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真核细胞重组表达的丙型肝炎病毒(HCV)E1和E2包膜蛋白为高甘露糖型糖蛋白,一般认为高甘露糖型E1、E2蛋白为复合寡糖型E1、E2蛋白的中间体.作者成功地用中国仓鼠卵巢(CHO)细胞分泌表达了去除羧基端疏水区的HCV E2复合寡糖型蛋白并分析了其抗原性.作者以重组HCV E2基因的质粒pC980为模板,作聚合酶链反应扩增编码HCV E2的DNA片段,插入质粒pSL1180,再合成编码6个组氨酸(his)残基的寡核苷酸,插入pSL1180中E2片段的羧基端,然后将E2-6hiS片段亚克隆入带二氢叶酸还原酶基因的表达质粒pCMV/DHFR,构成编码HCV E2第384～724位氨基酸和6个组氨酸残基的表达质粒pE2-724.为表达部分疏水羧基端缺如的E2蛋白,再构建出编码E2第384～681位氨基酸和6个组氨酸残基的表达质粒pE2-681.以磷酸钙共沉淀分别将两种表达
Recombinant eukaryotic expression of hepatitis C virus (HCV) E1 and E2 envelope protein is a high mannose glycoprotein, generally considered high mannose type E1, E2 protein complex oligosaccharide E1, E2 protein intermediates. The authors successfully expressed and analyzed the antigenicity of HCV E2 complex oligosaccharide with the carboxyl terminal hydrophobic region secreted by Chinese hamster ovary (CHO) cells.The recombinant plasmid pC980 of HCV E2 gene was used as a template for polymerase chain reaction The DNA fragment encoding HCV E2 was amplified by PCR and inserted into plasmid pSL1180. The oligonucleotide encoding 6 histidine residues was synthesized and inserted into the carboxyl terminus of the E2 fragment in pSL1180. The E2-6hiS fragment was then subcloned The expression plasmid pCMV / DHFR containing the dihydrofolate reductase gene forms an expression plasmid pE2-724 which encodes amino acids 384 to 724 of HCV E2 and 6 histidine residues, and is an expression plasmid which expresses a portion of the hydrophobic carboxy terminus E2 Protein, construct the expression plasmid pE2-681 which encodes amino acids 384-681 of E2 and histidine residues of 6. Two kinds of expression plasmids