丙型肝炎病毒包膜蛋白E1的分泌表达及性质  

Secretory Expression of Different C-terminal Truncated HCV E1 Proteins in Mammalian Cells and Characterization of the Expressed Products

作　　者：朱皑 孔玉英[1] 刘晶[1] 张祖传[1] 汪垣[1] 李光地[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2001年第6期634-640,共7页

基　　金：国家高科技"86 3"计划资助项目 (No .86 3 10 2 0 7 0 2 0 2 )~~

摘　　要：为了实现HCV包膜蛋白E1在哺乳动物细胞中的分泌表达 ,选用分泌表达载体pSecTagB ,构建了 3种E1羧端不同程度缩短的融合表达质粒 ,并在E1的上游融合乙型肝炎病毒前表面抗原S1中含肝细胞结合位点的免疫表位 preS1(2 1～ 47)序列作为标识。在HeLa细胞中进行了暂时表达 ,并对产物的性质进行了鉴定。去除羧端疏水区有利于E1融合蛋白的分泌 ,其中以S1E1t32 5分泌最佳。表达的融合蛋白都能被 preS1单抗及HCVE1多抗识别 ,是具有双重抗原性的糖蛋白 ,其糖链类型为高甘露糖型。建立了稳定表达S1E1t310、S1E1t32 5、S1E1t34 0的CHO细胞株 ,选择其中CHO/pSecS1E1t32 5对表达的分泌蛋白作了进一步研究。利用亲和纯化方法可由细胞培养液中富集和纯化分泌表达的HCVE1,为进一步开展E1性质的研究创造了条件。Three fragments of the HCV envelope 1 (E1) with different C terminal truncation at aa310, aa325, aa340 were cloned into the mammalian expression vector pSecTagB. An epitope in the hepatitis B surface antigen, preS1(21—47), were genetically engineered onto the N terminus of the recombinant protein and used as an affinity tag for detection and purification. The resulting pSec preS1 E1t310, pSec preS1 E1t325 and pSec preS1 E1t340 were transiently expressed in the HeLa cells and the antigenicity, secretory efficiency and glycosylation type of the recombinant E1 proteins were compared. All of the three recombinant proteins could be detected by both preS1 monoclonal antibody and E1 polyclonal antiserum. The expression products were secreted and highly mannose type glycosylated, with S1E1t325 being secreted, indicating the influence of the hydrophobic regions on the secretion of the E1 protein. Three CHO cell lines expressing the proteins, S1E1t310, S1E1t325 and S1E1t340, were established and the CHO/pSecS1E1t325 was chosen for further study. The secreted S1E1t325 could be enriched from cell culture medium by the preS1 antibody coupled Sepharose. The glycosylation analysis indicated the lack of complex glycogen even after the E1 was secreted via Golgi complexes. The established stable cell lines and anti preS1 affinity method could be utilized to enrich and purify the HCV E1 expressed in mammalian cells, and may be used for further characterization of this protein.

关 键 词：包膜蛋白E1 PRES1 双重抗原性 分泌表达 糖基化 丙型肝炎病毒 

分 类 号：Q786[生物学—分子生物学]