Objective To identify the sequence of cDNA of human leptin coding region, construct a prokaryotic expression vector, and express human leptin in E.coli. Methods RNA was extracted from human placenta tissue. Leptin gene was amplified from RNA by RT-PCR method. The PCR product was ligated with T vector. The ligation reaction was transformed to E.coli DH5? competent cells. The recombinant plasmid was checked by sequencing and restriction analysis. The human leptin gene was cloned into prokaryotic expression vector PET-28a and tranformed into E.coli BL21. The recombinant strain was constructed and induced by IPTG. The product was analyzed with SDS-PAGE and Western blotting. The expressive product was purified by sodium deoxycholate. Results Analysis indicated that the sequence of human leptin cDNA was the same with the reported sequence. Leptin gene was inserted into prokaryotic expression vector. The fusion protein expressed with high efficiency in recombinant E.coli BL21.The results of SDS-PAGE analysis indicated that the molecular weight of the fusion protein was about 20kD. Conclusion Hunam leptin gene was successfully identified from placenta. The leptin gene prokaryotic expression strain was constructed and a high expression of leptin was achieved in E.coli.

Objective To establish a rat model of hyperlipidemia and analyze the effect of conjugated linoleic acid (CLA) on lipid metabolism and expression of visfatin gene. Method High fat diet was used to establish the hyperlipidemia rat model. Blood was taken at four weeks after high fat diet feeding to analyze the level of glucose (GLU), cholesterol (CHO) and triglyeride(TG). The rats were divided into experimental group and control group after the hyperlipidemia rat model was established successfully. The experimental group rats were treated with CLA(0.8 mL /0.1 kg)orally for four weeks. The food intake and body weight were recorded. The rats were sacrificed, and both body fat and serum lipid levels were measured. Semi-quantitative RT-PCR was used to measure the expression level of visfatin mRNA. Result The hyperlipidemic rat models were induced by high fat diet successfully. The body weight, food intake and body fat in the rats of CLA experiment group were significantly decreased compared with that of the control group (P< 0.05). The level of GLU, CHO, TG and LDL in the experimental group were significantly lower than that in the control group (P< 0.05), but the serum HDL-C was increased in the experimental group. Semi-quantitative RT-PCR demonstrated that the expression level of visfatin gene of the experimental group was lower than that in the control group. Conclusion CLA can reduce the expression of visfatin gene and improve the lipid metabolism in hyperlipidemic rats.