this process, the processing results of the dominant epitopes by antigen presenting cells are autonomous, and finally form a high-yield MHC peptide complex, which can induce a wider range of downstream cellular immune response strength. Through genetic engineering technology, bovine Mycobacterium antigens CFP10, ESAT6 and Rv3615c were fused with LFn respectively. The LFn molecule is located at the N-terminus of the fusion protein, and the specific antigen CFP10, ESAT6 or Rv3615c is located at the C-terminus, forming LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein. The fusion protein is fermented and purified by a series of column chromatography using an E. coli expression system to obtain LFnCFP10 protein stock solution, LFnESAT6 protein stock solution and LFnRv3615c protein stock solution. A recombinant protein antigen is obtained by mixing the three fusion proteins. The recombinant protein antigen obtained by the present invention can induce a wider range of downstream cellular immune response intensity, thereby achieving the purpose of detecting bovine gamma interferon. The recombinant protein antigen obtained by the present invention is compared with avian and bovine PPD, and it is found that the recombinant protein antigen obtained by the present invention has higher bovine Mycobacterium specificity and sensitivity; and the recombinant protein culture tube prepared by the culture composition of the present invention has the advantages of immediate blood collection anticoagulation, simultaneous incubation and long-distance transportation of plasma after centrifugation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 construct maps for plasmids;

[0021] Figure 2 The agarose gel image of LFnCFP10 plasmid digestion, where M is KB Ladder, 1 is LFnCFP10 plasmid, and 2 is LFnCFP10 plasmid digested with Mlu1 and Xho1;

[0022] Figure 3 The agarose gel image of LFnESAT6 plasmid digested with enzymes, where M is KB Ladder, 1 is LFnESAT6 plasmid, and 2 is LFnESAT6 plasmid digested with Smal and Xho1;

[0023] Figure 4 The agarose gel image of LFnRv3615c plasmid digested with enzymes, where M is KB Ladder, 1 is LFnRv3615c plasmid, and 2 is LFnRv3615c plasmid digested with Nco1 and Xho1;

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

[0025] The present invention provides a recombinant protein antigen, which consists of a LFnCFP10 fusion protein, a LFnESAT6 fusion protein and a LFnRv3615c fusion protein in 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; the amino acid sequence of the LFnRv3615c fusion protein is shown in SEQ ID NO.3, and is specifically as follows:

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

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

[0028] SEQ ID NO.3:MVLSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLF TNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSGVLSAMTENLTVQPERLGVLASHHDNAAVDASSG VEAAAGLGESVAITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFSLRAFT。

[0029] In the present invention, 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 more preferably 1:1:1. The nucleotide sequence of the LFnCFP10 fusion protein is as shown in SEQ ID NO.4, specifically:; The nucleotide sequence of the LFnESAT6 fusion protein is as shown in SEQ ID NO.As shown in Figure 5, specifically: ATGGTGCTGAGCCGCGATATTCTGAGCAAAATTAACCAGCCGTATCAGAAATTTCTGGATGTGCTGAACACCATTAAAAACGCGAGCGATAGCGATGGCCAGGATCTGCTGTTTACCAACCAGCTGAAAGAACATCCGACCGATTTTAGCGTGGAATTTCTGGAACAGAACAGCAACGAAGTGCAGGAAGTGTTTGCGAAAGCGTTTGCGTATTATATTGAACCGCAGCATCGCGATGTGCTGCAGCTGTATGCGCCGGAAGCGTTTAACTATATGGATAAATTTAACGAACAGGAAATTAACCTGAGCAACGTGACCAGCATTATGACCGAACAGCAGTGGAACTTTGCGGGCATTGAAGCGGCGGCGAGCGCGATTCAGGGCAACGTGACCAGCATTCATAGCCTGCTGGATGAAGGCAAACAGAGCCTGACCAAACTGGCGGCGGCGTGGGGCGGCAGCGGCAGCGAAGCGTATCAGGGCGTGCAGCAGAAATGGGATGCGACCGCGACCGAACTGAACAACGC GCTGCAGAACCTGGCGCGCACCATTAGCGAAGCGGGCCAGGCGATGGCGAGCACCGAAGGCAACGTGACCGGCATGCAGCAGAAATGGGATTTTGCG; The nucleotide sequence of the LFnRv3615c fusion protein is as shown in SEQ ID NO.6, specifically: ATGGTGCTGAGCCGCGATATTCTGAGCAAAATTAACCAGCCGTATCA; the purity of the LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein are all ≥80%. .

