Polypeptide antigen composition of specific T cells of mycobacterium bovis and application of polypeptide antigen composition in preparation of kit for detecting bovine gamma interferon

Abstract

The invention provides a polypeptide antigen composition of mycobacterium bovis specific T cells and application of the polypeptide antigen composition in preparation of a kit for detecting bovine gamma interferon, and belongs to the technical field of biomedicine. The polypeptide antigen composition obtained by mixing 15 sections of short peptides with 20-50 amino acids designed on the basis of amino acid sequences of ESAT6, CFP10 and Rv3615c according to a specific ratio is good in specificity and can be applied to identification and detection of early bovine tuberculosis of BCG inoculated cattle and mycobacterium paratuberculosis positive cattle in NTM. Compared with poultry type and bovine type PPD, the polypeptide antigen composition obtained by the invention has the advantages that the polypeptide antigen composition obtained by the method has higher bovine mycobacterium specificity and sensitivity; the polypeptide culture tube prepared by the invention has the advantages of blood sampling and anticoagulation in use, and long-distance blood plasma transportation after incubation and centrifugation at the same time.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a polypeptide antigen composition of bovine mycobacterium-specific T cells and an application of the composition 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), and is a zoonotic infectious disease. 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] At present, the diagnosis of bovine tuberculosis mainly relies on the following methods: 1) Based on bacteriological detection methods, such as bacterial staining smear microscopy, bacterial culture and other methods. The isolation and culture of Mycobacterium bovis is the gold standard for the diagnosis of bovine tuberculosis, with good specificity, but low sensitivity, and it is difficult to promote on a large scale; 2) PCR detection method, this method is mainly used to isolate and culture pathogenic mycobacteria from diseased materials. The method has high technical requirements for operators and experimental operating environment, and it is easy to have false positives or false negatives during detection, and it is not easy to popularize; 3) Tuberculin intradermal allergy test, which was the first method used to diagnose bovine tuberculosis and is currently the most widely used standard method for detecting bovine tuberculosis in the world and recommended by WOAH. This method can only be used for in vivo testing, which is labor-intensive and not conducive to large-scale epidemiological surveys. It is also prone to false positives and false negatives. 4) Serological testing: This method is easy to operate, simple to use, has a high negative detection value, and is economical, but antibody testing is prone to false positives. 5) Gamma interferon release test: This method uses specific T cell antigens to stimulate bovine whole blood in vitro, and then detects the content of gamma interferon in the whole blood to determine whether the cattle are infected with mycobacteria. This method not only has high sensitivity and specificity, but also has more objective and reliable results. It only requires one contact with the animal, and has great advantages in detecting wild animals and large animals that are difficult to bind.

[0004] The gamma interferon release test includes a constant temperature incubation process after anticoagulation of fresh whole blood and specific T cell antigens are mixed in vitro, and a process for detecting the amount of gamma interferon secreted after stimulation by specific T cell antigens during the incubation process. Among them, the selection and preparation method of specific T cell antigens are key factors that determine the analytical performance of the detection method. In the early days of the establishment of the gamma interferon in vitro release test, PPD was used as a specific T cell antigen because the main component of PPD is a protein derivative of avian or bovine mycobacterium, which contains very complex protein antigen components. Although the sensitivity is acceptable, the specificity is far from ideal. Therefore, it is urgent to obtain an antigen with good specificity. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a polypeptide antigen composition of bovine Mycobacterium-specific T cells and its use in the preparation of a kit for detecting bovine gamma interferon. The polypeptide antigen composition obtained by mixing 15 short peptides of 9 to 30 amino acids designed based on the amino acid sequences of ESAT6, CFP10 and Rv3615c in a specific proportion has good specificity and can be used for the identification and detection of early bovine tuberculosis in BCG-vaccinated cattle and NTM-positive cattle for Mycobacterium avium paratuberculosis subspecies.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a polypeptide antigen composition for bovine mycobacterium-specific T cells. The polypeptide antigen composition consists of 15 polypeptides, wherein the amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.2; the amino acid sequence of polypeptide 3 is shown in SEQ ID NO.3; the amino acid sequence of polypeptide 4 is shown in SEQ ID NO.4; the amino acid sequence of polypeptide 5 is shown in SEQ ID NO.5; the amino acid sequence of polypeptide 6 is shown in SEQ ID NO.6; the amino acid sequence of polypeptide 7 is shown in SEQ ID NO.7; the amino acid sequence of polypeptide 8 is shown in SEQ ID NO.8; the amino acid sequence of polypeptide 9 is shown in SEQ ID NO.9; the amino acid sequence of polypeptide 10 is shown in SEQ ID NO.10; the amino acid sequence of polypeptide 11 is shown in SEQ ID NO.11; the amino acid sequence of polypeptide 12 is shown in SEQ ID NO.12; the amino acid sequence of polypeptide 13 is shown in SEQ ID NO.13; the amino acid sequence of polypeptide 14 is shown in SEQ ID NO.14; and the amino acid sequence of polypeptide 15 is shown in SEQ ID NO.15.

