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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 a Hepatitis C virus (HCV) mutant with histidine site mutation and test its infectivity. Methods The mutated H490A and H621A plasmids were constructed by mutating histidine at position 490 and position 621 of Alanine to Alanine using site - directed mutagenesis. The viral RNA was prepared by T7 in vitro transcription. Huh7.5.1 cells were transfected by electroporation, and the expression of viral proteins, the efficiency of electroporation and the infectivity of cell culture supernatants were detected by immunofluorescence. Results The constructed H490A and H621A mutant plasmids were identified by restriction enzyme digestion and sequencing. The wild-type and mutant virus RNAs were able to express viral proteins in Huh7.5.1 cells with the efficiency of electroporation up to 90% HCV positive cells were detected in the culture supernatant of infected cells, and the number of positive cells in the culture supernatant of H490A virus-infected cells was significantly decreased compared with the wild type. No positive cells were detected in the supernatant of H621A virus-infected cells. Conclusion The full-length expression plasmids H490A and H621A with histidine site mutation were successfully constructed, and the infectivity of the two mutant viruses was significantly reduced.