Chromosome Location, Molecular Cloning, Prokaryotic Expression, Antiserum Preparation Of Pig Adiponectin And Study On The MRNA Expression In Vivo

Adiponectin is a cytokine produced exclusively in adipocyte. After released from fat cells, it circulates along with blood cycle and regulates energy metabolism in vivo . Globular domain of adiponectin which present in plasma has similar function to full length adiponectin. Though adipose-specific, expression level and plasma concentrations of adiponectin decrease when animal has exess adipose tissue. In addition, plasma concentrations of adiponectin is significantly lower in type 2 diabetes and some cardiovascular disease patients than healthy one. Syndrome of these disease will be improved if plasma concentrations of adiponectin elevated. It was approved that adiponectin can increase insulin sensitivity and has anti-inflammatory effect.In this study, we try to establish assays to understand expression profile of pig adiponectin in vivo. First, coding sequence and globular domain of pig adiponectin were amplified with RT-PCR, and recombinant expression vector pGEX-Adiponectin and pGEX-gAdiponectin were constructed. Recombinant vector were induced in BL21(DE3) and fusion protein adiponectin-GST and globular adiponectin-GST were produced. Antiserum were raised against the recombinant proteins after immunized them to New Zealand rabbits. Second, a semi-quantitative PCR method was employed to measure adiponectin gene expession level of pig in vivo. Semi-quantitative PCR should be carried out at exponential phase. Cycles between 25 and 29 were appropriate for both adiponectin and 3 -actin. Then Semi-quantitative PCR was performed with 27 cycles and the result was that adiponectin mRNAs in lean pigs were higher than obese one. Next we established competitive PCR assay for quantification of pig adiponectin mRNA. Coding sequence of pig adiponectin was digested with restriction endonuclease Aval, 267bp fragment of N terminus and 372bp fragment ofC terminus were purified and ligated. PCR was perfomed with the ligation product as template and competitive template was produced.After series dilution of the competitive template, PCR was performed using different concentration of them and pig adiponectin mRNA as template. The PCR product was analyzed and pig adiponectin mRNA was calculated. Finally, we located pig adiponectin on chromosome 13 using Radiation hybrid panel.