戊型肝炎病毒开读框架(ORF2)1.3kb片断巴斯德毕赤酵母表达载体的构建

Constructing Pichia pastoris expression vectors of hepatitis E virus ORF2 1.3 kb gene

戊型肝炎病毒 (HEV)基因组为单链正股 RNA,整个基因组有 3个开放性阅读框架(ORFs) ,ORF2是主要结构基因编码区 ,将 HEV ORF2 1.3Kb基因与分泌性表达质粒 p HIL - S1和 p PIC9重组 ,构成受醇氧化酶 (AOX1)的启动子与转录终止区控制的酵母表达质粒 ,酶切线性化后转化入 GS115酵母菌 ,经表型筛选 ,PCR扩增筛选阳性克隆 。

Hepatitis E virus genome is a single-strained positive RNA that has three open reading frames(ORF S), ORF2 is the main structure gene code region. The Pichia Pastoris recombinant plastid pPIC9HEV and pHILS1HEV were constructed by subcloning the ORF2 1.3 kb gene of hepatitis E virus into secreted expression vector pPIC9 and pHILS1, and then transformed into EcolⅠ, selected positive recombinants. The constructed plamsid pPIC9HEV and pHILS1HEV were linearized by SalⅠ and BglⅡ,and then transformed into Pichia Pastoris GS115 by electroporation. The expression vector carrying interested gene integrated into GS115 chromone. MD and MM plates were used to select Pichia recombinants, and then positive clones were analyzed by PCR to confirm the integration of HEVORF2 gene.The expression of both HIS + MUTS and HIS + MUT + recombinants induced with methanol was tested.