Studies Of Transient And Stable Expression Of Goat Recombinant Follicle-Stimulating Hormone In Mammalian Cell Systems

Recombinant follicle-stimulating hormone has more advantages than the purified FSH, and would be beneficial to domestic reproduction. Expression of recombinant FSH in vitro might provide a chance to supply commercially. In this study, the eukaryotic expression vectors of goat FSHap were constructed and expressed in COS-7 cells or CHO cells on the basis of the clones of goat FSHa/p cDNA, which would have made preparations for recombinant FSH production.Construct eukaryotic expression vectors of gFSH a/p subunits and transiently express in COS-7 cells.The gene of FSHP was amplified with PCR as a template of a vector pGEM-T-FSHp, then expression vector pcDNAS.O was inserted by FSHP gene through double enzymatic digestions and DNA ligation. So the expression vector pcDNA-FSHp was constructed successfully. The total RNA were extracted from tissues of goat pituitary, and the FSHcc gene was acquied with RT-PCR, then the vector pcDNA-FSHa was constructed by the means of enzymatic digestions and DNA ligation. Both of constructed vectors had been sequenced and were transfected into COS-7 cells by PolyFect transfection reagent. The supernate of culture media and cell lysates were analyzed for recombinant goat FSH a/p subunits and heterodimer by radioimmunoassay. The results showed there were minimal dose of rgFSH, but efficiency of expression was lower. These results would give a hint that FSH a/P must be inserted into a same expression vector in order to obtain higher secretion.Construct dual expression vector of gFSHap and stably express in Chinese hamster ovary (CHO) cells.The process was as follows: specific primers of FSH a/P genes were designed thatcontained unique enzymatic sites and Kozak sequence, and genes of FSH a/P were amplified by PCR with the templates of pcDNA-FSHcc or pcDNA-FSHp, respectively; a/P genes were digested and ligated with dual expression vector pVITRO-2 using restriction endonucleases and DNA ligase. Consequently, FSH a/P genes were inserted into plasmid pVITRO-2. Enzymatic identification and gene sequencing indicated the dual expression vector pVITRO-FSHap was successfully constructed. pVITRO-FSHap were transfected into CHO cells by PolyFect transfection reagent, and stably expressing cells were selected by hygromycin B. The genomic DNA extracted from stable expression CHO cells were identified by PCR, and the result showed genes of gFSH a/P were integrated into genome of CHO cells. There was also secretion of recombinant goat FSH in culture media supernate using radioimmunoassay.In conclusion, we successfully constructed eukaryotic expression vectors of gFSH a/P ?pcDNA-FSHa/pcDNA-FSHp, and dual expression vector ?pVITRO-FSHap. Those vectors were expressed in COS-7 cells or CHO cells respectively. Moreover, CHO cells expressed goat FSH stably were acquired. These results would be the basis of production of recombinant FSH and study of long-acting hormone for the future.