Exendin-4真核表达载体的构建及其在毕赤酵母中的表达

目的:构建Exendin-4基因真核表达载体,并在巴斯德毕赤酵母GS115中进行表达,为大量获得Exendin-4奠定基础。方法:采用重叠聚合酶链反应法扩增出Exendin-4的完整序列,将其亚克隆到表达载体pPIC9K上,得到重组质粒pPIC9K-Exendin-4。重组质粒经SacI线性化后用电穿孔法导入到GS115中,经组氨酸缺陷筛选、G418高拷贝筛选及摇瓶表达筛选,甲醇诱导表达后用凝胶电泳法分析Exendin-4的表达。结果:序列测定结果表明成功地构建了毕赤酵母表达载体,电泳结果证明Exendin-4在毕赤酵母中能高效表达。结论:实现了Exendin-4单体在毕赤酵母中的表达,为Exendin-4的规模化生产奠定了基础。

Construction of Exendin - 4 Eukaryotic Expression Vector and Its Expression in P. Pastoris

OBJECTIVE： To construct eukaryotic expression vector of Exendin - 4, and study its expression in P. Pastoris strain GSl15 so as to lay foundation for gaining large quantity of Exendin- 4. METHODS： The whole sequence of Exendin- 4 gene was amplified by overlapping PCR.Then the gene was subcloned into the pPICgK expression vector to obtain recombinant plasmid pPIC9K - Exendin - 4, which was transformed into GSl15 by electroporation after Sac I linearization. Through histidine auxotroph screening, G418 high- copy screening, flask- shaking expression screening, and methanol- induced ex- pression, the expression of Exendin- 4 was analyzed by gel electrophoresis（GE） . RESULTS： The sequencing revealed the successful construction of the expression vector of P.pastoris and the GE results confirmed high expression of Exendin- 4 in P.pastoris. CONCLUSION： The expression of Exendin- 4 monomer in P.pastoris has been achieved and the results laid a foundation for large scale production of Exendin- 4.