幽门螺杆菌hpaA基因克隆及其蛋白的表达纯化

Cloning of hpaA Gene from Helicobacter pylori and Its Expression and Purification

目的克隆幽门螺杆菌hpaA基因,构建hpaA-pET-32a重组质粒,并对其重组蛋白进行表达纯化,为其进一步的免疫研究提供实验基础.方法采用PCR方法,从幽门螺杆菌临床分离菌株中扩增hpaA基因,T-A克隆后测定核苷酸序列,通过表达载体pET-32a构建的hpaA的重组质粒,在E.coliBL21(DE3)宿主菌中用不同浓度的IPTG诱导表达,最后对重组蛋白进行纯化.结果重组质粒hpaA-pET-32a经双酶切、PCR及测序证明构建成功,经诱导重组蛋白成功表达,以包涵体蛋白的形式存在,对其纯化后获得了高纯度的重组蛋白.结论成功构建幽门螺杆菌hpaA的原核表达系统,并对其重组蛋白进行了表达纯化.

Objective Cloning the hpa A gene of Helicobacter pylori,constructing hpa A-pET-32 a recombinant plasmid,expressing and purifying the recombinant protein to provide experimental basis for further immune research. Method The hpa A gene was amplified from clinical isolate of H. pylori by PCR. The nucleotide sequence was determined after T-A cloning. The recombinant plasmid of hpa A constructed by expression vector p ET-32 a was used to induce the expression of hpa A in E. coli BL21( DE3) host bacteria with different concentrations of IPTG. Finally,the target protein was purified. Results The recombinant plasmid hpa A-p ET-32 a was successfully constructed by double enzyme digestion,PCR and sequencing. The recombinant protein was successfully expressed in the form of inclusion body protein. High purity target protein was obtained after purification. Conclusion The prokaryotic expression system of H. pylori hpa A was successfully constructed,and its recombinant protein was expressed and purified.