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
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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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