P-152: Expression of Recombinant Human Luteinizing Hormone (hLH) in CHO Cells

Background: Human luteinizing hormone (hLH) stimulates steroid biosynthesis of the ovary, triggers ovulation and prepares androgen production of testicular Leydig cells. LH belongs to the family of glycoprotein hormones that are heterodimers consisting of a common α-subunit and specific β-subunit. hLH is necessary for clinical and infertility treatment. Recombinant DNA technology provides a useful tool for hLH production with high specific activity and purity. The objective of the present study was gene cloning of hLH subunits simultaneously in the innovative multigenic plasmid for gaining high levels of expression. Materials and Methods: In this study, open reading frame (ORF) sequences of subunits were separately amplified by PCR and cloned into pTZ57R/T vector and then they were subcloned simultaneously into pVITRO2- neo-mcs expression vector. All mechanisms were confirmed by PCR, digestion reaction and sequencing. Recombinant pVITRO2-neo-mcs expression vector was linearized by using restriction enzyme, the products were transfered into Chinese hamster ovary (CHO) cells by electroporation and examined by PCR. Expression of recombinant hLH was confirmed by SDS-PAGE and Western blotting techniques. Results: The recombinant vector pVITRO2-neo-mcs was able to insert subunits with hFerH and hFerL composite promoters in CHO cells genome and both subunits were expressed. Conclusion: The carbohydrate moieties of glycoprotein hormones perform main roles in the biopotency, so mammalian cells could prepare appropriate position for accurate glycosylation of hLH. Cloning of both subunits in one expression vector is good idea to insert them at the same time and near the each other. These functions makes more probability to associate subunits after expression processing in mammalian cells.