丙型肝炎病毒E2糖蛋白糖基化作用的研究

Construction of the three N-glycosylation site-specific mutants of HCV E2 glycoprotein

本研究构建丙型肝炎病毒(HCV)糖蛋白E2的N-糖基化位点定点突变体。采用高保真性的PfxDNA聚合酶设计两对引物分别引入两个突变位点通过PCR体外定点突变使E2第535、583位核苷酸由A突变为T从而使AAC编码的天冬酰氨突变为TAC编码的酪氨酸,使得N-糖苷化位点NNT、NST突变为YNT、YST。结果得到两个单位点以及一个双位点突变体,并将突变型E2连接到真核表达载体pcDNA3.1-/Myc-HisB上。成功获得的3个HCVE2糖蛋白糖基化位点定点突变体,为进一步进行HCVE2糖蛋白糖基化位点与分子伴侣之间的相互关系以及突变体对机体的免疫功能的影响的研究奠定了基础。

To construct three N-glycosylation site-specific mutants of HCV E2.Pfx DNA polymerase were used and four synthetic oligonucleotide primers were designed.By the use of PCR method,two single and one double N-gly-cosylation mutation were obtained.In these N-glycosylation sites,Asn-X-Thr /Ser,Asn encoding codon were replaced by-Tyr-encoding codon.Two single site(A535T and A583T)mutants and one double sites(A535T,A583T)mutant were obtained,and they were ligated in-frame into the vector pcDNA3.1(-)/Myc-HisB.