汉坦病毒G2膜蛋白基因在毕赤酵母GS115中的表达

Cloning and expression of G2 glycoprotein gene of Hantavirus in Pichia pastoris

目的 研究汉坦病毒G2膜蛋白基因在甲醇营养型巴斯德毕赤酵母中的克隆表达。方法 采用RT PCR扩增G2基因 ,转化感受态大肠杆菌DH5α,DNA序列分析后 ,SnaBI、NotI两次酶切下G2 ,再连接到经同样酶切的pPIC9K表达载体上 ,构建表达载体 pPIC9K G2 ,SalI线形化后电转化导入毕赤酵母宿主菌GS115 ,G418筛选转化子 ,1%甲醇诱导、摇瓶培养。结果 序列分析表明 ,克隆序列与设计相符 ,12 %SDS PAGE显示重组蛋白为5 0KD ,Western Blot证实其生物学活性。

Objective: To study the cloning and expression of G2 glycoprotein gene of Hantavirus in methylotropic yeast Pichia Pastois. Methods: G2 glycoprotein gene was amplified by RT-PCR and tranfected into the competent E.Coli DH5α, which was confirmed by DNA sequence analysis.The G2 was cut by SnaBI and Not I and then ligated into pPIC9K phagemid vector which was digested with the same restriction enzymes. The expression vector pPIC9K-G2 was constructed and introduced into Pichia Pastois GS115 by electroporation after linearized with Sal I. The transformants were screened by G418, fermented in flasks and induced by 1% methanol. Results: The sequence analysis indicated that the sequence of cloned G2 gene was consistent with the design. The recombinant protein was 50KD by 12%SDS-PAGE.The high specificity by Western-Blot assay was proved. Conclusion: The G2 glycoprotein gene is successfully expressed in Pichia Pastoris GS115 expression system.