幽门螺杆菌标准株NCTC11639 HSPA基因的克隆、表达及其抗原性的鉴定

目的:构建含人幽门螺杆菌(Helicobacterpylori,Hp)热休克蛋白A(heatshockproteinA,HSPA)编码基因的重组质粒,测定、分析其核酸序列,并在E.coli中表达,研究其抗原性。方法:应用PCR技术从HpDNA染色体中扩增HSPA编码基因片段,将其T-A克隆和测序,并与GenBank公布的其他Hp菌株基因序列比较,再将目的基因克隆至表达载体pGEX-4T-1上进行表达和纯化,用Westernblot检测产物的抗原性并用ELISA法对29株小鼠抗Hp全菌单克隆抗体进行鉴定。结果:HSPA基因全长351bp(GenBank登录号为DQ141574),核酸同源性为96%～98%,表达的HSPA融合蛋白分子量约为41ku,表达产物可被Hp感染患者血清识别,29株抗小鼠Hp全菌单克隆抗体中有4株是针对HSPA抗原的。结论:重组HSPA具有较好的抗原性,为Hp疫苗的研究奠定基础。

Cloning,expression and antigenicity identification of HSPA of Helicobacter pylori

Objective To construct recombinant plasmid containing HSPA of helicobacter pylori (Hp),sequence nucleic acid,express it in E.coli and study its antigenicity. Methods The gene encoding HSPA of Hp was amplified from Hp chromosomal DNA by PCR,then operated T-A cloned and sequenced. The gene was cloned into pGEX-4T-1 vector and expressed in E.coli. Expressed product was analyzed by Western blot and used to identify 29 strains of mouse monoclonal antibodies (mAbs) against Hp by ELISA. Results HSPA fragments were...