Cloning And Prokaryotic Expression Of Gnrh And Transporter Gene Sequence

Gonadotropin-releasing hormone (GnRH) are the key regulators of reproductive function in all vertebrate organisms. The GnRH molecule is synthesized in a small number of neurons in rostral hypothalamic regions of the brain. In mammals, these neurons release the GnRH decapeptide into the portal capillary system leading to the anterior pituitary gland. There, GnRH acts on acceptor of GnRH cell mambrane and causes the release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn act upon the gonads to stimulate their maturation, and to cause synthesis of sex steroid hormones, estrogen, progesterone and testosterone.GnRH can be use in Endometriosis, induction of ovulation and ART, uterine fibroids and benign gynecology, contraception (male and female), prostatic cancer, breast cancer, ovarian cancer, other malignancies, pediatric endocrinology.A pair of primers were designed according to the Published sequence of GnRH2'S gene mRNA and transporter gene with Oligo version 4.1 softwarre, they were annealed by their 3'terminal brief complementary base sequence to form small segment double chain, therery ,they serve mutually as template and primer ,and their extension were carried out to synthesize 90 bp' GnRH/TRS. GnRH/TRS gene was cloned into the pMD18-T vector, the recombinant pYCl plasmid was identified and analyzed by corresponding restriction endonuclease and nucleotid sequence,lt is indicated that the GnRH/TRS gene has been cloned successfully, then this gene was cloned into the expression vector pGEX-6p-l, The recombinant pYC2 plasmid was identified and analyzed by corresponding restriction endonuclease,It is indicated that the GnRH/TRS gene has been cloned successfully.The recombinant plasmid pYC2 was transformed into the BL21(ED3) plysS prokaryotic expression system. The expression product was induced with IPTG. SDS-PAGE analysis showed an induced expression product band about 28ku, which correspond to the sizes of GST-GnRH/TRS, reported in the literature. The amount of the goal protein was evaluated by densitometric scanning. It indicated that the product of the GST-GnRH/TRS gene was 33% of total bacterial protein of BL21(ED3). Then the product was purified by Glutathione Sepharose 4B chelation affinity chromatography, and upper purified GST-GnRH/TRS fusion proteins was received for further the foundations established in the scientific research and the real application.