[0030] The present invention provides a method for preparing the recombinant protein antigen, comprising the following steps:

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

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

[0033] In the present invention, 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 present invention fuses Mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c with LFn respectively through genetic engineering technology, the LFn molecule is located at the N-terminus of the fusion protein, and the specific antigen CFP10, ESAT6 or Rv3615c is located at the C-terminus to form fusion proteins LFnCFP10, LFnESAT6 and LFnRv3615c, and the fusion proteins are fermented and purified by a series of column chromatography using an Escherichia 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. The specific antigens in the LFn fusion protein prepared by LFn can be effectively delivered by LFn 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), thereby activating specific T cell responses.

[0034] In the present invention, the obtained LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein are mixed in a mass ratio of 1:0.5-1.5:0.5-1.5 to obtain a recombinant protein antigen; 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 more preferably 1:1:1.

[0035] The present invention provides a culture composition, which comprises the recombinant protein antigen.

[0036] In the present invention, the concentrations of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein are all 2-3 mg / mL, preferably 2.3-2.8 mg / mL, and more preferably 2.5 mg / mL.

[0037] In the present invention, the culture composition also includes 0.5% to 1.5% heparin sodium, 4% to 6% trehalose, and 0.5% to 1.5% glycine. The concentration of the heparin sodium is preferably 0.7% to 1.2%, and more preferably 1%. The concentration of the trehalose is preferably 4.5% to 5.5%, and more preferably 5%. The concentration of the glycine is preferably 0.7% to 1.2%, and more preferably 1%.

[0038] The invention provides a recombinant protein culture tube, which comprises the culture composition.

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

[0040] The present invention also provides a kit for detecting bovine interferon-gamma, 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 invention 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 interferon gamma.

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example

[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 the Mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c were fused with LFn by genetic engineering technology. The LFn molecule was located at the N-terminus of the fusion protein, and the specific antigen CFP10, ESAT6 or Rv3615c was located at the C-terminus to form the fusion proteins LFnCFP10, LFnESAT6 and LFnRv3615c. The fusion proteins were fermented and purified by a series of column chromatography using the E. coli expression system to obtain the LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein, which are three recombinant protein antigens, and their amino acid sequences are shown below:

[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 were translated into nucleotide sequences and codons were optimized for the E. coli expression system. NcoI and XhoI restriction sites were added to the 5' and 3' ends of the nucleotide sequences, respectively. The nucleotide sequences were constructed in the pET-28b(+) (with His tag at the C-terminus) expression vector ( Figure 1). The constructed expression plasmids of the three recombinant protein antigens were transformed into the expression strain E. coli Rosetta (DE3) pLysS, and the expression engineering bacteria of LFnCFP10, LFnESAT6 and LFnRv3615c were obtained respectively. The construction of expression vectors and expression strains were entrusted to Nanjing GenScript Biotech Co., Ltd., and the vectors and strains were provided by GenScript Biotech Co., Ltd.

[0049] The three fusion protein antigen expression engineering bacteria were inoculated into 2mL LB medium containing kanamycin (35μg / mL) and cultured at 37℃ until OD600nm=1.0. Then, they were inoculated into 400mL LB medium containing the corresponding antibiotics and cultured at 37℃ until OD600nm=0.6. Then, IPTG with a final concentration of 1mmol / L was added. After induction for 1.5h, the bacteria were centrifuged and harvested. The bacteria were resuspended in purified water and added with loading buffer. One part was analyzed by 12% SDS-PAGE, and the other part was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis was performed using mouse anti-His-tag monoclonal antibody. The results are shown in Figure 2. Figures 2 to 5 . Figures 2 to 5 The results showed that all three proteins were effectively expressed.

