庚型肝炎病毒E2区cDNA在毕赤酵母中的表达及抗原性鉴定

Expression and Characterization of Envelope Protein 2 Gene of Hepatitis G Virus in Pichia pastoris

利用PCR技术 ,从含有庚型肝炎病毒 (GBV C HGV)包膜蛋白E2cDNA(5 5 9bp)的质粒pGEX E2中 ,扩增得到能够编码日本血吸虫谷胱甘肽硫转移酶 (GST)和GBV C HGV包膜蛋白E2的融合基因片段。将此长度为 132 4bp的DNA片段插入到酵母表达载体pPIC9K中 ,使之位于α 因子信号肽下游 ,且与之同框。通过电激转化将构建的重组表达质粒pPIC9K GST E2插入到PichiapastorisGS115菌株染色体中。筛选His+Muts 表型的转化子 ,震荡培养 ,用0 5 %甲醇诱导表达 5d后 ,在培养液中得到表达的GST E2融合蛋白。经过表达条件的优化 ,GST E2蛋白可占培养液中总蛋白的 5 0 %。通过谷胱甘肽亲和层析柱纯化 ,GST E2融合蛋白的纯度可达 95 %左右。以庚型肝炎病人血清为探针 ,进行免疫印迹及ELISA实验 。

A cDNA fragment locating at the putative envelop protein 2(E2) region of GBV\|C/HGV fused with \%Schistosoma japonicum\% glutathione S\|transferase(GST) was amplified with PCR from plasmid pGEX\|E2.The amplified DNA fragment was inserted into plasmid pGEX\|5X\|1,at the downstream of the coding sequences of GST,in the same reading frame with the gene of GST.The fusion gene fragment of GST\|E2 was amplified with PCR,using the recombinant plasmid pGEX\|5X\|1\|E2 as the template.The amplified 1324 bp DNA fragment of GST\|E2 was inserted into Pichia pastoris expression vector pPIC9K in reading frame with α\|factor secreting signal peptide.The plasmid pPIC9K\|GST\|E2 was transformed into \%Pichia pastoris\% GS115 with electroporation.The transformants (His\++Mut\+s) were selected and induced to express the 54kD GST\|E2 fusion protein,which could be specially recognized by both the antisera directed against E2 and against GST.The GST\|E2 fusion protein was purified with Sepharose 4B glutathione affinity chromatography to a purity of 95%.The expression was optimized to achieve the highest expression level of GST\|E2 fusion protein which was accumulated up to 50% of total proteins in the culture supernatant.The GST\|E2 protein derived from the recombinant \%Pichia pastoris\% was proved possessing antigenicity and high specificity by ELISA,probed with sera from the patients infected by GBV\|C/HGV.