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Method for constructing high temperature stress-resistant recombinant Mycobacterium smegmatis

A kind of technology of Mycobacterium smegmatis and construction method, applied in the field of construction of recombinant Mycobacterium smegmatis

Pending Publication Date: 2018-12-18
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Increasing the culture temperature is not conducive to the growth of M. smegmatis and the hsp60 promoter needs to increase the culture temperature to increase the expression activity. It is a pair of contradictory important parameters for the expression of specific genes using M. smegmatis as a host. has been solved

Method used

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  • Method for constructing high temperature stress-resistant recombinant Mycobacterium smegmatis
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  • Method for constructing high temperature stress-resistant recombinant Mycobacterium smegmatis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Expression of HtpG protein using mycobacterium-Escherichia coli shuttle plasmid pMV261

[0024] (1) Construction of HtpG expression plasmid

[0025] With the genomic DNA of mycobacterium tuberculosis Mycobacterium tuberculo sisH37Rv as template, carry out PCR by forward primer (shown in SEQ ID No. 2 in the sequence listing) and reverse primer (shown in SEQ ID No. 3 in the sequence listing) Amplify to obtain the HtpG gene fragment, connect the HtpG gene fragment to the pMV261 vector (New use of BCG for recombinant vaccines, Nature, 1991.351(6326): 456-60; public available from Foshan Institute of Science and Technology), and the HtpG expression plasmid pMV261-HtpG was obtained.

[0026] (2) HtpG expression plasmid introduced into Mycobacterium smegmatis

[0027] 1) Mycobacterium smegmatis MC 2 155 is a mutant strain derived from Mycobacterium smegmatis ATCC 607, which has high transformation efficiency and is the most common strain of Mycobacterium smegmati...

Embodiment 2

[0031] Example 2: Detecting the impact of HtpG protein on the viability of Mycobacterium smegmatis under high temperature stress conditions

[0032] (1) Recombinant Mycobacterium smegmatis pMV261-HtpG / MC 2 155 and control bacteria pMV261 / MC 2 155 LB solid medium containing antibiotic kan 30 μg / mL were respectively streaked, cultured at 37°C for 3 days, and 3 different single colonies were picked for each strain and inoculated with antibiotic kan 30 μg / mL, 0.5% glycerol and 0.05% spit Warm LB liquid medium at 80°C and culture with shaking at 37°C.

[0033] (2) The above-mentioned 6 single colonies grow to the logarithmic growth phase (OD600 is about 0.5), and carry out gradient dilution (10- 1 to 10- 5 ), the specific dilution layout is shown in Table 1 below.

[0034] Table 1 Dilution Layout

[0035]

[0036] (3) For each dilution, pipette 5 μl of bacterial liquid into LB solid medium containing antibiotic kan 30 μg / mL, and blow dry until there is no liquid on the surf...

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Abstract

The invention discloses a method for constructing high temperature stress-resistant recombinant Mycobacterium smegmatis. The method is characterized in that an HtpG gene fragment is introduced to makethe recombinant Mycobacterium smegmatis have the function of expressing an HtpG protein. The HtpG gene fragment is introduced to Mycobacterium smegmatis to obtain the high temperature stress-resistant recombinant Mycobacterium smegmatis, so a problem that an hsp60 promoter in the Mycobacterium smegmatis expression system needs a high temperature to increase the expression activity and the high temperature is not conducive to the growth of the Mycobacterium smegmatis is solved. The method has great reference values in the biology research of mycobacteria for expressing specific genes with theMycobacterium smegmatis as a host strain and the development of new anti-tuberculosis vaccinse and microbial reactors. The invention also discloses the high temperature stress-resistant recombinant Mycobacterium smegmatis obtained by the construction method, and a primer pair for the PCR amplification of the Mycobacterium tuberculosis HtpG gene fragment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a recombinant mycobacterium smegmatis resistant to high temperature stress. Background technique [0002] Mycobacterium smegmatis was first reported by Lustgarten in 1884, and then Alvarez and Tavel also found the bacteria in genital secretions (smegma, smegma), so it was named mycobacterium smegmatis (mycobacterium smegmatis). Mycobacterium smegmatis belongs to the genus Actinomycetes and is also commonly found in soil, marine and freshwater environments. [0003] Mycobacterium smegmatis is a non-pathogenic microorganism that shares more than 2000 homologous genes with Mycobacterium tuberculosis, the pathogenic bacterium that causes tuberculosis, and has the same cell wall structure as Mycobacterium tuberculosis and other mycobacteria. M. smegmatis is readily cultured in most synthetic or complex laboratory media, forming visible colonies in 3 to 5 days. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/11C12R1/34
CPCC07K14/35C12N15/74
Inventor 周盈魏文静张晓莉王绪德周亚凤刘芳毕利军
Owner FOSHAN UNIVERSITY
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