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目的构建汉滩病毒(HTNV)包膜糖蛋白(GP)糖基化位点全突变重组假病毒。方法应用一次性多位点突变技术同时突变HTNV GP上5个N-糖基化位点(Gn的N134、N235、N347、N399和Gc的N928),克隆入慢病毒表达载体,与包装质粒共转染293T细胞,包装出重组假病毒颗粒,感染HEK293细胞,采用免疫荧光法检测和鉴定构建的重组假病毒。结果基因序列测定显示,突变体上5个N-糖基化位点均由天冬酰胺(Asn)的编码基因(AAT)突变为谷氨酰胺(Gln)的编码基因(CAG);制备的重组假病毒滴度为4.0×107 TU/ml;免疫荧光检测显示构建的重组假病毒可正确表达HTNV的Gn和Gc蛋白。结论成功构建了含有HTNV包膜糖蛋白5个N-糖基化位点全突变的重组假病毒颗粒,为研究糖基化对HTNV包膜糖蛋白生物学功能的影响创造了条件。
Objective To construct a recombinant mutant pseudovirus full of glycoprotein (GP) glycosylation site of Hantaan virus (HTNV). Methods A total of 5 N-glycosylation sites (N134, N235, N347, N399 and N928 of Gc) of HTNV GP were mutated simultaneously by a one-time multiple site mutation technique and cloned into a lentiviral vector. 293T cells were transfected, and the recombinant pseudovirions were packaged and infected into HEK293 cells. The constructed recombinant pseudovirus was detected by immunofluorescence assay. Results The sequence analysis showed that all five N-glycosylation sites on the mutants were mutated from asparagine (AAT) to glutamine (Gln) encoding gene (CAG) The titer of pseudovirus was 4.0 × 107 TU / ml. Immunofluorescence showed that the constructed recombinant pseudovirus could express Gn and Gc protein of HTNV correctly. Conclusion The recombinant pseudovirions containing 5 full-length N-glycosylation sites of HTNV envelope glycoprotein were successfully constructed, which provided the conditions for studying the effect of glycosylation on the biological function of HTNV envelope glycoprotein.