重组小鼠凝血因子Ⅶ毕赤酵母表达载体的构建及整合体的筛选

Construction of recombinant mouse coagulation factor Ⅶ expression vector and screening of integrants in Pichia pastoris

目的构建无凝血活性的重组小鼠凝血因子Ⅶ(rmFⅦ)毕赤酵母分泌型表达载体,获得高拷贝稳定整合菌株,为大量获得rmFⅦ蛋白,进行功能研究及其临床应用奠定基础。方法PCR扩增得到mFⅦ基因片段,插入酵母表达载体pPIC9K的α分泌信号开放阅读框的下游,再经定点突变(K341A)得到pPIC9KrmFⅦ,测序正确的质粒用SacⅠ线性化后电穿孔转化毕赤酵母GS115,G418梯度筛选转化菌,PCR鉴定目的基因的整合。结果获得了rmFⅦ毕赤酵母表达载体pPIC9KrmFⅦ,G418抗性筛选得到高拷贝菌株,PCR证实目的基因已整合到毕赤酵母染色体中。结论成功构建了rmFⅦ毕赤酵母表达载体,获得了高拷贝稳定整合菌株,为大规模表达纯化rmFⅦ蛋白及后续研究奠定了基础。

Objective To construct secretory recombinant mouse coagulation factor Ⅶ （rmFⅦ） expression vector and to obtain stable multicopy integrants in Pichia pastoris. Methods The fragment of mFⅦ cDNA was amplified from a PCDNA3-mFⅦ plasmid and subcloned into α factor secretion signal open reading frame of pPIC9K secretory expression vector, then the target amino acid was mutated by Site-Direct Mutation （K341A） and verified by DNA sequence. The recombinant expression vector bearing rmF Ⅶ gene was linearized with Sac Ⅰ and transformed into Pichia pastoris strain GS115, and stable multicopy integrants were obtained by screening in medium containing different concentrations of G418. Results The secretory rmFⅦ expression vector was successfully constructed and stable multicopy integrated strains were obtained. Conclusion This result offers efficient Pichia pastoris strains for mass production of rmFⅦ protein.