戊型肝炎病毒结构蛋白基因的真核表达

Eukaryotic expression of hepatitis E virus structural gene

目的使用酵母表达系统表达戊型肝炎病毒结构蛋白基因。方法构建含戊型肝炎病毒(HEV)结构蛋白嵌合基因HEVORF23(由HEVORF2C端800bp片段与ORF3全基因组成)的重组表达质粒pPICZαA_HEVORF23,并通过电转移的方法将该基因整合入甲醇营养型酵母的基因组DNA中,利用甲醇进行诱导表达,表达产物经纯化后,进行Western印迹鉴定。结果pH6.0环境下诱导培养72h后,在细胞沉淀中获得一个分子量大约为69kDa的可溶性目的蛋白,纯化蛋白经Western印迹检测后发现能与戊型肝炎患者血清发生较强的特异性免疫反应。结论采用酵母表达系统诱导表达可以获得抗原性较强的HEV重组表达抗原,为进一步开发研制有效的诊断抗原和疫苗奠定了基础。

Objective To express HEV structural gene by using pichia pastoris expression system. Methods The chimeric gene segment HEV ORF23（an g00bp gene segement close to the HEV ORF2 C end and the whole ORF3 gene segement） of HEV structural gene is cloned into pPICZ a A pichia expression vector by double digest with Not I and Xba Ⅰ. The reeombinant plasmid pPICZ a A-HEV ORF23 is transformed appropriate pichia pastoris strain by using electroporation and then the interest gene is integrated into the genome DNA by a single crossover. The recombinant pichia strains are selected for induced expression by methanol, purifying the expression products and doing western-blot detection. Results We obtain a 69kDa protein in the cell lysates after induced 72 hours under pH6.0 circanstance, The protein can be purified with His＋ Bind Resin, and the western-blot results is positive. Conclusions Using pichia pastoris expression system and His purification system, a soluble, high antigenic and immunogenic HEV recombinant protein can be obtained, and it makes a foundation for the development of the efficient hepatitis E diagnostic reagent and vaccine.