Tandem Expression Of GnRH Analogue In Escherichia Coli

Background:Gonadotropin-releasing hormone(Gn RH) is a decapeptide hormone synthesized and secreted by the hypothalamic neurons. It gets into the lobus anterior of hypophysis by the hypothalamus hypophyseoportal system, acts on the gonadotropic hormone secretingcells in the lobus anterior hypophysis to secrete Luteinizing hormone(LH) and Follicle-stimulating hormone(FSH), and therefore regulates the gonadogenesis and gametogenesis.Gn RH analogue is synthetic derivatives of Gn RH, which has a biopolar effect on pituitary. Gn RH analogue can compete receptor with Gn RH.Small doses and short-term applicantion of Gn RH can stimulate the scretion of Follicle-stimulating hormone(FSH) and Luteinizing hormone(LH),while large doses and long-term application of Gn RH analogue, the body will produce inhibition effect of gonadal function. Because of the regulation to hypophyseal hormone, The research and application field of Gn RH analogue mainly includes the reproduction of animals in agriculture and estrogen-dependent disease now, such as endometriosis, breast cancer, prostate cancer, myoma of uterus, endometrial cancer, polytrichosis and ovulation induction. Due to the requirement of Gn RH analogue in agriculture and Clinical, the price of Gn RH analogue has been always high. Seeking for a new method of the acquirement of Gn RH analogue is therefore extremely important. Objective:Due to the high costs of chemical synthesis,we use genetic engineering techniques to express the multiple-copies gene of Gn RH analogue in E. coli. Our work is helpful to further study of the acquirement of Gn RH analogue.Methods:(1) Compare the structure of Gn RH gene. Design and synthesis the reasonable multi-copies gene.(2) Contruct recombinant prokaryotic expression vector p GEX-2T-8Gn RH analogue, andtransfer the recombinant vector into the E.coli BL21, the recombinant p GEX-2T-8Gn RH analogue vector was identified by PCR ?restriction enzyme digestion and gene sequencing.(3) Contruct recombinant prokaryotic expression vector p GEX-2T-16 Gn RH analogue, and transfer the recombinant vector into the E.coli BL21, the recombinant p GEX-2T-16 Gn RH analogue vector was identified by PCR ?restriction enzyme digestion and gene sequencing.(4) The identified recombinant p GEX-2T-8Gn RH analogue?p GEX-2T-16 Gn RH analogue vectorwas transformed into E.coli BL21.(5) The Multiple-copies Gn RH analogue gene was highly expressed after optimizing the IPTG induced conditions.The Multiple-copies Gn RH analogue was pufired from E.coli BL21 which highly expressed Gn RH analogue using Glutathione Sepharose 4B.Results:(1) Choose the amino acid EHWSYGLRPGKR(Gn RH analogue) as the multi-copies in the expression of gene engineering after contrast. This structure is propitious to purification and Enzyme digestion.(2) Successfully constructed the recombinant prokaryotic expression vector p GEX-2T-8Gn RH analogue and p GEX-2T-16 Gn RH analogue.(3) E.coli BL21 expressed the Multiple-copies Gn RH analogue was inducted by IPTG.The SDS-PAGE electrophoresis results and the Western-Blotting results revealed that the fusion protein GST-Gn RH analogue was successfully expressed.(4) The fusion protein was successfully purified by Glutathione Sepharose 4B.Conclusion: Recombinant plasmid were successfully constructed and highly expressed in E.coli BL21, The fusion protein was successfully purified by Glutathione Sepharose 4B, laiding a foundation for further studying Gn RH analogue using biological synthesis production.