风疹病毒E2包膜糖蛋白亮氨酸突变对细胞融合的影响  

Effects of mutations at the E2 protein leucine sites on specific membrane fusion in rubella virus

作　　者：吴冰[1] 刘晓丽[1] 王志玉[1,2] 

机构地区：[1]山东大学公共卫生学院病毒学研究室,济南250012 [2]实验畸形学教育部重点实验室,济南250012

出　　处：《山东大学学报（医学版）》2009年第4期125-128,132,共5页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30570065);山东大学创新团队项目资助

摘　　要：目的探讨风疹病毒E2包膜糖蛋白中部分亮氨酸的突变对特异性细胞融合的影响。方法于E2蛋白中选取9个亮氨酸位点,采用基因定点突变的方法分别将其突变成丙氨酸,采用流式细胞术检测各突变株蛋白在细胞表面的表达效率,Giemsa染色法与指示基因法检测特异性细胞融合,血吸附实验检测其吸附活性。结果与野毒株蛋白相比,除L144A与L225A之外,其它突变株蛋白在细胞表面的表达效率均有不同程度的降低。排除各突变株蛋白在细胞表面表达效率变化的影响后,其引起的细胞融合效率均有不同程度的降低,其中位于E2蛋白氨基酸序列中间靠近氨基端的亮氨酸(L58、L105、L128与L144)突变时,其细胞融合效率降低更为明显,相应的血吸附效率也有所降低。结论E2蛋白中,其氨基酸序列中间靠近氨基端的蛋白区域(L58～L144)对引起的特异性细胞融合具有重要的作用。Objective To explore the effects of mutations at the E2 protein leucine sites on specific membrane fusion in rubella vires （RV）. Methods Nine leucine sites were chosen from E2 protein, and site-directed mutagenesis was used to obtain corre- sponding mutants. All mutants and wild type proteins were expressed in BHK21 cells and treated with acid media to induce spe- cific cen fusions. Expression efficiencies of mutant proteins on cell surfaces were quantified with fluorescence-activated cell sorter （FACS）. The fusion functions were separately assayed with Giemsa staining and a reporter gene method for qualitative and quan- titative analysis. Hemadsorption assays were performed to qualitatively and quantitatively detect binding activity of mutant pro- teins. Results The FACS indicated that expression efficiencies of all mutant proteins except for L144A and L225A were lower than those of the wild type proteins. When effects of lower cell surface expression efficiencies were eliminated, the cell fusion ac- tivities of all mutant proteins were lower than those of wild type proteins, especially those of mutants L58A, LIOSA, L128A and L144A. Hemadsorption assays demonstrated that binding abilities of mutants L58A, L105A, L128A and L144A were decreased, but those of the other five mutant proteins were almost the same as wild type proteins. Conclusion The 1.58-L144 region in E2 protein is important for specific membrane fusion.

关 键 词：风疹病毒 细胞融合 包膜糖蛋白 

分 类 号：R373.1[医药卫生—病原生物学]