幽门螺杆菌hp1188基因的原核表达

Prokaryotic expression of Helicobacter pylori gene hp1188

目的:重组表达幽门螺杆菌(Helicobacter pylori,H.pylori)hp1188基因,为研究该基因所编码蛋白在H.pylori的黏附过程中的作用奠定基础。方法:以H.pylori标准株NCTC11637基因组DNA为模板,PCR扩增hp1188基因片段,克隆至质粒pQE-30中获得重组质粒pQE30-hp1188,经DNA测序确认后将重组质粒转化大肠杆菌DH5α,IPTG诱导表达。Ni2+-NTA树脂亲和层析纯化表达蛋白,SDS-PAGE检测表达,Westernblot鉴定相应抗原表位。结果:扩增的hp1188基因片段全长810bp,与基因文库中的hp1188基因同源性达98%。SDS-PAGE显示表达产物相对分子量约为30600Dalton,与预期相符;大肠杆菌裂解液中表达产物接近可溶性蛋白总量的一半,但沉淀中仍能检测到表达蛋白。目标蛋白纯化后均一性接近90%,Westernblot证实与多克隆抗体有良好结合能力。结论:成功克隆了hp1188基因,并在大肠杆菌DH5α中获得高效表达。

Objective：To express the recombinant protein of the hp1188 gene of Helicobacter pylori NCTC 11637 in order to lay a foundation for the study on adhesion. Methods：The hp1188 gene was amplified from NCTC 11637 chromosomal DNA by PCR.The PCR product was inserted into the expression vector pQE-30.The recombinant vector pQE30-hp1188 was identified by DNA sequencing and transformed to E.coli DH5α for expression under IPTG induction.The recombinant protein was purified by Ni^2＋-NTA agarose and identified by SDS-PAGE and western blot. Results：The sequence of hp1188 gene amplified was about 810 bp with a 98% sequence homology with hp1188 genes in GenBank. SDS-PAGE determined that the molecular weight of its expression products was 30 600 Dalton. The expression products of E.coli DH5α lyzate was about 50% of total soluble proteins,and the pellet also had expressed. Its purity was over 90% after purified with Ni^2＋-NTA agarose and Western blot showed that the recombinant protein possesses fine binding capacity with the rabbit anti-H.pylori. Conclusion：hp1188 gene was successfully cloned and highly expressed in E.coli DH5α.