乙型脑炎病毒E蛋白毕赤酵母表达系统的构建

目的：构建乙型脑炎病毒E基因毕赤酵母表达系统。方泼：应用RT-PCR法扩增乙型脑炎病毒E蛋白表达基因，定向克隆入载体pPICZαA，将重组质粒以PineⅠ线性化并电转化入毕赤酵母GS115感受态细胞，用Zeocin筛选转化子进行鉴定。结果：PCR扩增产物约为1350bp，定向克隆获得pPICZαA-E表达载体，经测序结果与Genbank相应序列比较，核苷酸序列同源性为100％，电转化得到巴氏毕赤酵母重组菌株GS115-E，提取其基因组DNA经PCR鉴定与预期相符。结论：成功构建乙型脑炎病毒E基因毕赤酵母表达系统，为进一步进行E蛋白真核表达研究奠定基础。

The construction of Pichia pastoris expression system with E gene of Japanese encephalitis virus

Objective： To construct the Pichia pastoris expression system including E gene segment of Japanese encephalitis virus（JEV）. Methods： Amplifying the E gene of the SA-14 strain by RT-PCR, which encodes for E protein. Then the RT-PCR products were cloned into the pPICZαA vector after being cutted by two enzymes. After linerized by Pme Ⅰ enzyme, the recombinant plasmid was transformed into the competent cell of Pichia pastoris GS115 strain, and the transforments were screened with zeocin. Results： The RT-PCR product was about 1 350 bp. We compared the sequence of the E gene which inserted into the pPICZαA vector with the ones reported in Genbank, their similarity was 100%. After the recombinant plasmid pPICZαA-E was transformed into Pichia pastoris, the recombinant strain GS115-E was obtained, and their genome could be proved to be correct by PCR. Conclusion： We construct the Pichia pastoris expression system including JEV E gene successfully and lay a foundation for further reaserches.