CHO细胞表达的Ⅱ型单纯疱疹病毒膜糖蛋白G  

Envelope Glycoprotein G of Herpes Simplex Virus Type Ⅱ Expressed by CHO Cells

作　　者：金华明 魏丙卓 JIN Huaming;WEI Bingzhuo(Jiemin Biotech(Tianjin),Inc.,Tianjin 300450,China)

机构地区：[1]捷敏(天津)生物技术有限公司,天津300450

出　　处：《病毒学报》2020年第2期281-290,共10页Chinese Journal of Virology

摘　　要：Ⅱ型单纯疱疹病毒(Herpes simplex virus type Ⅱ,HSV-Ⅱ)的膜糖蛋白G(HSV-Ⅱ gG)在生物合成过程中被切割为两个片段,然而,HSV-Ⅱ gG蛋白的切割位点迄今未被实验测定。HSV-Ⅱ gG上带有大量的糖基化,然而糖基修饰在蛋白链上的分布还没有详细的实验确定。在本研究中,中国仓鼠卵巢细胞(CHO细胞)重组表达的HSV-Ⅱ gG的胞外区(HSV-Ⅱ gG ECD)的两个切割产物(一个39kD,一个64kD)被用串联液相色谱-质谱(LC/MS)进行肽图分析。LC/MS肽图分析显示,39kD的产物是HSV-Ⅱ gG ECD蛋白切割后的N端片段,LC/MS肽图分析可以确定HSV-Ⅱ gG ECD的肽链切割点在第335位氨基酸。亲和层析和蛋白免疫印迹实验显示64kD产物是HSV-Ⅱ gG ECD蛋白切割后的C端片段。对两个片段的糖基化分析显示,39kD和64kD蛋白产物带有相似程度的N-连接的糖基修饰(各片段带两个N-连接的糖基化),而O-连接的糖基化仅发生在64kD片段上,而且糖基化程度非常高(几乎相当于该段蛋白链本身的分子量)。本研究的结果支持HSV-Ⅱ gG胞外区的C端部分属于粘蛋白家族。HSV-Ⅱ gG胞外区的C端部分存在于病毒外膜上,这一粘蛋白属性与其参与病毒与宿主细胞的黏附的假设相符合。During its biosynthesis,the envelope glycoprotein G of herpes simplex virus type Ⅱ(HSV-Ⅱ gG)is cleaved into two fragments. The cleavage site has not been determined experimentally. Also,the distribution of glycosylation in the two cleavage products has not been analyzed in great detail. In the present study,the extracellular domain of HSV-Ⅱ gG(HSV-Ⅱ gG ECD)was expressed in the ovary cells of Chinese hamsters.Two resultant protein cleavage products(39 kD,64 kD) were subjected to peptide mapping using liquid chromatography–mass spectrometry. The 39 kD product was determined to be the N-terminal cleavage product and the cleavage site was mapped to the arginine residue at position 335. Immobilized metal-ion affinity purification and Western Blot indicated that the 64 kD product was the C-terminal cleavage product. Analyses of protein glycosylation(by enzymatic and chemical hydrolysis)of the two fragments showed that both had similar degrees of N-linked glycosylation(two N-linked sugar chains on each fragment),whereas the O-linked glycosylation pattern was very different between the two fragments. The O-linked glycosylation of HSV-Ⅱ gG appeared to be concentrated entirely in the C-terminal 64 kD fragment,and the degree of glycosylation was very high(the molecular weight of the sugar chains was almost equal to that of the protein itself). The present study suggested that the C-terminal portion of the HSV-Ⅱ gG extracellular domain belonged to the mucin family of proteins. The mucin-like characteristics was consistent with the hypothesis that the C-terminal segment of the HSV-Ⅱ gG extracellular domain was involved in cell-virus adhesion,as it remained on the membrane of the HSV-Ⅱ.

关 键 词：Ⅱ型单纯疱疹病毒(HSV-Ⅱ) 糖蛋白G(gG) 

分 类 号：R373.9[医药卫生—病原生物学]