The Primary Application Research On Recombinant Ovine FSH Expressed In The CHO Cell

FSH is a glycoprotein gonadotropin synthesized and secreted by basophils in anterior pituitary.It is formed by non-covalent bonding of alpha and beta subunits.It is often used to promote rapid reproduction and induce superovulation in female animals.Veterinary FSH is mostly extracted from pituitary gland.Human FSH is usually extracted from urine of menopausal women,but the production cost is still high,and the purity can not reach 99%.If purity is further improved,the cost will only be more expensive.In this study,recombinant DNA technology was used to secrete and express recombinant sheep FSH.Firstly,the recombinant FSH sequence of sheep was designed and the recombinant expression vector pcDNA3.1-FSH-?-? was constructed.The constructed recombinant expression vector pcDNA3.1-FSH-?-? was electrotransfected into CHO cells.After transfection,monoclonal cells(M-FSH-??-B31A4)were screened by G418.The content of recombinant sheep FSH expressed by monoclonal cells reached 350 ng/mL.Monoclonal cells were cultured in a 5 L bioreactor for 68 hours to collect cell supernatant.Then the supernatant was concentrated by ultrafiltration with 10 kD membrane,and the recombinant sheep FSH-?-? was obtained at 531 ng/mL.After the preparation of recombinant FSH-?-? was completed,the experiment was carried out by subcutaneous injection of female mice for several times.After one injection,the rats were injected again one day apart.After 72 hours,eyeball blood was collected from female mice.Serum LH and Pg were detected by ELISA.The content of them was significantly increased.The ovaries of female rats were dissected after death,and the size of ovaries was observed and weighed.The weight of ovaries increased significantly.In conclusion,the recombinant expression vector pcNDA3.1-FSH-?-? was successfully constructed and synthesized.The expression vector was successfully transfected into CHO cells,and monoclonal cells were obtained after screening.The recombinant protein expressed by monoclonal cells was concentrated by ultrafiltration and a higher concentration of recombinant sheep FSH was obtained.It was found that the recombinant sheep FSH had certain biological activity in mice,promoted ovarian enlargement and increased reproductive factors significantly,which laid a foundation for further industrial application.