A polypeptide antigen composition of mycobacterium bovis-specific t cells and its use in the preparation of a kit for detecting bovine gamma interferon

Abstract

The application provides a polypeptide antigen composition of bovine Mycobacterium tuberculosis specific T cells and application thereof in preparation of a bovine gamma interferon kit, and belongs to the field of biomedical technology. The polypeptide antigen composition is obtained by mixing 15 short peptides with 20-50 amino acids designed based on the amino acid sequences of ESAT6, CFP10 and Rv3615c in a specific ratio, and has good specificity and can be applied to the differential detection of early bovine tuberculosis of BCG vaccinated bovine and NTM Mycobacterium avium subspecies paratuberculosis positive bovine. The polypeptide antigen composition obtained by the application is compared with avian and bovine PPD, and it is found that the polypeptide antigen composition obtained by the application has higher bovine Mycobacterium tuberculosis specificity and sensitivity; and the polypeptide culture tube prepared by the application has the advantages of ready-to-use blood sampling, simultaneous incubation and long-distance transportation of plasma after centrifugation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a polypeptide antigen composition of Mycobacterium bovis specific T cells and application thereof in preparing a bovine gamma interferon kit. BACKGROUND

[0002] Bovine tuberculosis is mainly caused by Mycobacterium bovis infection of Mycobacterium tuberculosis complex (MTBC) members, and is a zoonosis. Bovine tuberculosis not only can be transmitted to humans through inhalation of bacteria-containing aerosols or consumption of raw milk, but also can affect animal production performance, food supply stability and public health safety. The World Organization for Animal Health (WOAH) lists it as a notifiable disease and an international trade compulsory disease.

[0003] At present, the diagnosis of bovine tuberculosis mainly relies on the following methods: 1) bacteriological detection methods, such as bacterial staining smear microscopy and bacterial culture. Mycobacterium bovis isolation and culture is the gold standard for the diagnosis of bovine tuberculosis, and has good specificity but low sensitivity, which is difficult to popularize on a large scale; 2) PCR detection method, which is mainly used for typing research of pathogenic Mycobacterium isolated and cultured from the disease material. This method has high technical requirements for the operator and high experimental operation environment requirements, and is prone to false positives or false negatives during detection, and is not convenient for popularization; 3) tuberculin intradermal allergic reaction test, which is the earliest method used to diagnose bovine tuberculosis and is the most widely used standard method for detecting bovine tuberculosis recommended by WOAH. This method can only be used for in vivo test, which is not conducive to large-scale epidemiological investigation and is prone to false positives and false negatives; 4) serological detection, which is convenient, simple, easy to use, high negative detection value and economical, but antibody detection is prone to false positives; 5) gamma interferon release test, which uses specific T cell antigens to stimulate bovine whole blood in vitro, and then detects the content of gamma interferon in the whole blood, so as to judge whether the bovine is infected with Mycobacterium. This method not only has high sensitivity and specificity, but also has more objective and reliable results, and only needs to be in contact with the animal once, which has great superiority for the detection of wild animals and large animals that are difficult to bind.

[0004] The gamma interferon release test includes the process of incubation at constant temperature after mixing the anti-coagulation fresh whole blood with specific T cell antigen in vitro, and the process of detecting the amount of gamma interferon secreted after being stimulated by specific T cell antigen during the incubation. The selection and preparation method of the specific T cell antigen is a key element to determine the analysis performance of the detection method. In the early stage of establishing the gamma interferon release test in vitro, PPD is used as the specific T cell antigen. The main component of PPD is the protein derivative of avian or bovine mycobacterium, and the protein antigen component contained therein is very complex. Although the sensitivity is acceptable, the specificity is very poor. Therefore, it is urgent to obtain an antigen with good specificity. SUMMARY

[0005] Therefore, the present application aims to provide a polypeptide antigen composition of bovine mycobacterium specific T cells and its application in preparing a bovine gamma interferon kit for detection, which is based on 15 short peptides with 9-30 amino acids designed according to the amino acid sequences of ESAT6, CFP10 and Rv3615c, and the polypeptide antigen composition obtained after mixing the short peptides in a specific ratio. The polypeptide antigen composition has good specificity and can be applied to the early differential detection of bovine tuberculosis in BCG vaccinated bovine and NTM positive bovine with paratuberculosis sub-species of mycobacterium avium.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a polypeptide antigen composition of bovine mycobacterium specific T cells, which is composed of 15 polypeptides. 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] As preferred, the mass ratio of the 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 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.

