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/mLZeocin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/mLZeocinYPD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。
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.
出处
《科学技术与工程》
2007年第8期1577-1581,共5页
Science Technology and Engineering
基金
国家自然科学基金(30170048
30470090)
军队"十一五"计划面上项目(06M255)资助
