Prokaryotic Expression And Activity Analysis Of Chicken Visfatin

Visfatin was identified as a novel adipocytokine that appeared to mimic the action of insulin, which was preferentially produced by human abdominal visceral fat. Recently it has been received much attention due to its potential roles in obesity, dyslipidemia, type II diabetes mellitus and immune disorders. Visfatin has been intensively studied in mammalian species, while few researches have been carried out in avian species.In this study, amplified from the liver of an 6-week-old AA broiler chicken, Visfatin cDNA was used as a template when the clone plasmid of chicken Visfatin was constructed. To create an in-frame fusion protein with a N-terminal 6×His-tag, the Visfatin DNA fragment was digested with Nco I and Xho I restriction enzymes and ligated into pET30a (Novagen, Germany) vector which predigested with the same enzymes. After that the recombinant plasmid pET30a-Visfatin was transformed into E.coli BL21(DE3) (TIANGEN) competent cells for expression and its authenticity was confirmed by sequencing. A soluble expression of recombinant Visfatin protein was obtained in E. coli BL21(DE3) after optimizing induction time, temperature, isopropyl-β-D-thiogalactopyranoside(IPTG) concentration and the pH of medium. SDS-PAGE was performed to visualize protein expression, and the recombinant protein was purified by His TrapTM HP of American GE according to the manufacturer’s instructions. The results showed that the soluble expression of the recombinant Visfatin protein was obtained after the 12-hour induction under 30℃, which was in log phase (OD600= 0.6-1.0) and the final concentration of IPTG was 0.4 mmol/L. Different concentrations of imidazole (50 mmol/L and 150 mmol/L) were used in the binding, washing and elution buffers, respectively. The results of western blotting showed that the recombinant Visfatin protein could react specifically with 6×His·His-Tag Monoclonal Antibody according to manufacturer’s recommendations.To validate the biological role of fusion Visfatin protein further, we investigated the effect of fusion Visfatin protein on the differention of 3T3-L1 adipocytes. The results showed that the fusion Visfatin protein significantly increased and lowered the expression of adipocyte differential marker genes (PPARγ,aP2, C/EBPa, FAS) when compared with the control and insulin groups, respectively. And thereby fusion Visfatin protein promoted the differentiation of preadipocytes into mature adipocytes partially. This essay also examined the effect of fusion Visfatin protein on the uptake of glucose in 3T3-L1 adipocytes, the results showed that 10 nmol/L fusion Visfatin protein could significantly promote and lower the glucose uptake of 3T3-L1 adipocytes compared with the control and insulin groups, respectively. This function of 5 nmol/L fusion Visfatin protein was significantly higher than these treatment groups of 10 nmol/L,25 nmol/L,50 nmol/L,100 nmol/L.The mimic-insulin role of Visfatin was confirmed by these two trials ultimately.