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

[0010] The application provides a culture composition comprising the polypeptide antigen composition.

[0011] As preferred, the polypeptide antigen composition is mixed by 15 polypeptide solutions with initial concentration of 0.5-1.5 mg / mL.

[0012] As preferred, 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.

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

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

[0015] The application further provides a kit for detecting bovine gamma interferon, which comprises the polypeptide antigen composition or the culture composition.

[0016] The application further provides the use of the polypeptide antigen composition or the culture composition in the preparation of a kit for detecting bovine gamma interferon.

[0017] Compared with the prior art, the present application has the following beneficial effects: based on the amino acid sequences of ESAT6, CFP10 and Rv3615c, after the prediction and analysis of the dominant antigenic determinant, 15 short peptides of 20-50 amino acids are obtained by artificial synthesis, the process of obtaining short peptides by antigen presenting cells through uptake and digestion is saved, high-concentration short peptides directly form MHC peptide complexes with MHC receptors on the surface of antigen presenting cells, causing a more extensive cell immune response intensity. The present application 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 application has good specificity and can be applied to the differential detection of early bovine tuberculosis of BCG-vaccinated cattle and NTM Mycobacterium avium subspecies paratuberculosis positive cattle. By comparing the polypeptide antigen composition obtained by the present application with avian and bovine PPD, it is found that the polypeptide antigen composition obtained by the present application has higher Mycobacterium bovis specificity and sensitivity; and the polypeptide culture tube prepared by the present application has the advantages of ready-to-use blood sampling, simultaneous incubation and long-distance transportation of plasma after centrifugation. DETAILED DESCRIPTION

[0018] The present application provides a polypeptide antigen composition of Mycobacterium bovis specific T cells, which is composed of 15 polypeptides, wherein the amino acid sequence of polypeptide 1 is shown as SEQ ID NO. 1; the amino acid sequence of polypeptide 2 is shown as SEQ ID NO. 2; the amino acid sequence of polypeptide 3 is shown as SEQ ID NO. 3; the amino acid sequence of polypeptide 4 is shown as SEQ ID NO. 4; the amino acid sequence of polypeptide 5 is shown as SEQ ID NO. 5; the amino acid sequence of polypeptide 6 is shown as SEQ ID NO. 6; the amino acid sequence of polypeptide 7 is shown as SEQ ID NO. 7; the amino acid sequence of polypeptide 8 is shown as SEQ ID NO. 8; the amino acid sequence of polypeptide 9 is shown as SEQ ID NO. 9; the amino acid sequence of polypeptide 10 is shown as SEQ ID NO. 10; the amino acid sequence of polypeptide 11 is shown as SEQ ID NO. 11; the amino acid sequence of polypeptide 12 is shown as SEQ ID NO. 12; the amino acid sequence of polypeptide 13 is shown as SEQ ID NO. 13; the amino acid sequence of polypeptide 14 is shown as SEQ ID NO. 14; and the amino acid sequence of polypeptide 15 is shown as SEQ ID NO. 15, which are 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] SEQ ID NO. 15: LGESVAITHGPYCSQFNDTLNVYLTAHNAL.

