优化分枝杆菌重组工程系统构建分枝杆菌突变株筛选方法

王楚涵; 马鹏娇; 罗涛; 鲍朗

优化分枝杆菌重组工程系统构建分枝杆菌突变株筛选方法

Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants

摘要

摘要:
目的通过为分枝杆菌重组工程系统(pJV53)加筛选标记，建立一种分枝杆菌突变株的筛选方法。

方法为pJV53加入蔗糖反筛选基因SacB和突变的潮霉素抗性基因hyg^S，通过潮霉素抗性恢复指示耻垢分枝杆菌(Ms)体内同源重组的成功，为突变株的筛选提供标记；通过蔗糖反筛选挑选脱去质粒的突变株。

结果成功构建重组质粒pJV53-SacB-hyg^S，筛选出MSMEG_4487 G188A突变株以及利福平耐药的Ms rpoB D516Y和Ms rpoB H526Q突变株，并成功将以上突变株脱去质粒。

结论 pJV53-SacB-hyg^S能够有效帮助构建筛选突变株，并对突变株进行脱质粒操作，具有普遍应用价值；结核分枝杆菌rpoB基因D516Y和H526Q的突变与该菌对利福平的耐药有关。

Abstract:
Objective To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53.

Methods The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hyg^S were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants.
Results The recombinant plasmid pJV53-SacB-hyg^S were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully.

Conclusion pJV53-SacB-hyg^S can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.

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