Studies Of Expression Of Recombinant Goat Long-acting FSH Agonist In Chinese Hamster Ovary Cells

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone. It is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is offen used in embryo transfer (ET) and superovulatory. Because half-life of FSH is very short in circulation, so it need repeated injection in therapy. To address this point, this study construct chimeric genes containing the sequence encoding the C-terminal peptide (CTP) fused to the translated sequence of the goat FSH β-subunit. CTP is unique COOH-terminal region of the β subunit of human choriogonadotropin. It has 28-residue peptides from positions 118 through 145. There are four carbohydrates via O-linkage to serine residues in this peptide backbone. Sialic acid of carbohydrates can prolong the half-life of FSH in circulation.Designing specific primers containing Kozak sequence, FSH β and hCG β as templates, amplify the chimeric gene FSH β-CTP through two PCRs. Digest and ligate FSH a, FSH β-CTP with dual espression vector pVOTRO using restriction endonucleases and DNA ligase. Consequently, FSH a and FSH ft genes were inserted into plasmid pVITRO. Gene sequencing indicated the expression vector pVITRO FSH αβ-CTP was successfully constructed. pVITRO FSHαβ-CTP were transfected into CHO cells by Lipofectin transfection reagent and stable expressing cells were selected by hygromycin. The genomic DNA extracted from stable expression CHO cells were identifiedby PCR and gene sequence. The results showed that genes of FSHαβ-CTP were integrated into genome of CHO cells. Using the same method, the chimeric genes FSHβ-CTP-a were constructed by fusing FSHα gene into FSHβ-CTP gene. FSHβ-CTP-α were inserted into plasmid pVITRO. The stable expressing cells were acquired by transfecting pVITRO FSHβ-CTP-a into CHO cells.The cells culture media supernate were detected by radioimmunoassay. Expression of FSHαβ-CTP is about 0.105mIU/ml. Expression of FSHP-CTP-a is about 0.120mIU/ml. It indicated that the presence of CTP enhances secretion.In conclusion, the results gained indicated that the long-acting FSH agonist genes of FSHP-CTP /FSHp-CTP-a were successfully constructed.Two genes were expressed in CHO cells respectively. This study lay the groundwork for the application of long-acting FSH agonists.