[0050] The engineered bacteria of three fusion protein antigens were inoculated in 100 mL of LB medium containing 35 μg / mL kanamycin, and cultured at 30±2°C and 215±10rpm for 12-16 hours, and then 10 mL of seed solution was inoculated in 1000 mL of fermentation medium containing 2% peptone, 1% yeast powder, and 0.5% glucose, and 1 mL of kanamycin with a concentration of 35 mg / mL was added at the same time. After fermentation at 37±2°C and 200rpm for about 3 hours, 2 mL of 1M IPTG was added for induction, and culture was continued for 3 hours, and then centrifuged at 9000rpm for 20 minutes to collect the bacteria. The bacteria were thawed and resuspended at 37°C with a resuspension buffer (40mM sodium phosphate, 0.1M sodium chloride, pH 7.0) 5 times the wet weight of the bacteria, circulated 3 times in a homogenizer at 827bar (1200psi), and then centrifuged at 2-8°C for 60 minutes to collect the precipitate (inclusion body). The inclusion bodies were resuspended with 4 times the weight of washing buffer (40mM sodium phosphate, 0.1M sodium chloride, 0.5M urea, pH7.0), centrifuged again to collect the precipitate, and the washing process was repeated once. The inclusion bodies were dissolved by stirring with 2 times the weight of inclusion body dissolution buffer (2% N-Lauroyl Sarcosine, 8M urea, 500mM sodium chloride, 10mM imidazole, 100mM triethanolamine, pH8.0), and then centrifuged at 20°C for 30min. The supernatant was filtered and purified by one-step affinity chromatography using Chelating Sepharose FastFlow medium. The eluted target protein was replaced with G-25 Medium Sephadex medium in the final buffer (25mM sodium phosphate, 140mM sodium chloride, pH8.0) for renaturation. Finally, the fusion protein antigen stock solution was obtained after sterilization filtration with a 0.22μm filter and stored in a refrigerator ≤-70°C.

[0051] 2. Manufacturing process of recombinant protein culture tubes made from recombinant protein antigens

[0052] Take the original solutions of LFnCFP10, LFnESAT6 and LFnRv3615c proteins, dilute them to 2.5 mg / mL with buffer (containing 0.82% sodium chloride, 0.02% sodium dihydrogen phosphate dihydrate, and 0.85% sodium dihydrogen phosphate dodecahydrate), and then add them to a culture tube containing a mixed solution of sodium heparin, trehalose and glycine, wherein the final concentration of sodium heparin is 1%, the final concentration of trehalose is 5%, and the final concentration of glycine is 1%. The mixed solution needs to be filtered through a 0.22 μm filter membrane or filter before being added to the culture tube for use.

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

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

[0055] The culture tube raw material used in the manufacturing process of the recombinant protein culture tube of the present invention is a sterile siliconized blood collection tube containing separation gel. Heparin sodium is added in the preparation process, and the solution is dried by vacuum drying technology with a vacuum degree of 1 mL. The recombinant protein culture tube can be directly used to draw blood from cattle, and has the advantages of anticoagulation, simultaneous incubation and long-distance transportation of plasma after centrifugation. The amount of bovine heparin anticoagulated whole blood stimulated by each segment of recombinant protein is 50 μg / mL whole blood.

[0056] 3. How to use the recombinant protein culture tube

[0057] Purified bovine tuberculin PPD3000 (Cat. No. 7600060, hereinafter referred to as bPPD) and purified avian tuberculin PPD 2500 (Cat. No. 7600065, hereinafter referred to as aPPD) prepared by Prionics were purchased and used to compare the differences in immunogenicity of the bovine Mycobacterium-specific T cell antigens provided by the present invention.

[0058] Currently, the commercial products of aPPD and bPPD can only be purchased as solutions or high-concentration dry powder reagents. When used, they need to be diluted in a biological safety cabinet and added to a cell culture plate to mix with anticoagulated bovine whole blood. The plasma after incubation needs to be additionally drawn into an appropriate container for storage and transportation. The recombinant protein culture tube provided by the present invention can be directly used for blood collection, anticoagulation, incubation, plasma separation and long-distance transportation.