[0008] Preferably, the mass ratio of polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 8, polypeptide 9, polypeptide 10, polypeptide 11, polypeptide 12, polypeptide 13, polypeptide 14, and polypeptide 15 is 1: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5.

[0009] Preferably, the purity of the 15 polypeptides is ≥95%.

[0010] The present invention provides a culture composition, which comprises the polypeptide antigen composition.

[0011] Preferably, the polypeptide antigen composition is formed by mixing 15 polypeptide solutions each having an initial concentration of 0.5 to 1.5 mg / mL.

[0012] Preferably, the volume ratio of the 15 polypeptide solutions in the polypeptide antigen composition is polypeptide 1: polypeptide 2: polypeptide 3: polypeptide 4: polypeptide 5: polypeptide 6: polypeptide 7: polypeptide 8: polypeptide 9: polypeptide 10: polypeptide 11: polypeptide 12: polypeptide 13: polypeptide 14: polypeptide 15=1:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5.

[0013] Preferably, the culture composition further comprises a mixed solution containing trehalose with an initial concentration of 35wt% to 45wt% and glycine with an initial concentration of 7wt% to 9wt%, and a heparin sodium solution with an initial concentration of 0.5wt% to 1.5wt%.

[0014] Preferably, the volume ratio of the mixed solution of the polypeptide antigen composition in the culture composition is 6-8:1; the volume ratio of the polypeptide antigen composition to the heparin sodium solution with an initial concentration of 0.5wt%-1.5wt% is 1:0.1-0.3.

[0015] The present invention also provides a kit for detecting bovine interferon-gamma, wherein the kit comprises the polypeptide antigen composition or the culture composition.

[0016] The present invention also provides the use of the polypeptide antigen composition or the culture composition in preparing a kit for detecting bovine interferon gamma.

[0017] Compared with the prior art, the present invention has the following beneficial effects: based on the amino acid sequences of ESAT6, CFP10 and Rv3615c, the present invention predicts and analyzes the dominant antigenic determinants, and obtains 15 short peptides of 20 to 50 amino acids by artificial synthesis, eliminating the process of obtaining short peptides by antigen presenting cells through uptake and digestion. High-concentration short peptides directly form MHC peptide complexes with MHC receptors on the surface of antigen presenting cells, causing a wider range of downstream cellular immune response intensity. The present invention obtains a polypeptide antigen composition by mixing the obtained 15 short peptides according to a specific ratio. The polypeptide antigen composition obtained by the present invention has good specificity and can be applied to the identification and detection of early bovine tuberculosis in BCG-vaccinated cattle and avian Mycobacterium paratuberculosis subspecies-positive cattle in NTM. The polypeptide antigen composition obtained by the present invention is compared with avian and bovine PPD, and it is found that the polypeptide antigen composition obtained by the present invention has higher specificity and sensitivity for Mycobacterium bovis; and the polypeptide culture tube prepared by the present invention has the advantages of immediate anticoagulation of blood collection, simultaneous incubation and long-distance transportation of plasma after centrifugation. DETAILED DESCRIPTION

[0018] The present invention provides a polypeptide antigen composition for bovine mycobacterium-specific T cells. The polypeptide antigen composition consists of 15 polypeptides, wherein the amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.2; the amino acid sequence of polypeptide 3 is shown in SEQ ID NO.3; the amino acid sequence of polypeptide 4 is shown in SEQ ID NO.4; the amino acid sequence of polypeptide 5 is shown in SEQ ID NO.5; the amino acid sequence of polypeptide 6 is shown in SEQ ID NO.6; the amino acid sequence of polypeptide 7 is shown in SEQ ID NO.7; the amino acid sequence of polypeptide 8 is shown in SEQ ID NO.8; the amino acid sequence of polypeptide 9 is shown in SEQ ID NO.9; the amino acid sequence of polypeptide 10 is shown in SEQ ID NO.10; the amino acid sequence of polypeptide 11 is shown in SEQ ID NO.11; the amino acid sequence of polypeptide 12 is shown in SEQ ID NO.12; the amino acid sequence of polypeptide 13 is shown in SEQ ID NO.13; the amino acid sequence of polypeptide 14 is shown in SEQ ID NO.14; the amino acid sequence of polypeptide 15 is shown in SEQ ID NO.15, specifically as follows:

