Optimization Of Expression Of Recombinant Follicle-Stimulating Hormone Via Vesicles Transfection In Pichia Pastoris

Follicle-stimulating hormone(FSH) plays an important part in the process of gametogenesis,differentiation,maturation and the appearance of behaviors related with the reproduction. Recombinant follicle-stimulating hormone (rFSH) can avoid the deficiency of FSH prepared from pituitaries of domestic animals.Finding out vector suitable for expression of Recombinant Follicle-Stimulating Hormone transfection in Pichia Pastoris is important.Non-viral vectors are widely used for their advantages over viral vectors in recent years, because they are safe and cheap, easy to manufacture inlager scale, and they can also deliver large pieces of DNA. But current failure and frustration is the inadequacy of efficiency of cationic lipids. There are many parameters affecting efficiency of cationic lipid, kind of the cationic lipid and lipid to DNA ratio (or charge ratio of positive and negative) are two key factors governing efficiency among them. The synthesis of new cationic lipids and changing lipid to DNA ratio for DNA transfer has become an interesting field attracting more and more researchers.The yeast expression vector pPIC9K/EGFP-FSHβ-CTP-αcontaining long-acting objective gene was digested by Sal I. According to the vesicles'characteristics and preparation methods, we also use cationic surfactant as the main material for preparation of vesicles to induce the linear vector forming vesicles.In the present study, we complex linear vector pPIC9K/EGFP-FSHβ-CTP-αwith three diffeent cationic surfactant CTAB,DTAB,DEAB at different DNA/ cationic surfactant mixing ratios 1:0.008,1:0.3,1:1.4,1:4,1:7.2,1:18,1:40 and use them transfecting Pichia pastoris competent cell GS115.We also use electric shock method, commercialization transfection reagents Lipofectamine? 2000,Sofast as the control to compare the transfection efficiency and select the best cationic surfactant and DNA/ cationic surfactant mixing ratios.The results of the experiment showed that:1. Complex linear vector pPIC9K / FSHβ-CTP-αwith three diffeent cationic surfactant CTAB, DTAB, DEAB can form vesicle for transfering gene;2. The transfection efficiency of cationic surfactant is different if they have different alkyl chains and head groups.The transfection efficiency decrease with the increase of the alkyl chains. And for cationic surfactant with tha same alkyl chains and different head groups, the transfection efficiency of surfactant with methyls as head groups is higher than ethyls;3. The transfection efficiency is also influced by different DNA/ cation surfactant mixing ratio and the optimal ratio of three different cationic surfactant is all 1:7.2. Lower(1:0.008) or higer(1:40) DNA/ cationic surfactant mixing ratios induce lower gene expression level;4.We can synthesis new cationic surfactant system or changing DNA/ cationic surfactant mixing ratios for DNA transfer to upgrade the transfection efficiency compared with electric shock method,commercialization transfection reagents Lipofectamine? 2000 and Sofast.This study compare transfection efficiency by different cationic surfactant and at different DNA/ cationic surfactant mixing ratio, the results lay the the ground work for carryingout the further research on rFSH preparation and vesicle parceling in biological and medical applications.