丙肝抗原决定簇与CTB的融合、表达及融合蛋白的纯化

Gene Fusion and Expression of Cholera Toxin B Subunit with HCV Epitopes

通过固相化学合成法合成了编码丙型肝炎病毒(HCV)结构区和非结构区的4个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串联后与ctxB基因融合,构建了12种表达不同嵌合蛋白的重组质粒,各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量在10～50μg/ml之间,随所融合的抗原决定簇不同而不同,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化,达到了电泳纯,为进一步研究融合蛋白的抗原性及用作抗-HCV ELISA诊断试剂抗原打下了基础。

Four oligonucleotides which encode epitopes of structure and nonstructural antigen HCV, were chemically synthesized and fused with cholera toxin B subunit(CTB) gene in different ways of tanden repeat. Twelve recombinant plasmids were constructed and all of these plasmids could expressed fusion proteins of CTB and HCV epitopes. The expression level of fusion proteins ranged from 10~50 fig/ml depending on amino acid composition of the epitopes, and more than 95% of the chimeras were secreted into the medium. Purified fusion proteins were obtained by affinity chromatography. The work described provided a sound basis for the application of the fusion protein in assambling anti-HCV ELISA kit.