The Expression Of Coagulation Factor Ⅷ In P. Pastoris

Human coagulation factorⅧ(hFⅧ) plays an essential role in the human cascade of internal coagulation. And the factor defect can cause hemophilia A. Currently, the protein replacement therapy what is inputting the preparations containing hFⅧare adopted in the clinical treatment of this disease. In the world, many people get this disease, about 1/10000-3/10000. So the only way of extracting hFⅧfrom the plasma can not meet the requirement of such a large numbers. Therefore, using genetic engineering method to produce hFⅧundoubtedly have great commercial value. Considering the strong expression system of P. pastoris, this research use P. pastoris GS115 as the host strain, and have studied the initial fermentation conditions after obtaining the secretory strains which produce the recombinant human coagulation factorⅧ(rhFⅧ).This paper select pPIC9 secretory expression vector to construct the strain, and successfully obtain the recombinant vector pPIC9-FⅧ, which is linearized by SalI and integrate into the chromosome of GS115 by electrotransformation in the next step. Then, with the following steps of screening the positive clones by genomic PCR, selecting the His~+ Mut~+ phenotype by MM and MD plates, cultivating the P. pastoris in the BMGY/BMMY nutrient medium in shake flasks and identificating culture supernatant by SDS-PAGE, the positive strains which can express rhFⅧare finally obtained. By the research of the expression pattern, it is found that, the expression level of this protein increases with the induction time and reaches a peak in 144h.After the research of the fermentation conditions by BMGY/BMMY nutrient medium in shake flasks, the result shows that the rhFⅧcan been detected in a low expression level in the induction conditions of 28℃, 250rpm and each 24h with 1% methanol added.These results indicate that this paper can successfully express hFⅧin P. pastoris, though the expression level is low. But it is likely to offer a theoretical basis and provide further insight into the study of hFⅧhighly expressed in P. pastoris in the future.