汉坦病毒G1糖蛋白毕赤酵母表达系统的构建

Construction of Pichia pastoris expression system of G1 glycoprotein of hantavirus

目的构建汉坦病毒HTN型和SEO型G1糖蛋白的毕赤酵母表达系统。方法应用RT-PCR法扩增汉坦病毒Z10株和L99株编码G1包膜糖蛋白的基因,将扩增产物分别与pMD18-T simple载体连接,经序列分析后双酶切,定向克隆入载体pPICZαA,继而用BstX I酶切线性化重组质粒并电转化入P.pastoris X33感受态细胞,用Zeocin筛选转化子进行鉴定。结果 PCR扩增产物Z10株为1 937bp、L99株为1 991bp,与T载体相连测序结果与GenBank相应序列比较,核苷酸序列同源性分别为99.5%和99.7%,其编码蛋白质二级结构均无改变。定向克隆毕赤酵母表达载体获得pPICZαA-Z10G1和pPICZαA-L99G1,分别电转化得到巴氏毕赤酵母重组菌株PpX33-Z10G1和PpX33-L99G1,提取其基因组DNA经PCR鉴定与预期相符。结论成功构建汉坦病毒HTN型和SEO型G1糖蛋白毕赤酵母表达系统,为进一步研究奠定基础。

Objective To construct the Pichia pastoris expression system of G1 glycoprotein of hantaan virus and Seoul virus. Methods The gene of the Z10 strain and L99 strain were amplified by RT-PCR, which encoded for Glycoproterin G1. Then the RT-PCR products were cloned into the pMD18-T simple vector. After identifing by sequence analysis, the heterologous gene in the recombinant plasmid was cutted by two enzymes and inserted into pPICZctA respectively. After linerized by BstX 1 enzyme, the recombinant plasmid was transformed into the competent cell of Pichia pastoris-X33, and the multi-copy transformants was screened with Zeocin. Results The Z10 strain RT-PCR product was about 1 937 bp, and the L99 strain RT-PCR product was about 1 991 bp. The sequence of the gene which inserted into the pMD18-T simple vector were compared with the ones reported in Genbank, their similarity was 99.5% and 99.7% respectively. Their target protein＇s second structure was not changed. After the recombinant plasmid pPICZαtA-Z10G1 and pPICZαtA-L99G1 were transformed into Pichia pastoris, the recombinant strain Ppx33-Z10G1 and Ppx33-L99G1 were obtained, and their genome could be proved to be correct by PCR. Conclusion The Pichia pastoris expression system of hantavirus G1 glycoprotein has been constructed successfully in the present study.