戊型肝炎病毒结构区OFR2基因在毕赤酵母中的分泌型表达

Secreted Expression of Hepatitis E Virus ORF2 Gene in Pichia Pastoris

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Pichia pastoris表达系统表达戊型肝炎病毒（HEV）结构区ORF2 1.3 kb基因。对构建的酵母分泌型表达载体pHIL-S1/HEV和pPIC9/HEV重组体,酶切线性化后转化入GS115酵母菌,经表型鉴定,PCR扩增筛选阳性克隆,对不同表型,不同表达载体的重组株用含甲醇的培养基诱导表达,ELISA及SDS-PAGE筛选表达活性菌株,Western-blot证实表达蛋白与HEVORF2单克隆抗体有特异性反应。得到有效表达HEVORF2蛋白的菌株并初步优化表达条件。

We have used the methytotrophic yeast Pichia Pastoris to express the open reading frame2(ORF2) 1.3 kb gene of Hepatitis E virus(HEV). The recombinant secreted expression vector pPIC9HEV and pHUS1HEV were linearized by SaiI and BglII,and then transformed into Pichia Pastoris GS115 by electroporation. The expression vector carrying interested gene was integrated into GS115 chromone. MD and MM plates were used to selected Mut phenotype, and then positive clones were analyzed by PCR to confirm the integration of HEVORF2 gene. We test expression of both HIS+ MUTs and HIS+ MUT+ in BMGY/BMMY media. A high level of secreted protein could be detected by ELISA and SDS-PAGE. Westems-blot showed it could be recognized by the McAb of HEV ORF2.We get the strains that can express HEV ORF2 protein and we tried to find the best expression condition.