Expression And Functional Assay Of Recombination Human Follicle Stimulating Hormone

Human follicle-stimulating hormone(hFSH)is a kind of glycoprotein gonadotropin secreted by anterior pituitary basophils.hFSH plays an important role in the diagnosis and treatment of infertility.Currently,urinary follicle stimulating hormone(uFSH)and imported recombinant human follicle stimulating hormone(rhFSH)are the mainly products available on Chinese market,but the domestic rhFSH has not been found on market so far.The biological activity rhFSH produced by gene engineering is similar to the natural FSH,and rhFSH possesses features as high purity,uniform and safe source.In theory the high purity rhFSH can be prepared at large scale.Therefore it is valuable to prepare rhFSH by gene engineering and assay its biological function.In this study,human FSH? and FSH? subunit gene was amplified in polymerase chain reaction by designing specific primers.And the correctness of the gene is proven by the gene sequencing.Then the FSH? subunit gene was connected with the pIRES-Hyg3 plasmid to build the pIRES-Hyg3-FSH? expression vector.The FSH? subunit gene was inserted into the pIRES-Puro3 and pIRES-Neo plasmid to build the pIRES-Puro3-FSH? and p IRES-Neo-FSH? expression vector.The expression vectors which contain target gene were extracted from transformed E.coli and then verified by agarose gel electrophoresis and gene sequencing.The expression vectors with FSH? gene and FSH? gene were transfected into CHO and HEK293 cells.The positive clone cells lines were screened out by selective medium containing hygromycin B,puromycin and G418.Target protein rhFSH secreted by positive clone cells was identified by Western Blotting.At the same time,two ELISA methods were established to detect the expression of rhFSH and screened out the high expression rhFSH cell lines.In this project,an experimental model of the follicle stimulating hormone receptor mediated rhFSH into cell membrane which exhibited a phenomenon of fluorescent localized on cell surfaces internalized into cell membrane was established to verify biological activity of rhFSH.Moreover,the bioactivity of purified rh FSH was verified by miceovulationstimulation,which indicated that the ovulation number of mice in experimental group was 4 to 10 times compare with the control group.In this study,we successfully constructed p IRES-Hyg3-FSH?,pIRES-Puro3-FSH? and p IRES-Neo-FSH? eukaryotic expression vectors and obtained stable expression cell line.The positive biological activity of high purity rhFSH was verified by animal experiment in vivo and cell experiment in vitro.