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Construction of Recombinant Mice Factor Ⅶ-pPIC9K Vector and Expression in Pichia
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Abstract:
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.
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