[0019] SEQ ID NO.1: SAIQGNVTSIHSLLDEGKQSLTKLAAAW;

[0020] SEQ ID NO.2: MTEQQWNFAGIEAAASAIQGNVT;

[0021] SEQ ID NO.3:QQKWDATATELNNALQNLARTISEAGQAM;

[0022] SEQ ID NO.4:KLAAAWGGSGSEAYQGVQQKWDATA;

[0023] SEQ ID NO.5:

[0024] WDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA;

[0025] SEQ ID NO.6:RFQEAANKQKQELDEISTNIRQAGVQYS;

[0026] SEQ ID NO.7:GSLQGQWRGAAGTAAQAAVVRFQEAANK;

[0027] SEQ ID NO.8:

[0028] VRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF;

[0029] SEQ ID NO.9:

[0030] LGESVAITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRI AA;

[0031] SEQ ID NO.10:

[0032] NVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLF T;

[0033] SEQ ID NO.11:LAKSLRIAAKIYSEADEAWRKAIDGLFT;

[0034] SEQ ID NO.12:LLDEGKQSLTKLAAAWGGSGSEAYQGV;

[0035] SEQ ID NO.13:AATLAQEAGNFERISGDLKTQIDQVES;

[0036] SEQ ID NO.14:ATLAQEAGNFERISGDLKTQIDQV;

[0037] <h2 style=";text-align:left;direction:ltr">SEQ ID NO.15:LGESVAITHGPYCSQFNDTLNVYLTAHNAL。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0038] In the present invention, the nucleotide sequence of the polypeptide 1 is shown in SEQ ID NO.16, specifically: AGCGCAATCCAGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAG GGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGG; the nucleotide sequence of the polypeptide 2 is shown in SEQ ID NO.17, specifically: ATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAG CGCAATCCAGGGAAATGTCACG; the nucleotide sequence of the polypeptide 3 is shown in SEQ ID NO.18, specifically: CAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCA GAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATG; the nucleotide sequence of the polypeptide 4 is shown in SEQ ID NO.19, specifically: AAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGG TGTCCAGCAAAAATGGGACGCCACGGCT; the nucleotide sequence of the polypeptide 5 is shown in SEQ ID NO.20, specifically: TGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGC GCGGACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGCAACGTCACTGGGATGTTCGCA; the nucleotide sequence of the polypeptide 6 is shown in SEQ ID NO.21, specifically: CGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGAT CTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCG; the nucleotide sequence of the polypeptide 7 is shown in SEQ ID NO.22, specifically: GGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCA GGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAG; the nucleotide sequence of the polypeptide 8 is shown in SEQ ID NO.The nucleotide sequence of the polypeptide 9 is shown in SEQ ID NO.23, specifically: GTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGA CGAGGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTC; the nucleotide sequence of the polypeptide 9 is shown in SEQ ID NO.24, specifically: CTAGGCGAATCTGTGGCGATCACTCACGGTCCGTACTGCTCACAGTTCAACGACACGTTAAATGTGTACTTGACTGCCCACAATGCCCTGGGCTCGTCCTTGCATACGGCCGGTGTCGATCTCGCCAAAAGTCTTCGAATTGCGGCG; the nucleotide sequence of the polypeptide 10 is shown in SEQ ID NO.25, specifically: AATGTGTACTTGACTGCCCACAATGCCCTGGGCTCGTCCTTGCATACGGCCGGTGTCGATCTCGCCAAAAGTCTTCGAATTGCGGCGAAAATATATAG CGAGGCCGACGAAGCGTGGCGCAAGGCTATCGACGGGTTGTTTACC; the nucleotide sequence of the polypeptide 11 is shown in SEQ ID NO.26, specifically: CTCGCCAAAAGTCTTCGAATTGCGGCGAAAATATATAGCGAGGCCGAC GAAGCGTGGCGCAAGGCTATCGACGGGTTGTTTACC; the nucleotide sequence of the polypeptide 12 is shown in SEQ ID NO.27, specifically: CTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTG GGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTC; the nucleotide sequence of the polypeptide 13 is shown in SEQ ID NO.28, specifically: GCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGC GACCTGAAAACCCAGATCGACCAGGTGGAGTCG; the nucleotide sequence of the polypeptide 14 is shown in SEQ ID NO.29, specifically: GCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTG; the nucleotide sequence of the polypeptide 15 is as shown in SEQ ID NO.30, specifically: CTAGGCGAATCTGTGGCGATCACTCACGGTCCGTACTGCTCACAGTTC AACGACACGTTAAATGTGTACTTGACTGCCCACAATGCCCTG. .

