目的:构建去除凝血功能，但保留TF亲和力的rmFⅦ-pPIC9K表达载体并用毕赤酵母表达目的蛋白。方法：通过RT-PCR自小鼠肝脏获得FⅦcDNA，对其进行定点突变后连入pPIC9K质粒，电击转化Gs115酵母细胞，经G418筛选、BMGY/BMMY小量摇瓶培养表达目的蛋白，并进行初步的凝血、结合活性鉴定。结果： 成功构建了3种rmFⅦ-pPIC9K表达载体（M1：LCmFⅦ-pPIC9K；M2：K341AmFⅦ-pPIC9K；M3：QEAmFⅦ-pPIC9K），并通过酵母表达获得相应蛋白，经初步活性鉴定其中两种符合设计目的。

Objective：To construct the yeast expressive vector of rmFⅦ, in which mFⅦ was mutated to inhibit coagulation without affecting the affinity for TF, and express it in Pichia pastoris.Methods：The full length cDNA encoding mFⅦ was amplified from a mouse liver by RT-PCR method， site-direct mutated and restriction enzyme digested as design. Cloning into pPIC9K, electroporation of Gs115, in vivo screen of multiple inserts by G418 resistance, BMGY/BMMY are used for induction and expression of rmFⅦ in pichia pastoris. These proteins were also screened for functional activity.Results：Three different rmFⅦ-pPIC9K yeast expression vectors and it′s aim protein were obtained，two kinds of proteins were found to be functional active as design. Conclusion：rmFⅦ protein can be expressed in pichia pastoris and it might facilitate the development of tumor-target molecule, and novel anti-agiogenesis drug study.

重庆市院士基金（NO.6317)