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Screening and identifying method for cow mycobacteria immunogenic secreted protein

A technology of Mycobacterium bovis and secreted protein, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, analysis materials, etc. and other problems, to achieve the effect of high immunogenicity ratio, improved efficiency, and improved success rate

Inactive Publication Date: 2008-10-08
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0003] When studying MTB secreted proteins, traditional protein separation and analysis techniques have been used for many years. These technical means are limited by instruments, and the operation is complicated and costly, which restricts the discovery and functional research of Mycobacterium bovis secreted proteins
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  • Screening and identifying method for cow mycobacteria immunogenic secreted protein
  • Screening and identifying method for cow mycobacteria immunogenic secreted protein

Examples

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Embodiment 1

[0036] Example 1 Cloning and expression of Mb1761c gene and identification of its immunogenicity

[0037] According to the M.bovis Mb1761c gene sequence provided by GenBank, the following 2 pairs of primers were designed using the primer design software PrimerPremier 5.0: P15'-CTGGGATCCATGGCCGTTGAATCCAGC-3'

[0038] P25'-GGAAGCTTCTAGCGCCACTTGATGCC-3'

[0039] Add BamHI and HindIII restriction sites to the 5' and 3' ends of the upstream and downstream primers, respectively. Primers were synthesized by Shanghai Shenergy Biotechnology Co., Ltd.

[0040] The MB1761c gene was amplified by PCR using M. bovis DNA as a template and P1 and P2 as primers. The amplification conditions were: 94°C for 5 min; 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, a total of 30 cycles; 72°C for 10 min, and 4°C to terminate the reaction. The PCR product was analyzed by 1% agarose gel electrophoresis, and purified and recovered according to the instructions of the DNA G...

Embodiment 2

[0049] Cloning of embodiment 2 Mb 1216c gene

[0050] According to the screened candidate protein Mb1216c gene sequence, the following 2 pairs of primers were designed using the primer design software PrimerPremier 5.0: P1: 5′-GAGGATCCATGACACCGGGTTT-3′

[0051] P2: 5′-AGGAAGCTTTCAATCGTCCC-3′

[0052] Add BamH I and HindIII restriction sites to the 5' and 3' ends of the upstream and downstream primers, respectively. Primers were synthesized by Shanghai Shenergy Biotechnology Co., Ltd.

[0053] Mb1216c gene was amplified by PCR using Mycobacterium bovis DNA as template and P1 and P2 as primers. The amplification conditions were: 94°C for 5 min; 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, a total of 30 cycles; 72°C for 10 min, and 4°C to terminate the reaction. The PCR product was analyzed by 1% agarose gel electrophoresis, and purified and recovered according to the instructions of the DNA Gel Recovery and Purification Kit. At the same time, t...

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Abstract

The invention relates to a screening and identifying method of bovine mycobacterium immunogenicity secretion type protein. The method comprises the following steps: firstly, analysis of gene sequence of the protein coding genes of the bovine mycobacterium gene group in the gene pool is performed, and then drainage membrane helix is found among the screened protein; the protein which contains the drainage membrane helix is determined as membrane protein, and the secretory protein and the membrane protein are distinguished accordingly. The bovine mycobacterium protein with homology smaller than 36 percent with the homology of fowl mycobacterium is screened according to the principle that the bovine mycobacterium and human mycobacterium tuberculosis have higher homology; as the candidate protein, polymerase chain reaction clone is performed on the candidate protein genes, the expression vector is established and immunogenicity measurement is performed on the expression product, then bovine mycobacterium secretion type protein with immunogenicity is obtained. The probability of screening and obtaining the molecule with immunogenicity is increased greatly by the method, thus new candidate vaccine molecules are provided for prevention and cure of the bovine tuberculosis in our country.

Description

technical field [0001] The invention relates to a method for screening and identifying immunogenic secretory proteins of Mycobacterium bovis, which uses bioinformatics technology and experimental biology technology to screen and identify secretory protein molecules with strong immunogenicity in Mycobacterium bovis, which can To provide new candidate vaccine molecules for the control of bovine tuberculosis. Background technique [0002] At present, the biggest problem in the control of bovine tuberculosis is that there is no practical vaccine application. Vaccines currently being studied include subunit vaccines, genetic vaccines, live attenuated vaccines, and BCG. BCG is banned from domestic cattle herds due to the high rate of false positives in its detection process. Therefore, the development of new safe and efficient vaccines has become a research hotspot in the control of bovine tuberculosis. Studies have shown that the body mainly relies on cellular immunity to resi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12Q1/68G01N33/68G01N33/558
Inventor 朱建国严懿嘉华修国姜毅刘芳
Owner SHANGHAI JIAO TONG UNIV
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