幽门螺杆菌ahpC基因的克隆与原核表达

Cloning and expression of H.pylori ahpC in prokaryotic expression vector

目的构建幽门螺杆菌(Helicobacter pylori,Hp)ahpC基因的原核表达系统,表达并纯化Ahp蛋白。方法用PCR方法从中国分离株MEL-Hp27的染色体DNA中扩增出ahpC基因片段,将目的基因插入表达载体pET-30a中,构建重组质粒pET30a-ahpC。重组质粒经DNA测序鉴定后转化大肠杆菌E.coli BL21(DE3),IPTG诱导表达。采用镍离子亲和层析纯化蛋白,并用SDS-PAGE鉴定。结果PCR扩增的ahpC基因长度为594 bp,经酶切和测序分析,插入到载体的基因与GenBank公布的序列相似性达99%;SDS-PAGE显示,经IPTG诱导出分子质量为23 ku的目的蛋白,且产量较高。结论成功构建了ahpC表达载体pET30a-ahpC,并在大肠杆菌中高效表达。

Objective To construct a prokaryotic expression system of ahpC gene of Helicobacter pylori. Methods The ahpC gene was amplified from Hp chromosomal DNA by PCR technique and cloned into the expression vector pET-30a. The recombinant vector pET30a-ahpC was identified by DNA sequencing and transformed to E. coli BL21 （DE3） for expression under induction by IPTG. The expression product was analyzed by SDS-PAGE. Results PCR product showed that ahpC gene consisted of 594 bp. The gene fragment that was inserted into the recombinant vector was identified to GenBank for 99%. SDS-PAGE showed that the induced protein was expressed highly in the host bacterium. Conclusion A prokaryotic high-expression system for ahpC gene has been successfully constructed. It can highly express r-AhpC protein in E. coli.