[0038] In the present application, the nucleotide sequence of the polypeptide 1 is as shown in SEQ ID NO. 16, specifically: AGCGCAATCCAGGGAAATGTCACGTCCATTCATTCCCTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGG; the nucleotide sequence of the polypeptide 2 is as shown in SEQ ID NO. 17, specifically: ATGACAGAGCAGCAGTGGAATTTCGCGGGTATCGAGGCCGCGGCAAGCGCAATCCAGGGAAATGTCACG; the nucleotide sequence of the polypeptide 3 is as shown in SEQ ID NO. 18, specifically: CAGCAAAAATGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATG; the nucleotide sequence of the polypeptide 4 is as shown in SEQ ID NO. 19, specifically: AAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTCCAGCAAAAATGGGACGCCACGGCT; the nucleotide sequence of the polypeptide 5 is as shown in SEQ ID NO. 20, specifically: TGGGACGCCACGGCTACCGAGCTGAACAACGCGCTGCAGAACCTGGCGCGGACGATCAGCGAAGCCGGTCAGGCAATGGCTTCGACCGAAGGCAACGTCACTGGGATGTTCGCA; the nucleotide sequence of the polypeptide 6 is as shown in SEQ ID NO. 21, specifically: CGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCG; the nucleotide sequence of the polypeptide 7 is as shown in SEQ ID NO. 22, specifically: GGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAG; the nucleotide sequence of the polypeptide 8 is as shown in SEQ ID NO.23, specifically: GTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTC; the nucleotide sequence of the polypeptide 9 is shown as SEQ ID NO. 24, specifically: CTAGGCGAATCTGTGGCGATCACTCACGGTCCGTACTGCTCACAGTTCAACGACACGTTAAATGTGTACTTGACTGCCCACAATGCCCTGGGCTCGTCCTTGCATACGGCCGGTGTCGATCTCGCCAAAAGTCTTCGAATTGCGGCG; the nucleotide sequence of the polypeptide 10 is shown as SEQ ID NO. 25, specifically: AATGTGTACTTGACTGCCCACAATGCCCTGGGCTCGTCCTTGCATACGGCCGGTGTCGATCTCGCCAAAAGTCTTCGAATTGCGGCGAAAATATATAGCGAGGCCGACGAAGCGTGGCGCAAGGCTATCGACGGGTTGTTTACC; the nucleotide sequence of the polypeptide 11 is shown as SEQ ID NO. 26, specifically: CTCGCCAAAAGTCTTCGAATTGCGGCGAAAATATATAGCGAGGCCGACGAAGCGTGGCGCAAGGCTATCGACGGGTTGTTTACC; the nucleotide sequence of the polypeptide 12 is shown as SEQ ID NO. 27, specifically: CTCCTTGACGAGGGGAAGCAGTCCCTGACCAAGCTCGCAGCGGCCTGGGGCGGTAGCGGTTCGGAGGCGTACCAGGGTGTC; the nucleotide sequence of the polypeptide 13 is shown as SEQ ID NO. 28, specifically: GCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCG; the nucleotide sequence of the polypeptide 14 is shown as SEQ ID NO. 29, specifically: GCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTG; the nucleotide sequence of the polypeptide 15 is shown as SEQ ID NO.30, specifically: CTAGGCGAATCTGTGGCGATCACTCACGGTCCGTACTGCTCACAGTTC AACGACACGTTAAATGTGTACTTGACTGCCCACAATGCCCTG.

[0039] In the present application, the mass ratio of the 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 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, 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, further preferably 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1.

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

[0041] The present application provides a culture composition comprising the polypeptide antigen composition.

[0042] In the present application, the polypeptide antigen composition is mixed from 15 polypeptide solutions with an initial concentration of 0.5-1.5 mg / mL, preferably 0.7-1.2 mg / mL, further preferably 1 mg / mL.

[0043] In the present application, 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, 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, more preferably 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1.