[0039] In the present invention, the mass ratio of polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 8, polypeptide 9, polypeptide 10, polypeptide 11, polypeptide 12, polypeptide 13, polypeptide 14, and polypeptide 15 is 1:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5 1.5:0.5~1.5:0.5~1.5, preferably 1:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2, more preferably 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1.

[0040] In the present invention, the purity of the 15 polypeptides is ≥95%.

[0041] The present invention provides a culture composition, which comprises the polypeptide antigen composition.

[0042] In the present invention, the polypeptide antigen composition is formed by mixing 15 polypeptide solutions with initial concentrations of 0.5-1.5 mg / mL. The initial concentrations of the 15 polypeptide solutions are preferably 0.7-1.2 mg / mL, and more preferably 1 mg / mL.

[0043] In the present invention, the volume ratio of the 15 polypeptide solutions in the polypeptide antigen composition is polypeptide 1: polypeptide 2: polypeptide 3: polypeptide 4: polypeptide 5: polypeptide 6: polypeptide 7: polypeptide 8: polypeptide 9: polypeptide 10: polypeptide 11: polypeptide 12: polypeptide 13: polypeptide 14: polypeptide 15=1:0.5~1.5 ... 1.5:0.5~1.5:0.5~1.5:0.5~1.5, preferably 1:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2:0.7~1.2, more preferably 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1.

[0044] In the present invention, the culture composition also includes a mixed solution containing trehalose with an initial concentration of 35wt% to 45wt% and glycine with an initial concentration of 7wt% to 9wt%, and a heparin sodium solution with an initial concentration of 0.5wt% to 1.5wt%. The initial concentration of trehalose in the mixed solution is preferably 37wt% to 43wt%, and more preferably 40wt%; the initial concentration of glycine is preferably 7.5wt% to 8.5wt%, and more preferably 8wt%; the initial concentration of the heparin sodium solution is preferably 0.7wt% to 1.2wt%, and more preferably 1wt%. The addition of trehalose and glycine can play a role in protecting peptide segments and preventing peptide segment degradation; the addition of heparin sodium solution can play an anticoagulant role, prolonging the coagulation time, thereby ensuring that the plasma can be transported over long distances.

[0045] In the present invention, the polypeptide antigen composition, the mixed solution and the heparin sodium solution are divided into culture tubes, and the polypeptide culture tubes are obtained by vacuum drying, and the polypeptide culture tubes can be used to detect bovine gamma interferon. The volume ratio of the polypeptide antigen composition to the mixed solution in the culture composition is 6 to 8:1, preferably 6.5 to 7.5:1, and more preferably 7:1; the volume ratio of the polypeptide antigen composition to the heparin sodium solution with an initial concentration of 0.5wt% to 1.5wt% is 1:0.1 to 0.3, preferably 1:0.15 to 0.25, and more preferably 1:0.2 to 0.21.

[0046] The present invention also provides a kit for detecting bovine interferon-gamma, wherein the kit comprises the polypeptide antigen composition or the culture composition.

[0047] The present invention also provides the use of the polypeptide antigen composition or the culture composition in preparing a kit for detecting bovine interferon gamma.

[0048] 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.

[0049] Example

[0050] 1.15 Obtaining the peptide sequence

[0051] The bovine tuberculosis pathogen Mycobacterium bovis was sequenced with the BCG genome, and the BCG missing genes such as Mycobacterium bovis ESAT6 (Accession: YP_178023), CFP10 (Accession: NP_218391), and Rv3615c (Accession: NP_218132) were screened out, and polypeptides were designed based on their amino acid sequences. According to the following principles, 15 segments of polypeptides were designed to form a combination: First, the charge, hydrophobicity, length, and structural characteristics of the candidate polypeptides were analyzed to ensure their specificity and stability. Second, the selected candidate polypeptide combination has amino acids that can form a stable spatial conformation, which enhances the functionality and practicality of the polypeptide. The solid phase synthesis method (solid phase peptide synthesis technology) was adopted to fix and extend the amino acid chain one by one on the solid carrier, effectively remove the protecting group, and realize the efficient synthesis of the polypeptide, with a purity of more than 95%. The amino acid sequences of the 15 segments of polypeptide sequences obtained are shown in Table 1.

