摘要
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 pseudovirus carrying every N-linked glycosylation site from Hantavirus（HTNV）mutants. Methods First,five N-glycosylation sites（Gn N134,N235,N347,N399,and Gc N928）in HTNV glycoprotein were simultaneously mutated using multi site-directed mutagenesis and multi-gene mutants were then constructed.Recombinant lentiviral vectors were constructed and transfected into 293 Tcells with packaging plasmids to generate a recombinant pseudovirus.The recombinant pseudovirus was tested with an indirect immunofluorescence assay after transfection into HEK293 cells. Results Sequencing indicated that the gene CAG coding Gln was successfully substituted for the gene ATT coding Asn in multi gene mutants.The titer of the recombinant pseudovirus was 4.0×10^7 TU/ml and results of the indirect immunofluorescence assay indicated that the recombinant pseudovirus correctly expressed Gn and Gc proteins. Conclusion Recombinant HTNV pseudoviral particles carrying 5multi N-linked glycosylation sites of glycoproteins were successfully constructed.This work proposed a method for investigating the influence of glycosylation levels on biological functions of HTNV glycoprotein.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第3期204-208,共5页
Journal of Pathogen Biology
基金
国家重大传染病防治专项课题(No.2013ZX10004609-002)
国家自然科学基金面上项目(No.30801037)
关键词
汉滩病毒
包膜糖蛋白
糖基化位点全突变体
重组假病毒
Hantavirus
envelope glycoprotein
mutants with every glycosylation site
recombinant pseudovirus
