Study Of Cloning Of Ghrelin Gene, Prokaryotic Expression And Preparation Of Polyclonal Antibodyo Of Ghrelin Protein In Swine

Ghrelin, composed of 28 amimo acids, is the first endogenous ligand of GH secretogogue receptor. It was reported first by kojima from the stomach endocrine cells and nucleus arcuatus hypothalami from people and mouse. It was named of Ghrelin for the function of stimulating the secretation of adenohypophysis GH. Ghrelin is the second endogenous material for regulating the secretation of GH besides growth hormone releasing hormone (GHRH) and somatostatin (SS). Ghrelin is synthesized mainly in stomach and intestine, and also a little synthesis in hypophysis, lower thalamus, placenta, kidney and pancre et al. Ghrelin can not only regulate the secretation of GH, but also stimulate appetite, regulate energy balance and stimulate gastric acid secretation.According to the sequence of preproghrelin mRNA, a pair of primer of the expression system of prokaryotic was designed. Ghrelin gene was amplified by RT PCR and the PCR fragmens were cloned into the pMD 18-T Vector, subcloned into the expression vector pET28a(+), constrcted the expression system of prokaryotic respectively. The polypeptides of the prokaryotic expression were obtained. Purified inclusion proteins and incomplete Freund adjuvant and aluminum adjuvant are mixtured and grinded, then are injected to New Zealand albino rabbit. The rabbit-anti-swine ghrelin immune serum is obtained. And the proteins are proved to be the polyclonal antibodies of swine by Western-blot. The research is base for the further step of Ghrelin research.