[0052] Table 1 Amino acid sequence of polypeptide sequence

[0053] Peptide 1 SAIQGNVTSIHSLLDEGKQSLTKLAAAW Peptide 2 MTEQQWNFAGIEAAASAIQGNVT Peptide 3 QQKWDATATELNNALQNLARTISEAGQAM Peptide 4 KLAAAWGGSGSEAYQGVQQKWDATA Peptide 5 WDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA Peptide 6 RFQEAANKQKQELDEISTNIRQAGVQYS Peptide 7 GSLQGQWRGAAGTAAQAAVVRFQEAANK Peptide 8 VRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF Peptide 9 LGESVAITHGPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAA Peptide 10 NVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFT Peptide 11 LAKSLRIAAKIYSEADEAWRKAIDGLFT Peptide 12 LLDEGKQSLTKLAAAWGGSGSEAYQGV Peptide 13 AATLAQEAGNFERISGDLKTQIDQVES Peptide 14 ATLAQEAGNFERISGDLKTQIDQV Peptide 15 LGESVAITHGPYCSQFNDTLNVYLTAHNAL

[0054] 2. Manufacturing process of polypeptide culture tubes made from polypeptide antigens

[0055] The synthesized polypeptide was purified, the organic solvent was removed, and a concentrated solution was obtained, which was sterile filtered and freeze-dried into polypeptide dry powder. The 15 segments of polypeptide dry powder were dissolved in sterile PBS to 1 mg / mL, and mixed in equal proportions to obtain a polypeptide mixed solution. A mixed solution containing 40wt% trehalose, 8wt% glycine and 1wt% heparin sodium solution was prepared, and the solution was filtered through a 0.22μm filter membrane or filter. After the polypeptide mixed solution was mixed with a mixed solution containing 40wt% trehalose and 8wt% glycine in a ratio of 7:1, it was dispensed into a culture tube containing 20μL 1wt% heparin sodium solution at 110μL / tube.

[0056] Then the culture tube is placed in a vacuum dryer for vacuum drying to obtain a polypeptide culture tube, and the vacuum degree should be between 46000 Pa and 56000 Pa. The method for vacuum drying protein refers to patent ZL201010294090.1.

[0057] The raw material of the culture tube used in the manufacturing process of the polypeptide culture tube of the present invention is a sterile siliconized blood collection tube containing separation gel, sodium heparin is added in the preparation process, and the solution is dried by vacuum drying technology with a vacuum degree of 1 mL. The polypeptide 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 each short peptide to stimulate bovine heparin anticoagulation whole blood is greater than 8 μg / mL whole blood.

[0058] 3. Methods of using polypeptide culture tubes made from polypeptide antigens and avian PPD and bovine PPD

[0059] 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.

[0060] At present, the commercial products of aPPD and bPPD can only be purchased as solution or dry powder high-concentration reagents. When used, they need to be diluted in a biological safety cabinet and added to the 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 polypeptide culture tube provided by the present invention can be directly used for blood collection, anticoagulation, incubation, plasma separation and long-distance transportation.

[0061] 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:

[0062] 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 polypeptide 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.

[0063] 2) After incubation at 37°C for 23±1h, the supernatant plasma was collected to obtain aPPD, bPPD, N, P and T plasma, respectively.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] Experimental example

[0070] 29 dairy cows were selected for sampling and testing. According to the usage methods of aPPD, bPPD and polypeptide culture tubes (Examples), the bovine tuberculosis gamma interferon enzyme-linked immunosorbent assay kit of Hainan Weiwei Clinical Immunology Laboratory Co., Ltd., the negative control culture tubes whose main component is PBS, and the positive control culture tubes whose main component is lectin, the corresponding plasma specimens were prepared and the amount of bovine gamma interferon in the plasma was detected. The OD450nm-OD650nm of the samples are shown in Tables 2 and 3.

