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Construction and application of tubercle bacillus Pup gene deletion mutation strain

A gene deletion and mutant strain technology, applied in the construction of gene deletion mutant strains and the field of recombinant genes, can solve problems such as unclear drug resistance regulation mechanism

Inactive Publication Date: 2019-10-08
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the specific drug resistance regulation mechanism of Pup protein and MTB is still unclear.

Method used

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  • Construction and application of tubercle bacillus Pup gene deletion mutation strain
  • Construction and application of tubercle bacillus Pup gene deletion mutation strain
  • Construction and application of tubercle bacillus Pup gene deletion mutation strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction and identification method of Mycobacterium tuberculosis Pup gene replacement deletion mutation vector

[0048] 1. Materials and methods

[0049] 1.1 Main reagent materials

[0050] 1.1.1 Main materials: Mycobacterium tuberculosis international standard virulent strain (H37Rv) was purchased from China National Institute for the Control of Pharmaceutical and Biological Products and kept in our laboratory; Clinical isolates of Mycobacterium tuberculosis isolated from nicotinic acid (INH-MTB), clinical isolates of Mycobacterium tuberculosis resistant to rifampicin alone (RFP-MTB), clinical isolates of Mycobacterium tuberculosis resistant solely to streptomycin (SM -MTB), pure ethambutol-resistant Mycobacterium tuberculosis clinical isolates (EB-MTB), and multidrug-resistant Mycobacterium tuberculosis clinical isolates (MDR-MTB) were collected, identified and preserved by our laboratory ; E.coli Competent Cells DH5α and T-Vector pMD™19 (Simple) were ...

Embodiment 2

[0078] Example 2 Construction and screening method of mycobacterium tuberculosis Pup gene deletion mutant strain

[0079] 1. Experimental method

[0080] 1.1 Extraction and purification of deletion mutant vector T-Pup-N-K-Pup-C

[0081] Take 10 μl of the bacterial solution containing the deletion mutation carrier T-Pup-N-K-Pup-C stored in a -80°C refrigerator, and spread it on the LB of kanamycin (100 μg / ml) and ampicillin (50 μg / ml) Double-antibody solid medium, cultivate overnight (at least 12 hours) at 37°C. Pick a single positive colony and inoculate it in the LB double-antibody liquid medium of kanamycin (100 μg / ml) and ampicillin (50 μg / ml), culture overnight on a shaker at 37°C, and extract it with a small plasmid extraction kit. The plasmid was purified, identified and purified by DNA gel electrophoresis, and the concentration and purity of the plasmid was determined by an ultraviolet spectrophotometer. The purified plasmids were stored in a -20°C low-temperature re...

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Abstract

The invention discloses a construction method and application of a tubercle bacillus Pup gene deletion mutation strain. Based on a fusion PCR technique and a T carrier cloning technique, a replacementtype deletion mutation carrier T-Pup-N-K-Pup-C of a gene is constructed and identified for the first time, through an electrotransformation technique, the recombinant Pup gene deletion mutation carrier enters mycobacterium tuberculosis competent cells, and through chromosome homologous recombination, multiple screening and identification, the tubercle bacillus Pup gene deletion mutation strain containing a kanamycin resistant gene is obtained for the first time. According to the tubercle bacillus Pup gene deletion mutation strain constructed by the construction method disclosed by the invention, a Pup gene in an MTB Pup-proteasome system is knocked out, so that the drug tolerance of a clinical drug-tolerance tubercle bacillus separation strain can be reduced, the clinical drug-tolerance tubercle bacillus separation strain can restore sensibility to tubercle resisting medicines, the ubercle bacillus Pup gene deletion mutation strain can help for exploring the pathogenic mechanism of MTB and controlling propagation of tuberculosis, and a new direction is provided for seeking a tubercle resisting drug target.

Description

[0001] Construction and application of a mutant strain of Mycobacterium tuberculosis Pup gene deletion. technical field [0002] The invention belongs to the technical field of recombinant gene and gene deletion mutant strain construction, and relates to the construction and application of Mycobacterium tuberculosis Pup gene deletion mutant strain. Background technique [0003] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium Tuberculosis (MTB) infection. About 1 / 3 of the world's people are infected with MTB. TB is a worldwide infectious disease that seriously endangers human health, and it is also an infectious disease with the highest mortality rate caused by a single bacterial infection. In recent years, due to the emergence of drug-resistant MTB, coupled with the rise of MTB and HIV dual infection, the global tuberculosis prevention and control situation has become very severe. For a long time, the drug resistance mechanism and pathogenic mechani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/32
CPCC07K14/35C12N15/74
Inventor 张万江邵萌张舜文吴江东吴芳张辉张杰董江涛徐芳柳小玲朱荟云梁粟
Owner SHIHEZI UNIVERSITY
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