[0044] In the present application, the culture composition further comprises a mixed solution containing trehalose at an initial concentration of 35wt%-45wt% and glycine at an initial concentration of 7wt%-9wt%, and a heparin sodium solution at an initial concentration of 0.5wt%-1.5wt%, wherein the initial concentration of trehalose in the mixed solution is preferably 37wt%-43wt%, more preferably 40wt%; the initial concentration of glycine is preferably 7.5wt%-8.5wt%, more preferably 8wt%; and the initial concentration of the heparin sodium solution is preferably 0.7wt%-1.2wt%, more preferably 1wt%. The addition of trehalose and glycine can protect the peptide fragments and prevent their degradation; and the addition of the heparin sodium solution can prevent blood coagulation and prolong the coagulation time, thereby ensuring that the blood plasma can be transported over a long distance.

[0045] In the present application, the polypeptide antigen composition, the mixed solution and the heparin sodium solution are separately placed into culture tubes, and vacuum drying is performed to obtain polypeptide culture tubes, which can be used for detecting bovine gamma interferon. The volume ratio of the polypeptide antigen composition to the mixed solution in the culture composition is 6-8:1, preferably 6.5-7.5:1, more preferably 7:1; and the volume ratio of the polypeptide antigen composition to the heparin sodium solution at an initial concentration of 0.5wt%-1.5wt% is 1:0.1-0.3, preferably 1:0.15-0.25, more preferably 1:0.2-0.21.

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

[0047] The application also provides the polypeptide antigen composition or the culture composition in the preparation of a kit for detecting bovine gamma interferon.

[0048] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0049] Examples

[0050] 1. Obtaining of the polypeptide sequences of 15 segments

[0051] The bovine tuberculosis pathogen Mycobacterium bovis is compared with the genome of the BCG vaccine, and BCG vaccine deletion genes such as the Mycobacterium bovis ESAT6 (Accession: YP_178023), CFP10 (Accession: NP_218391), Rv3615c (Accession: NP_218132) genes are screened out, and polypeptides are designed according to the amino acid sequences. According to the following principles, 15 polypeptides are designed and combined: 1. Analyze the charge, hydrophobicity, length, and structural characteristics of the candidate polypeptides to ensure their specificity and stability. 2. The selected candidate polypeptide combination has amino acids that can form a stable spatial conformation, enhancing the functionality and practicality of the polypeptides. Solid-phase synthesis (solid-phase peptide synthesis technology) is adopted to fix and extend the amino acid chain one by one on a solid support, effectively remove the protecting group, and achieve efficient synthesis of polypeptides with a purity of more than 95%. The amino acid sequences of the 15 polypeptide sequences are shown in Table 1.

[0052] Table 1 Amino acid sequences of polypeptide sequences

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

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

[0055] The synthesized polypeptides are purified and organic solvents are removed to obtain a concentrated solution, which is sterile-filtered and freeze-dried into polypeptide dry powder. The 15 polypeptide dry powders are each dissolved in sterile PBS to 1 mg / mL, and mixed in equal proportions to prepare a polypeptide mixed solution. A mixed solution containing 40 wt% trehalose, 8 wt% glycine, and a 1 wt% heparin sodium solution are prepared, and the solutions are filtered through a 0.22 μm filter membrane or filter. The polypeptide mixed solution and the mixed solution containing 40 wt% trehalose and 8 wt% glycine are mixed in a ratio of 7:1, and then divided into culture tubes containing 20 μL of a 1 wt% heparin sodium solution at 110 μL / tube.

[0056] Then the culture tubes are placed in a vacuum drying machine for vacuum drying to obtain polypeptide culture tubes. The vacuum degree should be between 46000 Pa and 56000 Pa. The method for vacuum drying proteins is described in patent ZL201010294090.1.

[0057] The raw material of the polypeptide culture tube is a sterile siliconized blood collection tube with a separation gel. In the preparation process, sodium heparin is added, and vacuum drying technology is used to dry the solution with a vacuum degree of 1 mL. The polypeptide 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 amount of short peptide used for stimulating bovine heparin anticoagulation whole blood is greater than 8 μg / mL of whole blood.

[0058] 3. Use method of the polypeptide culture tube prepared from the polypeptide antigen and avian PPD and bovine PPD

[0059] Bovine purified tuberculin stimulating antigen (Bovine Tuberculin PPD 3000, 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 to control the immunogenicity difference of the Mycobacterium bovis specific T cell antigen provided by the present application.

