人幽门螺杆菌热休克蛋白A基因的克隆、表达及初步应用

Cloning, expression and preliminary clinical application of heat shock protein A gene of human Helicobacter pylori

目的 获取重组表达人幽门螺杆菌 (Helicobacterpylori ,Hp)热休克蛋白A(HspA)亚单位 ,并应用于临床诊断。方法 利用PCR技术扩增HpHspA基因 ,将其构建至PinPointTMXa 3载体中进行诱导表达 ,并对其产物进行SDS PAGE、免疫印迹等检测。同时 ,将纯化的重组蛋白结合ELISA方法对幽门螺杆菌感染患者清除治疗前后血清中的抗HspA抗体进行检测。结果 所克隆的HspADNA由 3 5 4个碱基组成 ,可编码 118个氨基酸残基的多肽 ,SDS PAGE和免疫印迹显示所表达的蛋白分子量约为 14× 10 3 ,并可与相应的抗血清发生特异反应 ;ELISA结果发现 ,消化性溃疡患者在清除治疗后早期只出现明显的抗HspA抗体滴度下降 ,而抗幽门螺杆菌抗体要在治疗后 6月才出现明显下降。结论 重组表达HspA为诊断。

Objective To obtain the recombinant human Helicobacter pylori (Hp) heat shock protein A(HspA) and apply it to clinical diagnosis. Methods The HspA DNA was amplified with PCR from the chromosomal DNA of clinically isolated H. pylori and then inserted into plasmid PinPoint TM Xa 3. The vector pPin HspA was transfected to E.coli JM109 for recombinant protein which were later analyzed with SDS PAGE, Western blot. The purified protein was used with ELISA to test HspA antibody in patients with Hp infection before and after eradication treatment. Results The cloned DNA consisted of 354 base pairs, and encoded 118 amino acid residues with a molecular weight of 14×10 3. Recombinant HspA was confirmed by the sera of Hp infected patients and rabbits with Western blot. The levels of serum anti recombinant HspA antibodies were declined significantly after eradication treatment, but changes of Hp antibodies were not apparent until almost 6 monthes later. Conclusion The identified recombinant HspA protein estabtishes a basis for experimental study on diagnosis, prevention and treatment of Hp infection.