Tuberculosis protein, and preparation and application thereof

A protein and tuberculosis technology, applied in the field of genetic engineering, can solve problems such as the research and application of Mycobacterium bovis BCG_2653c protein that have not been seen

Inactive Publication Date: 2013-10-23
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no relevant reports on the research and application of Mycobacterium bovis BCG_2653c protein in China

Method used

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  • Tuberculosis protein, and preparation and application thereof
  • Tuberculosis protein, and preparation and application thereof
  • Tuberculosis protein, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of Genetically Engineered Bacteria

[0029] 1.1 Design of primers

[0030] According to the BCG_2653c sequence of the BCG Pasteur strain genomic DNA in GenBank, a pair of specific primers were designed. The primers were synthesized by Beijing Liuhe Huada Gene. The primers were as follows:

[0031] BCG_2653c-F: 5'-TTAATC GGTACC ATGACCACCGCACGCGA-3' (SEQ ID NO: 3); the underline represents the Kpn I restriction site;

[0032] BCG_2653c-R: 5'-ATAACC AAGCTT GATGATCCGAGGTCGCTAG-3' (SEQ ID NO: 4); the underline represents the Hind III restriction site.

[0033] 1.2 PCR amplification of target gene, product recovery and construction of cloning vector

[0034] Genomic DNA of the BCG Pasteur strain was extracted with the Genomic DNA Extraction Kit of Tienensis Bacteria, and PCR amplification was carried out with BCG_2653c-F and BCG_2653c-R as primers using it as a template. The PCR reaction system is 50 μL:

[0035]

[0036] The PCR reaction con...

Embodiment 2

[0039] Example 2. Expression, purification and verification of BCG_2653c protein

[0040] 2.1 Expression and purification of recombinant protein

[0041] Transform the recombinant plasmid pET-30a-BCG_2653c constructed in Example 1 into Escherichia coli BL21(DE3) competent cells, and after verifying correctness by PCR, pick positive colonies (DE3-pET-30a-BCG_2653c) to kana-resistant In LB medium, culture overnight at 37°C, 180rpm. The next day, the cells were inoculated into 400 mL of fresh ampicillin-resistant LB medium at a ratio of 1:100. When bacteria grow to OD600 At 0.5, add IPTG with a final concentration of 0.5mmol / L, induce at 37°C for 6h, and centrifuge at 9000rpm for 10min to collect bacteria, resuspend the bacteria with 100mL of PBS, wash twice in this way, and finally resuspend with 40mL of PBS, Under the condition of ice bath, after fully ultrasonication, at 4°C, 10000rpm, 15min, the supernatant and precipitate were collected, and detected by 12% SDS-PAGE, the r...

Embodiment 3

[0044] ELISA and ELISPOT detection of embodiment 3 bovine IFN-γ

[0045] 3.1 ELISA test of bovine IFN-γ

[0046] Go to a dairy farm in Jiangsu, collect 5ml of heparin anticoagulated whole blood from the cow to be tested, and transport it to the laboratory at room temperature. The test was carried out according to the instructions of the Mycobacterium bovis Gamma Interferon Test Kit for Cattle kit: the specific steps were as follows: first, add 1.5ml of blood samples to each well of a 24-well tissue culture plate, divide each blood sample into 5 wells, add PBS and poultry PPD respectively , stimulated by bovine PPD, and incubated in a 37°C incubator for 24h. Pipette the supernatant into a pretreated 96-well plate, add enzyme-labeled chromogenic antibody and incubate at room temperature for 1 hour; add TMB chromogenic substrate after washing 6 times, incubate at room temperature in the dark for 30 minutes, then add stop solution; within 5 minutes after termination, use OD 450...

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Abstract

The invention discloses a protein having immunogenicity to bovine tuberculosis and application thereof. The protein having immunogenicity to bovine tuberculosis in the invention is a recombinant protein of mycobacterium BCG_2653c; and the BCG_2653c has a nucleotide sequence as represented by SEQ ID No: 1 and an amino acid sequence as represented by SEQ ID No: 2. The recombinant protein provided by the invention is applicable to a detection reagent for bovine tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a preparation method of mycobacterium bovis antigenic protein. Background technique [0002] Bovine tuberculosis (BTB) is a zoonotic chronic infectious disease mainly caused by Mycobacterium bovis. It is characterized by loss of appetite, weight loss, anemia, and decreased milk production in sick cattle. To date, losses from bovine tuberculosis are greater than all other diseases of cattle combined. The World Organization for Animal Health (OIE) lists it as a B-type animal infectious disease, and my country lists it as a B-type animal disease. Every year, about 3 million people in the world are infected with Mycobacterium bovis, which causes tuberculosis. Therefore, the World Health Organization (WHO) pointed out: "In countries where bovine tuberculosis still exists, human beings are always threatened by it. Unless efforts are made to eliminate bovine tuberculosis, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12N15/31C12N15/70G01N33/569
Inventor 焦新安殷月兰高云飞付红徐正中陈祥潘志明孙林
Owner YANGZHOU UNIV
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