[0060] 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 bovine whole blood. The plasma after incubation needs to be additionally sucked into a proper container for storage and transportation. The polypeptide culture tube provided by the present application can be directly used for blood collection, anticoagulation, incubation, plasma separation, and long-distance transportation.

[0061] The bovine tuberculosis gamma interferon enzyme-linked immunoassay kit and the negative control culture tube with PBS as the main component and the positive control culture tube with phytohemagglutinin as the main component are provided by Hainan Weiniayi Clinical Immunological Test Co., Ltd. The test process is as follows:

[0062] 1) Collect at least 5 mL of heparin (lithium) anticoagulant bovine whole blood samples, mix 250 μL of anticoagulant whole blood and 25 μL of aPPD and bPPD in a cell culture plate according to the aPPD and bPPD instructions; add 1 mL of anticoagulant whole blood to the polypeptide culture tube (T), negative control culture tube (N), and positive control culture tube (P) and shake or directly use the culture tube to collect blood into the tube and shake.

[0063] 2) After 23±1 h of incubation at 37℃, the supernatant plasma is obtained, and aPPD, bPPD, N, P, and T plasma are obtained.

[0064] 3) The test plasma sample and the bovine tuberculosis gamma interferon enzyme-linked immunoassay kit should be equilibrated at room temperature (22±5℃) for more than 30 min.

[0065] 4) Add 50 μL diluent and 50 μL aPPD, bPPD, N, P and T plasma into the wells of the enzyme-labeled plate; replace the plasma with the diluent in the negative quality control wells, and add the same amount of recombinant bovine gamma interferon quality control as the plasma in the positive quality control wells, and incubate at room temperature (22±5°C) for 1 h.

[0066] 5) After incubation, discard the liquid in the wells, add 200 μL of 1x washing solution to each well, 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 of step 5).

[0068] 7) Add 100 μL of TMB color developing 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, and read and record the double wavelengths (450 nm, 650 nm) within 5 min using an enzyme-labeled instrument, and calculate OD450nm-OD650nm.

[0069] Experimental Example

[0070] 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 polypeptide culture tubes (embodiment) and the bovine tuberculosis gamma interferon enzyme-linked immunoassay kit of Hainan Weiniye Clinical Immune 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 2 and 3.

[0071] Table 2 Detection results of bovine gamma interferon in the plasma samples of the anticoagulated whole blood of 29 cows after stimulation by different antigens (OD450nm-OD650nm)

[0072]

[0073]

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

[0075]

[0076]

[0077] In the sampling detection of 29 cows, the immunogenicity of different types of antigens was analyzed, and the polypeptide 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. 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.

[0078] In summary, the bovine Mycobacterium specific polypeptide antigen provided by the application has higher specificity and sensitivity than aPPD and bPPD as a novel T cell antigen; and the prepared polypeptide culture tube has the advantages of ready-to-use blood sampling, simultaneous incubation and long-distance transportation of plasma after centrifugation.

[0079] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A polypeptide antigen composition for Mycobacterium bovis-specific T cells, characterized in that, The polypeptide antigen composition comprises 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. 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 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:

1.

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

3. A culture composition, characterized in that, The culture composition comprises the polypeptide antigen composition according to claim 1 or 2.

4. The culture composition according to claim 3, characterized in that, The polypeptide antigen composition is prepared by mixing 15 polypeptide solutions with the same initial concentration, wherein the initial concentration of each of the 15 polypeptide solutions is 0.5~1.5 mg / mL.

5. The culture composition according to claim 4, characterized in that, The culture composition also includes a mixed solution containing trehalose at an initial concentration of 35wt%~45wt% and glycine at an initial concentration of 7wt%~9wt%, and a heparin sodium solution at an initial concentration of 0.5wt%~1.5wt%.

6. The culture composition according to claim 5, 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.

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