Escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof

A technology of Mycobacterium tuberculosis and rv2837c, which is used in the preparation of tuberculosis diagnosis and preventive preparations or medicines, recombinant expression in Escherichia coli and the field of obtaining Mycobacterium tuberculosis Rv2837c active protein, which can solve the problem of limited effect of tuberculosis antigens and no tuberculosis New vaccines and other issues

Active Publication Date: 2016-05-11
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0012] Although the above-mentioned protective antigens have shown certain vaccine potential, so far, no new tuberculosis vaccine tha

Method used

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  • Escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof
  • Escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof
  • Escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0040] Construction of Rv2837c prokaryotic expression vector:

[0041] Primers were designed according to the genome Rv2837c gene sequence of Mycobacterium tuberculosis H37Rv strain:

[0042] P1: 5'-CGGCATATGGTGACGACGATCGACCCAAG-3', NdeI site

[0043] P2: 5'-TTTAAGCTTGCCAGCTGCGCGATCTGACG-3', HindIII site

[0044] Primers were synthesized by Shanghai Sunny Biotechnology Co., Ltd. The target gene was amplified by PCR using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template. The reaction parameters were: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, renaturation at 57°C for 30 seconds, extension at 72°C for 100 seconds, 35 cycles, and 72 cycles after the last cycle. ℃ extension 5min. 1% agarose gel electrophoresis analysis showed that the 1023bp target band was amplified (see figure 1 ). The target fragment was recovered by the gel recovery kit, double-digested with NdeI and HindIII, and the digested product was recovered ...

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Abstract

The invention relates to an escherichia coli recombinate expression method of mycobacterium tuberculosis Rv 2837c active protein and applications thereof. The mycobacterium tuberculosis Rv 2837c protein gene is cloned into a prokaryotic expression vector Pet28a+, and the gene is named as pET-CnpB. A Ni ion affinity chromatography method is adopted to obtain purified Rv2837c active protein. The recombinant Rv2837c protein can be used to detect the serum of animals and patients affected by mycobacterium tuberculosis. The recombinant Rv2837c protein can induce high level body fluid and cellular immunologic response of mouse, and can be used to develop novel subunit vaccine of tuberculosis. So Rv2837c protein has a good application prospect. The invention relates to the applications of the active recombinant protein in the preparation of drugs or preparations for diagnosing and preventing tuberculosis.

Description

technical field [0001] The invention belongs to the field of tuberculosis diagnostic reagents and vaccines. The invention relates to a method for recombinantly expressing and obtaining active protein of mycobacterium tuberculosis Rv2837c in Escherichia coli. The invention also relates to the application of the recombinant protein in the preparation of tuberculosis diagnosis and prevention preparations or medicines. Background technique [0002] Tuberculosis Epidemic Status [0003] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, and it is still the most serious infectious disease worldwide. According to the WHO report, about 1 / 3 of the world's population has been infected with Mycobacterium tuberculosis, and there were about 9 million new patients in 2013. Among all infectious diseases, the death rate of tuberculosis is second only to AIDS. WHO lists TB, together with AIDS and malaria, as the most important infectiou...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/70G01N33/569A61K39/04A61P31/06
CPCA61K39/04A61K2039/575C12N9/22C12N15/70C12N2800/101G01N33/5695G01N2333/922G01N2469/20
Inventor 柏银兰徐志凯王丽梅王立飞康健路延之褚阳光刘知鑫高俊吉思雨曹田宇
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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