Researching On The Human Leptin By Transgenic Chicken Bioreactors

The leptin is the product of obese gene which can inhibit the food intake, regulat the fat metabolism and the energy deposition function. In addition to the role of Leptin and Obesity-related things, there are a variety of physiological processes regulating role, such as heat the role of regulation of the immune system, action before puberty, and reproductive functions. The recombinant human leptin also has some effect for people who was lack of leptin or fat atrophy of individuals with diabetes.In order to set up the chicken bioreactor system in vivo and produce natural medicinal protein production,we recombinant the human leptin protein gene-specific vector of fallopian tube and let it express specifily in eggs .1.The total RNA was extracted from human white adipose tissue.we design specific primers according to a published literature, and amplified human leptin protein gene OB with RT-PCR, and cloned to the pMD19-T vector for sequence analysis. Results: Compared with the GenBank released by human leptin gene (NM000230) the homology is 100%, indicating that we got the right sequence.2.we successfully Construct the recombinant expression plasmid PGEX-OB with the OB gene which was got from the pmd-OB. After double-digestion, PCR identification and analisis of the DNA sequence, we transformed E.coli BL21 to construct strain BL21-PGEX-OB. human leptin fusion protein was highly expressed in the form of inclusion bodies in 37℃through induction by IPTG.SDS-PAGE analysis revealed that the BL21-PGEX-OB expressed the correct fusion protein of recombinant human leptin and the expression of fusion protein molecules is 20 KD which is as similar as the theoretical value size. The fusion protein was highest expressed when induced with IPTG by 6h, and in the concentration of inducer IPTG in 1mmol/L. Genetool analysis revealed that the greatest expression of the target protein of total cell protein amounted to 30%.3.we Constructed the recombinant human leptin eukaryotic expression vector pcDNA-OB which was successfully expressed in 3T3 cells and verified the biological activity of human leptin protein through weight reducing experiments in mice. RT-PCR results showed that transfection cells with the recombinant plasmid successfully expressed human leptin protein; Injection of recombinant plasmid pcDNA-OB mice decreased body weight which the statistical analysis showed significant differences (P <0.01)which showed that recombinant human leptin protein has biological activity. 4.The ob gene which was amplified followed with pmd-ob through PCR was correctely cloned to the tubal specific expression vector POV3,and let it express in hens in temporary. Western Blot test results showed that that recombinant human leptin protein was successfully expressed in the eggs .To sum up, in this experiment the cloned human leptin protein gene can be a successful in vitro expression, and weight reduce in mice verified its biological activity and we successfully let it express in hens. it laid the foundation of further study of biological activity of leptin, as well as a number of diseases.