【摘要】 利用逆转录聚合酶链式反应(RT-PCR)的方法克隆肠道病毒71型SHZH98株外壳蛋白VP3基因,连接T载体,经序列测定后,构建酵母分泌型表达质粒pPIC9K/VP3,经序列测定证实-factor信号肽和VP3的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导,筛选到5株高效表达VP3蛋白的工程菌株.ELISA实验表明:重组蛋白VP3具有较好的免疫原性.更多还原

【Abstract】 The genes encoding the capsid protein VP3 were amplified by RT-PCR.After con-firmed by DNA sequence analysis,the VP3 was inserted into the Pichia.Pastoris expression vector pPIC9K.The sequence of ORF of-factor and VP3 were confirmed by DNAsequencing with primer 5’-AOX1 and 3’-AOX1.Then the pPIC9K/VP3 weretransformed into yeast GS115 by the of electro-transformation.After the PCR analysis and the screening for Mut+ phenotype of the transformants,Then multiple inserted transformants were fermented in flasks.After induction by methanol(96 hours),recombinant protein could be effectively secreted into media.SDS-PAGE showed that the gene VP3 could expressas 31kD protein as same as thenatural protein,ELISA analysisof the culture supernatants confirmed that they had the desired immunogenicity of EV71.更多还原