葡萄糖调节蛋白GRP78与HBV的PreS1相互作用位点的初步研究

Preliminary study of interaction sites on glucose-regulated protein GRP78 with PreS1 of hepatitis B virus

目的:初步研究78 k D的葡萄糖调节蛋白(the 78 k D glucose-regulated protein,GRP78)与乙型肝炎病毒(hepatitis B virus,HBV)的前S1蛋白(Pre S1)的相互作用位点。方法:利用PCR技术扩增GRP78的基因,将扩增的目的基因克隆至p W28载体质粒,在大肠杆菌(Escherichia coli,E.coli)B834中表达,经过镍离子亲和层析柱纯化GRP78蛋白;将Pre S1 3个截短片段的重组质粒(p GST-Pre S1-X1/X2/X3)在B834中表达后,经过GST亲和层析柱纯化相应蛋白;利用蛋白质体外结合实验(pull down)、微量热泳动(microscale thermophoresis,MST)检测GRP78与Pre S1 3个截短片段的相互作用。结果:成功构建重组质粒p W28-GRP78;获得GRP78蛋白及Pre S1 3个截短片段的融合蛋白;pull down及MST实验验证了GRP78可以与Pre S1的3个片段结合,且GRP78与GST-Pre S1-X1结合效果最好。结论:利用分子克隆技术及蛋白质表达纯化技术,获得GRP78蛋白及Pre S1截短片段的融合蛋白,并初步筛选了Pre S1与GRP78的相互作用位点,为后续研究打基础。

Objective：To study Lnteraction sites of the glucose-regulated protein 78 kD（GRP78） with pre-S1 protein（PreS1） of hep- atitis B virus（HBV）. Methods：Being amplified by PCR,the Open reading frame of GRP78 was subcloned into pW28 and expressed in Escherichia coli（E.coli） B834. The GRP78 protein was purified by nickel affinity chromatography column. Three truncated plasmids of HBV PreS1, including pGST-PreS1-X1, pGST-PreS1-X2, pGST-PreS1-X3, were expressed in B834 and then the bacterial protein was purified by GST affinity chromatography column. Interactions between GRP78 and PreS1 truncations were detected by pull down and microscale thermophoresis（MST）. Results：The recombinant plasmid of pW28-GRP78 was successfully constructed. The GRP78 protein and the fusion protein（GST-PreS1-X1, GST-PreS1-X2,GST-PreS1-X3） were obtained. The directly interaction between GRP78 and fusion protein were detected by pull down and MST,suggesting that the 1-65 amino acid residues of PreS1 play a key role for GRP78 binding to PreS1. Conclusion：Thanks to the molecular cloning and purified technology of protein purification,the fusion protein of GRP78 protein and the PreS1 truncated seg- ments is formed. And interaction sites of GRP78 protein and the PreS1 is primarily compared,providing preliminary data for future study.