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目的定点突变幽门螺杆菌标准菌株ATCC26695 hsp60基因的1398、1399位点,并获得基因突变后hsp60基因的原核表达蛋白。方法采用Primer Premier5.0软件设计hsp60基因的2对引物,引入2个突变位点,通过重叠延伸PCR法进行3次扩增,获得突变型hsp60基因,克隆至原核表达载体pEASY-E1,重组质粒转化至E. coli BL21(DE3),对其进行测序验证,经IPTG诱导表达后,SDS-PAGE电泳检测表达产物。结果将幽门螺杆菌hsp60基因1398、1399位点AG突变为GC,构建了突变型hsp60基因的表达载体,转化大肠埃希菌后高效表达可溶性hsp60蛋白。结论构建了突变型hsp60基因表达载体,并能表达可溶性目的蛋白,为对其功能研究奠定了基础。
Objective To mutate 1398,1399 hsp60 gene of the standard strain of H. pylori ATCC26695 and obtain the prokaryotic expression protein of hsp60 gene after gene mutation. Methods Primer Premier5.0 software was used to design two pairs of primers of hsp60 gene and two mutations were introduced. The mutant was amplified three times by overlap extension PCR. The mutant hsp60 gene was cloned into the prokaryotic expression vector pEASY-E1. The recombinant plasmid The recombinant plasmid was transformed into E. coli BL21 (DE3) and verified by sequencing. After induced by IPTG, the expressed product was detected by SDS-PAGE. Results The AG of 1398 and 1399 of Helicobacter pylori was mutated to GC, and the expression vector of mutant hsp60 gene was constructed. After transformed into Escherichia coli, the soluble hsp60 protein was highly expressed. Conclusion The mutant hsp60 gene expression vector was constructed and expressed soluble target protein, which laid the foundation for the study of its function.