丙型肝炎病毒包膜蛋白E2中关键组氨酸位点突变体的构建与感染性  

Construction and infectivity of hepatitis C virus mutant containing key histidine in envelope protein E2

作　　者：刘霜[1,2] 鞠鹤鹏[2] 戚中田[2] 边中启 秦照玲[2] 

机构地区：[1]解放军昆明总医院传染病中心,昆明650032 [2]第二军医大学微生物学教研室 上海市生物防护重点实验室,上海200433

出　　处：《中国生物制品学杂志》2012年第5期531-535,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30900066;81171564);国家自然科学基金委创新研究群体基金(30921006);上海市重点学科建设项目资助(B901)

摘　　要：目的构建组氨酸(Histidine)位点突变的丙型肝炎病毒(Hepatitis C virus,HCV)突变体,并检测其感染性。方法利用定点突变技术,分别将HCV包膜蛋白E2第490位和第621位的组氨酸突变为丙氨酸(Alanine),构建突变质粒H490A和H621A,采用T7体外转录法制备病毒RNA,通过电穿孔法导入Huh7.5.1细胞,免疫荧光法检测病毒蛋白的表达、电穿孔效率及细胞培养上清的感染性。结果 H490A和H621A突变型质粒经酶切及测序鉴定构建正确;体外转录获得的野生型与突变型病毒RNA均能在Huh7.5.1细胞中表达病毒蛋白,电穿孔效率高达90%以上;野生型病毒感染细胞培养上清中可检测到HCV阳性细胞,H490A病毒感染细胞培养上清中的阳性细胞数量比野生型明显减少,H621A病毒感染细胞培养上清中未检测到阳性细胞。结论成功构建了组氨酸位点突变的全长表达质粒H490A和H621A,两种突变体病毒的感染性均显著降低。Objective To construct the hepatitis C virus（HCV） mutants containing point mutation of histidine and determine its infectivity.Methods The histidine at sites 490 and 621 of HCV envelope protein E2 were mutated into alanine by site-directed mutagenesis to construct plasmids H490A and H621A respectively.Viral RNAs were generated by T7 transcription in vitro and electroporated into Huh7.5.1 cells.The expression of viral protein,the efficiency of electroporation and the infectivity of cell culture supernatant were determined by IFA.Results Restriction analysis and sequencing proved that mutants H490A and H621A were constructed correctly.Viral proteins were expressed in the cells electroporated with either wild-type or mutant RNA transcribed in vitro,and the efficiencies of electroporation were as high as more than 90%.HCV positive cells were detected in cell culture supernatant infected with wild-type HCV,while decreased significantly in those with H490A mutant,and undetectable in those with H621A mutant.Conclusion Full-length expression plasmids,H490A and H621A,containing point mutation of histidine were successfully constructed,of which the infectivity decreased significantly.

关 键 词：丙型肝炎病毒 病毒包膜蛋白质类 组氨酸 点突变 

分 类 号：R373.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]