[0071] Table 2 Detection results of bovine gamma interferon in plasma samples of 29 cows' anticoagulated whole blood stimulated with different antigens (OD450nm-OD650nm)

[0072]

[0073]

[0074] Table 3 Comparison of immunogenicity of the polypeptide antigen composition provided by the present invention with aPPD and bPPD

[0075]

[0076]

[0077] In the sampling test of 29 dairy cows, the immunogenicity of different types of antigens was analyzed respectively. The polypeptide 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. In the sample groupings of aPPD<0.1 and bPPD<0.1, the mean and median of the novel T cell antigen provided by the present invention are not significantly different from those of aPPD and bPPD; but in the sample groupings of aPPD≥0.1 and bPPD≥0.1, the mean and median of the novel T cell antigen provided by the present invention are significantly different from those of aPPD and bPPD.

[0078] In summary, the bovine Mycobacterium-specific polypeptide antigen provided by the present invention as a new type of T cell antigen has higher specificity and sensitivity than aPPD and bPPD; the prepared polypeptide culture tube has the advantages of immediate anticoagulation of blood collection, simultaneous incubation and long-distance transportation of plasma after centrifugation.

[0079] 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 polypeptide antigen composition for Mycobacterium bovis-specific T cells, characterized in that: The polypeptide antigen composition consists of 15 polypeptides, wherein the amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.2; the amino acid sequence of polypeptide 3 is shown in SEQ ID NO.3; the amino acid sequence of polypeptide 4 is shown in SEQ ID NO.4; the amino acid sequence of polypeptide 5 is shown in SEQ ID NO.5; the amino acid sequence of polypeptide 6 is shown in SEQ ID NO.6; the amino acid sequence of polypeptide 7 is shown in SEQ ID NO.7; the amino acid sequence of polypeptide 8 is shown in SEQ ID NO.8; the amino acid sequence of polypeptide 9 is shown in SEQ ID NO.9; the amino acid sequence of polypeptide 10 is shown in SEQ ID NO.10; the amino acid sequence of polypeptide 11 is shown in SEQ ID NO.11; the amino acid sequence of polypeptide 12 is shown in SEQ ID NO.12; the amino acid sequence of polypeptide 13 is shown in SEQ ID NO.13; the amino acid sequence of polypeptide 14 is shown in SEQ ID NO.14; the amino acid sequence of polypeptide 15 is shown in SEQ ID NO.

15.

2. The polypeptide antigen composition according to claim 1, characterized in that: The mass ratio of polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 5, polypeptide 6, polypeptide 7, polypeptide 8, polypeptide 9, polypeptide 10, polypeptide 11, polypeptide 12, polypeptide 13, polypeptide 14 and polypeptide 15 is 1: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.5: 0.5-1.

5.

3. The polypeptide antigen composition according to claim 1 or 2, characterized in that: The purity of the 15 polypeptides is ≥95%.

4. A culture composition, characterized in that The culture composition comprises the polypeptide antigen composition according to any one of claims 1 to 3.

5. The culture composition according to claim 4, characterized in that The polypeptide antigen composition is prepared by mixing 15 polypeptide solutions with initial concentrations of 0.5 to 1.5 mg / mL.

6. The culture composition according to claim 5, characterized in that The volume ratio of 15 polypeptide solutions in the polypeptide antigen composition is polypeptide 1: polypeptide 2: polypeptide 3: polypeptide 4: polypeptide 5: polypeptide 6: polypeptide 7: polypeptide 8: polypeptide 9: polypeptide 10: polypeptide 11: polypeptide 12: polypeptide 13: polypeptide 14: polypeptide 15=1:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.5:0.5~1.

5.

7. The culture composition according to claim 6, characterized in that The culture composition also includes a mixed solution containing trehalose with an initial concentration of 35wt% to 45wt% and glycine with an initial concentration of 7wt% to 9wt%, and a heparin sodium solution with an initial concentration of 0.5wt% to 1.5wt%.

8. The culture composition according to claim 7, characterized in that The volume ratio of the polypeptide antigen composition to the mixed solution in the culture composition is 6-8:1; the volume ratio of the polypeptide antigen composition to the heparin sodium solution with an initial concentration of 0.5wt%-1.5wt% is 1:0.1-0.

3.

9. A kit for detecting bovine gamma interferon, characterized in that: The kit comprises the polypeptide antigen composition according to any one of claims 1 to 3 or the culture composition according to any one of claims 4 to 8.

10. Use of the polypeptide antigen composition according to any one of claims 1 to 3 or the culture composition according to any one of claims 4 to 8 in the preparation of a kit for detecting bovine interferon-gamma.

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