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Application of msmeg_6171 in regulating the susceptibility of Mycobacterium smegmatis to antibiotics

A technology of mycobacterium smegmatis and antibiotics, applied in the field of application in regulating the sensitivity of mycobacterium smegmatis to antibiotics

Active Publication Date: 2022-03-04
广东省结核病控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the regulation of MSMEG_6171 and its homologous protein Rv3660c on bacterial antibiotic sensitivity

Method used

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  • Application of msmeg_6171 in regulating the susceptibility of Mycobacterium smegmatis to antibiotics
  • Application of msmeg_6171 in regulating the susceptibility of Mycobacterium smegmatis to antibiotics
  • Application of msmeg_6171 in regulating the susceptibility of Mycobacterium smegmatis to antibiotics

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Using the mycobacterium-Escherichia coli shuttle plasmid pMV261-3X flag (New use of BCG forrecombinant vaccines, Nature, 1991.351 (6326): 456-60; the public can obtain it from the Guangdong Provincial Tuberculosis Control Center) to express MSMEG_6171 protein, and obtain overexpressed MSMEG_6171 of Mycobacterium smegmatis.

[0028] 1. Construction of MSMEG_6171 expression plasmid

[0029] Mycobacterium tuberculosis mc 2 Genomic DNA of 155 as a template, through the forward primer 5'-a gaattc atgccgtcgagtcgtaagg-3' (SEQ ID No.3) and reverse primer 5'-taa aagctt ccgccgcctcccgcacag-3' (SEQ ID No.4) was amplified by PCR to obtain the MSMEG_6171 gene coding fragment, and the gel electrophoresis detection results were as follows figure 1 As shown, the MSMEG_6171 gene coding fragment was connected to the pMV261-3X Flag vector by NEB's EcoRI, HindIII and T4 ligases to obtain the MSMEG_6171-3X Flag fusion protein expression plasmid pMV261-HtpG-3X Flag. The recombinant pla...

Embodiment 2

[0038] The MSMEG_6171 gene knockout strain was constructed by homologous gene recombination.

[0039] Table 1. Recombinant plasmids related to homologous gene recombination

[0040]

[0041] 1. Construction of suicide plasmid

[0042] (1) PCR amplification of MSMEG_6171 upstream and downstream homology arms: design primer 6171-UF: 5'-ccc from MSMEG_6171 gene and its upstream and downstream DNA sequences aagctt atgtagtcacgcatccggtcc-3' (SEQ ID No.5) (the underlined sequence indicates the HindIII site), 6171-UR: 5'-gagtgcctcgtcgagacccggttcgtcgcatccgatcac-3' (SEQ ID No.6), obtained by amplifying M.smegmatis genomic DNA as a template MSMEG_6171 gene upstream homology arm (Up, 855bp). With 6171-DF: 5'-gtgatcggatgcgacgaaccgggtctcgacgaggcactc-3' (SEQ ID No. 7) and 6171-DR: 5'-t taattaa cggtgttgagcgcggcg-3' (SEQ ID No.8) (the underlined sequence indicates the PacI site), using M. smegmatis genomic DNA as a template to amplify the downstream homology arm (Down, 849bp) of the MSME...

Embodiment 3

[0056] MIC detection of wild-type Mycobacterium smegmatis, MSMEG_6171-deleted Mycobacterium smegmatis, complemented strain and MSMEG_6171-overexpressed Mycobacterium smegmatis to 10 antibiotics.

[0057] The sensitivity of MSMEG_6171 to antibiotics was detected by dilution method. Antibiotic solutions of different concentrations (rifampicin, isoniazid, streptomycin, vancomycin, ethionamide, cycloserine, meropenem, cefepime, cefoxitin, cefotaxime) after doubling dilution Add separately to sterile 96-well polystyrene plates. Drugs were added to the 1st to 11th wells, 10 μL per well, no drug was added to the 12th well as a growth control, and 3 parallel samples were taken. Dilute the bacterial solution to 0.5 McFarland turbidity, then dilute it with 7H9 liquid medium at a rate of 1:1000, add 90 μL of the diluted bacterial solution to each well, seal it and incubate on a shaker at 37°C for 2 to 3 hours. The lowest concentration that can inhibit the growth of bacteria is the MICs...

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Abstract

The invention discloses a method for regulating the sensitivity of Mycobacterium smegmatis to antibiotics, which is characterized in that it is realized by regulating the expression level of MSMEG_6171. Inhibiting the expression of MSMEG_6171 enhanced the sensitivity of Mycobacterium smegmatis to antibiotics, and promoting the overexpression of MSMEG_6171 weakened the sensitivity of Mycobacterium smegmatis to antibiotics. It provides a new method for regulating the sensitivity of Mycobacterium smegmatis to antibiotics.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to the application of MSMEG_6171 in regulating the sensitivity of Mycobacterium smegmatis to antibiotics. Background technique [0002] Mycobacterium smegmatis (M.smegmatis) belongs to Gram-positive saprophytic bacteria, which has the characteristics of rapid growth, no pathogenicity, high gene homology and similar cell structure with M. tuberculosis, and is widely used It is a relatively ideal experimental model for mycobacterial infection and related immunology research. At the same time, it also has expanded applications in non-mycobacterial infection and other related immune research. [0003] Mycobacterium smegmatis has a lipid-rich cell wall with complex components and diverse functions. It is an important barrier for mycobacteria to resist the pressure of the external environment and escape host immune attacks. The M. smegmatis cell wall consists of two parts: an inner ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/32
CPCC12N15/74C07K14/35
Inventor 魏文静巫株华周琳郭卉欣陈亮陈瑜晖陈燕梅
Owner 广东省结核病控制中心