鸡抗病毒Mx蛋白基因在毕赤酵母GS115中的表达

Expression of chicken Mx antiviral protein in Pichia pastoris GS115

为了利用酵母表达技术制备鸡抗病毒Mx蛋白,试验采用基因工程技术,将编码鸡抗病毒Mx蛋白基因亚克隆至含有分泌信号肽序列的毕赤酵母表达载体中,构建成分泌型重组表达载体pPIC9K-Mx;用电转化法将线性化的pPIC9K-Mx转化至毕赤酵母菌株GS115中,G418梯度筛选转化菌;再经MM与MD平板对比生长试验筛选,PCR鉴定目的片段,筛选出高拷贝重组子,该高拷贝菌株分别经0.5%、1%甲醇诱导,表达产物再经SDS-PAGE检测。结果表明:成功构建了鸡抗病毒Mx蛋白基因的毕赤酵母表达载体,经G418抗性筛选得到高拷贝菌株;在甲醇含量维持在1%时,鸡抗病毒Mx蛋白在毕赤酵母GS115中获得良好表达,表达产物大小约为75ku;表达蛋白约占表达上清液的39.2%。

To highly express chicken Mx antivirus protein in Pichia pastoris and lay the foundation for further study of the gene for resistance activity. The chicken Mx gene was cloned into the yeast - escherichia shuttle vector pPIC9K to construct secret recombinant expressing plasmid of pPIC9K - Mx. The pPIC9K - Mx was linearized by salland transformed into GS115 yeast by electroplation. And the recombinants were selected using MD culture plate, then, the higheopy clones respectively, induced by 0.5% i 1% methanol for5 days, and SDS - PAGE was used to detected the recombinant Pichia pastoriss trainwere selected by G418 resistance. In addition, MM/MD culture plate were used to select the His ^＋ transformants and be identified by PCR. The high -copy strains, expression products selected by G418 and identified by PCR were obtained. As aresult, after induced by 1% methanol, the antiviral protein of chicken Mx gene had been espressed successfully in Pichia pastoris GS115. SDS - PAGE identification showed that the expressed antiviral protein was 75 ku in size,which was the same as＇ forecast. And scanning results showed that the expression in the supernatant accounted for about 39.2%.