汉滩病毒包膜糖蛋白糖基化位点突变体重组假病毒的构建及初步鉴定

目的：为进一步研究汉坦病毒包膜糖蛋白的糖基化与病毒的感染性和免疫原性等的关系,构建含有汉滩病毒（HTNV）囊膜糖蛋白（GP）糖基化位点突变体的重组假病毒。方法：利用定点突变的方法,分别突变了HTNV 76-118株的5个N-糖基化位点并克隆入慢病毒表达载体,与包装质粒共转染293T细胞,构建5株重组假病毒。感染HEK293细胞后,进行RT-PCR鉴定及免疫荧光检测。结果：经测序显示构建的含有N-糖基化位点突变体的5个重组假病毒原序列中的天冬酰胺（N）均被置换为谷氨酰胺（Q）。RT-PCR结果显示5个重组假病毒均有HTNV GP基因的表达。免疫荧光检测5个重组假病毒均可表达HTNV的Gn和Gc蛋白。结论：成功构建了含有HTNV包膜糖蛋白糖基化位点突变体的5个重组假病毒,分别命名为rLV-M1、rLV-M2、rLV-M3、rLV-M4和rLV-M5。本研究为明确N-糖基化对汉坦病毒生物学活性的影响提供了有利的研究工具,并为汉坦病毒疫苗及致病机理的进一步研究打下了一定的基础。

Construction and Identification of Recombinant Pseudovirus Carrying N-linked Glycosylation-site Mutants of Hantaanvirus

Objective： To further study the effect of hantavirus envelope glycoprotein glycosylation on viral immunogenicity and infectious, five recombinant pseudovirus carrying N-linked glycosylation site mutants of Hantaanvirus（HTNV） were constructed.Methods： Mutated five N-glycosylation sites of GP in HTNV 76-118 strain using site-directed mutagenesis methods and constructed recombinant pseudovirus by cotransfecting the Lentiviral expression vector and packaging vectors into the HEK293 cells. Then the five recombinant pseudovirus were identified by RT-PCR and Immunofluorescence（IFA）. Results： Five N-linked glycosylation site mutants were contrasted and their sequencing showed the asparagines（N） residues at the N-linked glycosylation sites on Gn and Gc were replaced by glutamine（Q）. RT-PCR results showed that all of the five recombinant pseudovirus could express the HTNV GP gene. IFA results showed that the recombinant pseudovirus also could express the Gn and Gc protein of HTNV. Conclusion： The five recombinant pseudovirus carrying N-linked glycosylation site mutants of HTNV were successfully constructed and named rLV-M1, rLV-M2, rLV-M3,rLV-M4 and rLV-M5, respectively. The result of this study provided tools for study on the roles of N-linked glycosylation of HTNV glycoproteins in biological activity and laid the foundation for futher esearch of hantavirus vaccine and pathogenic mechanism.