[0059] The bovine tuberculosis gamma interferon enzyme-linked immunosorbent assay kit and the negative control culture tube whose main component is PBS and the positive control culture tube whose main component is phytohemagglutinin were provided by Hainan Weiwei Clinical Immunology Laboratory Co., Ltd. The test process is as follows:

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

[0061] 2) After incubation at 37°C for 23±1h, the supernatant plasma was collected to obtain aPPD, bPPD, N, P and T plasma, respectively.

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

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

[0064] 5) After incubation, shake off the liquid in the wells, add 200 μL of 1× washing solution to each well for washing, 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°C) for 60 min, and repeat the washing in step 5).

[0066] 7) Add 100 μL of TMB chromogenic substrate to each well, incubate at room temperature (22±5°C) in the dark for 15 min, add 50 μL of stop solution to each well, read and record dual wavelengths (450 nm, 650 nm) with a microplate reader within 5 min, and calculate OD450nm-OD650nm.

[0067] Experimental example

[0068] 29 dairy cows were selected for sampling and testing. According to the usage of aPPD, bPPD and recombinant protein culture tubes, the bovine tuberculosis gamma interferon enzyme-linked immunosorbent assay kit of Hainan Weiwei Clinical Immunology Laboratory Co., Ltd., the negative control culture tube whose main component was PBS, and the positive control culture tube whose main component was plant lectin, the corresponding plasma specimens were obtained and the bovine gamma interferon in plasma was detected. The OD450nm-OD650nm of the samples are shown in Tables 1 and 2.

[0069] Table 1 Detection results of bovine gamma interferon in plasma samples of 29 cows' anticoagulated whole blood stimulated with different antigens (OD450nm-OD650nm)

[0070]

[0071]

[0072] Table 2 Comparison of immunogenicity of the recombinant protein antigen provided by the present invention with aPPD and bPPD

[0073]

[0074] In the sampling test of 29 dairy cows, the immunogenicity of different types of antigens was analyzed respectively. The recombinant protein antigen provided by the present invention has significantly higher immunogenicity than aPPD and bPPD, and can induce stronger specific in vitro cellular immune response, which is reflected in the detection of higher bovine gamma interferon in plasma.

[0075] In the sample groups of aPPD<0.1 and bPPD<0.1, the mean and median of the novel T cell antigen provided by the present invention were not significantly different from those of aPPD and bPPD; but in the sample groups of aPPD≥0.1 and bPPD≥0.1, the mean and median of the novel T cell antigen provided by the present invention were significantly different from those of aPPD and bPPD.

[0076] In summary, the bovine Mycobacterium-specific recombinant protein antigen provided by the present invention has higher specificity and sensitivity than aPPD and bPPD as a new type of T cell antigen; the prepared recombinant protein culture tube has the advantages of immediate anticoagulation of blood collection, simultaneous incubation and long-distance transportation of plasma after centrifugation.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

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 in 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; the amino acid sequence of the LFnRv3615c fusion protein is shown in 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 are all ≥80%.

3. The method for preparing a recombinant protein antigen according to claim 1 or 2, characterized in that: The following steps are involved: (1) Mycobacterium bovis antigens CFP10, ESAT6 and Rv3615c were fused with LFn to obtain LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein; (2) The obtained LFnCFP10 fusion protein, LFnESAT6 fusion protein and LFnRv3615c fusion protein were mixed at a mass ratio of 1:0.5-1.5:0.5-1.5 to obtain a recombinant protein antigen.

4. A culture composition, characterized in that The culture composition comprises the recombinant protein antigen according to claim 1 or 2.

5. The culture composition according to claim 4, characterized in that The concentrations of the LFnCFP10 fusion protein, the LFnESAT6 fusion protein and the LFnRv3615c fusion protein are all 2-3 mg / mL.

6. The culture composition according to claim 4, characterized in that The culture composition also includes 0.5% to 1.5% heparin sodium, 4% to 6% trehalose, and 0.5% to 1.5% glycine.

7. A recombinant protein culture tube, characterized in that: The recombinant protein culture tube comprises the culture composition according to claim 6.

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

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

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