| Follicle-stimulating hormone plays an important part in the process of gametogenesis,differentifation,maturation and the appearance of behaviors related with the reproduction. The Follicle-stimulating hormone preparations are extensively applied to the reproductive control and improve reproductive potency . In human assisted reproduction clinical Follicle-stimulating hormone are widely used in the infertility, It can induce the ovulation of single healty oocyte or the induction of mulitiple ovulation or superovulation. In practice recombinant follicle-stimulating hormone could avoid the deficiency of follicle-stimulating prepared from pituitaries of domestic animals,which takes the chance to the extensively and efficienly utilized.In this study, Pichia pastoris expression vectors were constructed based on the goat follicle-stimulating hormone and It's analogous genes cDNA sequence were found and steddly expressed in Pichia pastoris,which laid the foundation for hyperexpression of recombinant follicle-stimulating hormone.The specific priemers which have corresponding enzymatic sites and without its cognate peptide were designed based on the follicle-stimulating hormone and its analogous genes sequence and the fezture of vector.Using the pVITRO-FSHα,β,pVITRO-FSHβ-CTP-α, as the template.FSHα,FSHβ,FSHβ-CTP, FSHβ-CTP-αwhich have specific enzymatic sites were amplified by PCR.The amplifiled objective genes and pPIC9K,pPICZαA expression vectors were double digestide by EcoRI and NotI then the objective genes were cloned into pPIC9K,pPICZαA vector at the EcoRI and NotI sites to obstain the Yeast epression vector including the objective genes.After identification by PCR and DNA sequencing, extract Yeast expression vector largely.Linearized the purified Yeast expression vector by enzymetic digestion.The Yeast vector including FSHαwas cotransformed with the Yeast vectors including FSHβ,FSHβ-CTP respectively into the genome of electrocompetent cells,the vector including FSHβ-CTP-αwas transformanted into the genome of electrocompetent cells singlely.The positive Yeast transformants His+ and multiple inserts were screened by auxotrophic medium,antibiotic medium and PCR,Then the Mut+ phenotypes were screened by Minimal Dextrose Medium(MD) and Minimal Methanol Medium(MM). His+ Mut+ phenotype transformants were inoculated Buffered Glycerol-complex Medium(BMGY) in baffled flask to get enough Yeast cells.Using Buffered Methanlo-complex Medium(MM) including Methanol which concentration is 0.5% to induce the expression.Adding 100%Methanol to a final concentration of 0.5% Methanol every 24 hours to maintain induction and transfer 1mLof the expression cultue to a 1.5mLmicrocentrifuge tube,which were used to do the SDS-Polyacrylamide Gel Electrophoresis and Wester blot analysis.The level of FSH were determined by Radio-immunoassy(RIA).The result show that the molecular of the objective proteins as follows: FSHαβ27KD,FSHαβ-CTP 28KD,FSHβ-CTP-α29KD,whichare consistent with the anticipative results ,but FSHβis 22KD,the glycosylation of it is high.The expression level of Multiple inserts secreted by high concentrstion G418 are marked increase than the transformants screened by low concentration G418.The expression level of follicle-stimulating hormone analogous genes are higher than the follicle-stimulating hormone gene significantly ,which is show that the CTP structure could increase the expression of objective genes. In conclusion,Pichiapastoris expression vector including follicle-stimulating hormone and its long-acting analogous genes have been successfully constructed in our lab and the expression in GS115 were induced successfully.This study lay the the ground work for carrying out the research on follicle-stimulating hormone and its long-acting analogous genes structure and application. |
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