Regulating Effect Of Recombinant Adiponectin On Glyconeogenesis In Hepatocytes Of Newborn Calf

In this experiment, adiponectin(ADPN) gene was cloned and then expressed successfully in Pichia Pastoris by genetic engineering principle,modern cytobiology and molecular biology technique.The biological activity of recombinant ADPN was detected. Regulating effect of the recombinant ADPN on the expression level of PC mRNA, PECPK mRNA and their enzyme activity in vitro bovine hepatocytes culture was evaluated by Real-time fluorescent quantitive RT-PCR and chromatometry.Total RNA was extracted from bovine hepatocytes and then adiponectin gene was amplified by RT-PCR.The product of PCR was cloned into pMD18-T vector.The positive recombinant plasmid was identified by double enzyme digestion and PCR.And then the sequence was analyzed by DNAMAN.The results showed that the gene fragment amplified was 669 bp and shard 100% of the homology with bovine adiponectin encoding sequence from GenBank. It is confirmed that the gene fragment obtained was a bovine adiponectin gene.A new pair of primers containing the required digestion sites as Pichia Pastoris expression system was designed and synthesized. 669bp of bovine adiponectin gene without signal peptide was amplified.The obtained gene was ligated with an expression vector pPICZαA to construct the expression plasmid pPICZαA-BovADPN.The recombinant plasmids were transformed into strain E.coli DH5α.The expression vector pPICZαA-BovADPN in yeast was constructed. After linealization of the plasmids, adiponectin gene was integrated into the genome of host yeast GS115 by electroporation. Adiponectin was induced to express by ethanol and was identified by SDS-PAGE, Dot-blot and Western blot. After the optimization of expression conditions the recombinant bovine adiponectin was expressed successfully in Pichia pastoris with the yield of 31.5 mg per liter culture.The biological activities of recombinant adiponectin expressed in Pichia pastoris were determined by in vivo experiment in mice. The result showed that recombinant adiponectin could decrease the concentration of plasma glucose and triglyceride in model of diabetes mice significantly.It showed that biological activities of recombinant adiponectin were satisfactory.According to the publishing sequence in GenBank,primers were designed and RT-PCR was employed to amplify the partial cDNA of PC and PECPK.The target sequences were recombinated into cloning vector,respectively.Homology of the target sequences were all 99%.The method of Real-time fluorescent quantitive RT-PCR was established successfully to detect the expression level of PC mRNA and PECPK mRNA.The hepatocytes of newborn calf were digested using a method of improved collagenase digestion. Cells were seeded (1×106) onto custodite cell culture dishes with polylysine. After attachment, fluorescent quantitative RT-PCR was employed to determine the expressing level of PEPCK mRNA and PC mRNA in primary hepatocytes which were treated with different concentrations of recombinant ADPN(0μg/mL,10μg/mL,20μg/mL,30μg/mL and 40μg/mL)for 24 hours.At same time, the activity of PEPCK and PC was determined. The results showed that the expression level of PEPCK mRNA in hepatocytes was gradually decreased with increasing concentration of recombinant ADPN in cell culture media.It showed a negative correlation between the expression level of PEPCK mRNA and concentration of recombinant ADPN. It showed a significant difference with control hepatocytes(P<0.05). The result showed that adiponectin has no significant effect on PC mRNA expression. The activity of PEPCK was gradually decreased with increasing concentration of recombinant ADPN. Comparing with the control group,enzymatic activity of PEPCK significantly decreased even in the low concentration of adiponectin.The more adiponectin concentration increased,the more enzymatic activity of PEPCK decreased(P < 0.05). It showed a significant dose-dependent between activity of PEPCK and concentration of recombinant ADPN.The enzymatic activity of PC had no significant decrease after being treated with adiponectin of different concentrations comparing with the control group.The results of animal experiment showed that enzymatic activity and mRNA expression level of PEPCK was decreased significantly comparing with the control group.It showed that adiponectin has no significant effect on PC mRNA expression,enzymatic activity.The level of plasma glucose decreased comparing with the control group. It is conclude that ADPN could decrease glyconeogenesis through inhibiting PEPCK mRNA abundance and its enzume activity in hepatocytes.