中文摘要：

构建了编码汉坦病毒囊膜糖蛋白G2基因的重组质粒，在毕赤酵母中表达，为汉坦病毒基因工程疫苗的研究提供实验基础。利用PCR法从含汉滩病毒76—118株M基因的M56质粒中扩增编码糖蛋白G2的基因片段，克隆入酵母分泌表达载体pPICZαA，构建重组质粒pPICZαA—G2。酶切鉴定挑取的阳性克隆，转化入GS115工程菌，在含100μg／mL Zeocin的YPD培养基上筛选。挑选单菌落，PCR鉴定阳性克隆，用0．5％甲醇诱导表达，并利用SDS-PAGE及Western-Blot鉴定表达产物。序列分析表明所获得的基因片段与编码汉滩病毒76—118株囊膜糖蛋白G2的基因一致；100μg／mL Zeocin YPD培养基上筛选出含pPICZαA—G2转化子，PCR鉴定为阳性克隆；SDS—PAGE可见约70kDa处有目的蛋白表达条带，经Western—Blot证实该条带为汉滩病毒囊膜糖蛋白G2。成功地构建了重组酵母表达载体pPICZαA—G2，并在毕赤酵母中初步表达成功，为今后汉坦病毒囊膜糖蛋白G2表达纯化以及基因工程疫苗的制备奠定了一定基础。

英文摘要：

To express the glycoprotein G2 of Hanta virus in the Pichia expression system, the G2 gene was amplified by PCR from the M56 plasmid which contains the M segment of Hantaan virus 76-118 strain. Then the G2 gene was cloned into the yeast expression vector pPICZαA. The recombinant was identified by restriction enzyme analysis. The positive clone named pPICZαA - G2 was introduced into the Pichia host strain GS115. The transformants were screened on the YPD medium containing 100 μg/mL Zeocin. After identified by PCR, the positive transformants were induced by 0.5% methanol, and the expressionproducts were detected by SDS-PAGE and Western-Blot. The sequence analysis demonstrated that the recombinant plasmid pPICZαA - G2 was constructed successfully and the sequence was consistent with the designing. PCR result showed that pPICZαA-G2 transformants were obtained. After induced expression, G2 protein could be recognized by the anti-his specific mAb and convalescent patient sera of HFRS. The successful expression of G2 in Pichia expression system lays the basis for further research on genetic engineering vaccine of HFRS.