重组幽门螺杆菌热休克蛋白A亚单位的高效表达及其初步纯化

High efficiency expression and preliminary purification of recombinant heat shock protein A subunit of Helicobacter pylori

目的 在大肠杆菌中高效表达幽门螺杆菌的热休克蛋白A基因 (hspA) ,并对其初步纯化。 方法 用巢式PCR扩增hspA基因。经测序证实后 ,克隆于表达载体 pIM 1中 ,转化大肠杆菌。以SDS PAGE和免疫印迹分析目的蛋白的表达 ,并测定N 端氨基酸的序列。采用固相镍离子亲和层析 ,对重组hspA进行初步纯化。结果 扩增的hspA基因为 35 7bp ,并在大肠杆菌中得到高效可溶性表达。重组蛋白的表达量最高可达细菌总蛋白的 6 0 .5 %。免疫印迹及氨基酸测序结果证实 ,表达产物为幽门螺杆菌hspA亚单位。固相镍离子亲和层析初步纯化的重组HspA的纯度为 87.8%。结论 hspA亚单位的高效表达与初步纯化 。

Aim To express the heat shock protein A (hspA) gene from Helicobacter pylori(Hp) in E.coli and purify the recombinant protein. Methods The hspA gene was amplified from Hp genome DNA by nest PCR. After sequencing, hspA gene was recombined with expression vector pIM 1 and transformed into E.coli . The expression of recombinant hspA protein was analyzed by SDS PAGE and Western blot. Recombinant hspA was purified through nickel affinity chromatography coloum. Results High efficiency expression of hspA gene was obtained in E.coli . The expressed protein amounted 60.5% of the total bacterial proteins. The expressed product was HspA subunit. The analysis of Western blot and N terminal amino sequencing proved that.The purity of recombinant HspA was 87.8% after preliminary purification through nickel affinity chromatography. Conclusion High efficiency expression and preliminary purification of recombinant hspA subunit of Helicobacter pylori lay the foundation for acquirment in bulk of Hp subunit antigens.