戊型肝炎病毒结构区ORF2蛋白在巴氏毕赤酵母中的胞内表达与纯化的初步研究

为寻求新型表达系统来研制戊型肝炎基因工程疫苗 ,利用甲醇营养型酵母 pichiapastoris表达系统表达戊型肝炎病毒 (HEV)结构区ORF2蛋白。采用PCR方法从HEVcDNA中扩增得到的ORF2基因克隆到酵母表达载体pPIC3.5K上 ,构建成重组质粒 pPIC3.5KORF2。该质粒转化酵母菌GS115 ,经G418筛选得到高拷贝转化子。转化菌株经Mut表型鉴定后 ,用含甲醇的培养基诱导表达 ,SDS PAGE及ELISA筛选高表达活性菌株 ,放大培养后进行亲合层析纯化。在该系统中成功地表达了高生物活性的HEVORF2蛋白 ,经亲和层析纯化后扫描分析重组蛋白分子量约为 5 9kD ,纯度可达 96 %。Westernblotting证实 ,它与HEVORF2单克隆抗体有特异性反应。HEV结构区ORF2蛋白在甲醇营养型酵母中的成功表达 ,以及初步纯化得到的具有强免疫学活性的重组蛋白 ,为研制新型戊型肝炎基因工程疫苗奠定了基础

Intracellular Expression of Hepatitis E Virus ORF2 Protein in pichia pastoris and Its Purification

We have used the methytotrophic yeast pichia pastoris toexpressthe open reading frame 2(ORF2) protein of hepatitis E virus (HEV). The gene which encodes amino acid residues 69～660 of ORF2, was amplified from cDNA of HEV by PCR and cloned into the pichia expression vector pPIC3.5K. The recombinant plasmid was transformed into GS115 and screened for G418 resistant and Mut phenotype. The high resistant Mut's clones were chosen to test the expression of HEV ORF2 protein in BMGY/BMMY media by SDS-PAGE and ELISA. The high expressing clone was scaled up and the recombinant products were purified by affinity chromatography. The expressed protein was estimated to be 59kDa with 96%purity by scanning analysis. Western blot showed it could be recognized by the McAb of HEV ORF2. The successful expression of HEV ORF2 protein in p. Pastoris and the production of recombinant protein provides basis for genetically engineered vaccine development of hepatitis E.