Study On Expression Of Human Follicle Stimulating Hormone Factor In Goat Mammary Epithelial Cells By Adenovirus

Follicle stimulating hormone(FSH)is one of the most important sex hormones in animals.It mainly acts on germ cells and plays a key role in the development,maturation and reproductive physiology of individuals.The FSH on the market is mainly divided into urinary FSH and FSH prepared by cell engineering.The former has low yield and uneven source.The latter has high production cost and high price,which cannot meet market demand.At present,animal mammary gland protein preparation drugs have the characteristics of high yield and low price.However,studies on the preparation of FSH using large animal mammary glands have not been reported,mainly because excessive FSH in animals can cause normal physiological disorders of animals,eventually leading to Animal death.FSH is a heterodimeric glycoprotein formed by non-covalent bonding of FSHαand FSHβsubunit protein.The FSHαand FSHβsubunits alone do not have biological activity in animals.Therefore,in this study,human FSHαand FSHβsubunit proteins were separately expressed in goat mammary epithelial cells(GMEC)by adenovirus,and then FSHαand FSHβsubunit proteins were recombined and purified in vitro to obtain intact recombinant human follicle stimulating hormone(rhFSH).The in vitro activity test was used to verify that rhFSH is biologically active.This study explored the expression of human FSH protein in goat mammary gland cells in vitro.This study would provide a technical basis for the preparation of recombinant human follicle stimulating hormone by using large animal mammary glands.The results showed that:1.The FSHαand FSHβsubunit genes were subcloned using PCR technology.The pAdTrack-CMV-FSHαand pAdTrack-CMV-FSHβadenovirus shuttle vectors were constructed by one-step cloning by ligating the gene of interest to the shuttle vector pAdTrack-CMV,respectively.The shuttle vector was identified and linearized by PmeI digestion,and homologously recombined with the backbone vector pAdEasy-1 in E.coli BJ5183 to obtain pAdEasy-FSHαand pAdEasy-FSHβrecombinant adenoviral vectors.2.The correct recombinant adenoviral vectors pAdEasy-FSHαand pAdEasy-FSHβwere linearized with PacI,purified by isopropanol precipitation,and then transfected into HEK293A cells by Lipofectamine 2000 to obtain the first generation recombinant adenovirus.After expansion,a high concentration of recombinant adenovirus was obtained.The concentration of Ad-FSHαand Ad-FSHβrecombinant adenovirus was 1×10~9 pfu/mL by LaSRT method.3.GMEC cells were infected with recombinant adenoviruses Ad-FSHαand Ad-FSHβ,respectively.The multiplicity of infection of the recombinant adenovirus Ad-FSHαwas determined to be 160,and the MOI value of the recombinant adenovirus Ad-FSHβwas200.The cells were lysed to obtain protein,and Western Blot assay showed that FSHαand FSHβsubunit proteins could be efficiently expressed in GMEC cells by recombinant adenovirus.4.The FSHαand FSHβsubunit proteins expressed by GMEC cells were recombined and purified in vitro,and the FSHαand FSHβsubunit proteins were successfully reconstituted into rhFSH in vitro by double-antibody sandwich ELISA and Western Blot.FSH targeting follicle stimulating hormone receptor(FSHR)binding assay and determination of cAMP value in HKE-293-FSHR cells showed that in vitro recombinant rhFSH and positive control drug Gonal-F also have in vitro biological activity.In summary,this study used adenovirus-mediated human follicle stimulating hormone gene FSHαand FSHβto express in goat mammary epithelial cells,and expressed FSHαand FSHβsubunit proteins,which were recombined and purified in vitro to obtain rhFSH.The rhFSH prepared in this study had biological activity.This study would lay the foundation for the next step of using large animal mammary glands and preparing transgenic animals to produce recombinant human follicle stimulating hormone.