Research On Eukaryotic Expression Of Bovine Resistin And Determination Of Its Biological Activities

Resistin is kind of novel small-molecule protein that is secreted by white adipose cells. It is the link that connects obesity to type 2 diabetes mellitus, and it can weak the sensitivity of fat cells, muscle cells, liver cells to insulin. The search that resistin induced insulin resistance mainly in mouse and human at home and abroad, and the mechanism is no consistent conclusion. Search of the relationship between resistin and insulin resistance in cattle is much little. Resistance gene prokaryotic expression in cattle and ketone cows resistin gene eukaryotic expression has been reported until now, but whether the product of resistin eukaryotic expression has activity or not, has not been reported.This study which provides a scientific basis to the treatment of Animal glucose metabolism disorders is on bovine resistin gene cloning, eukaryotic expression and biological activity of expression products.Extracting total RNA from abdominal adipose in cattle which is age of 18 months, and then reverse transcription and PCR amplification. Gel electrophoresis and ligated T vector with PCR and restriction enzyme digestion to identify,then take it to Bioengineering Co., Ltd. Shanghai for sequencing. We obtained a bovine resistin gene cDNA coding sequence that size of 343bp through RT-PCR amplification. Cloning the bovine resistin gene to pMD18-T vector successfully, and then constructs the recombinant Plasmid pMD-Res. The size of obtained target gene is 343bp, and the homology is 99% comparing to bovine resistance gene of Genbank. It indicates that the gene that we obtained is bovine resistin coding gene.Designing eukaryotic primers, conducting PCR amplification, connecting T vector, conducting double digestion,and then ligated eukaryotic expression vector pPICZaA. Sequencing after PCR and restriction enzyme digestion. Linearizing the eukaryotic expression plasmid to carry out genetic recombination in yeast,by both phenotype and PCR screening for expression, and then optimizing expression conditions through phenotype and PCR screening. Obtaining purified protein through Dot-blot, SDS-PAGE, western-blot identification and concentration and purification. The results show that we constructed the bovine resistin gene eukaryotic expression plasmid (pPICZaA-resistin) successfully, and obtained 5 strains of integration through phenotype and PCR screening. The optimizations of bovine resistance eukaryotic system are:1.0% methanol concentration, expression time of 72h, at the same time enhanced protein expression. We got 1 strain of excellent recombinant yeast expression integration by Dot-blot, and successfully expressed the bovine resistin protein, the size is 14KD.20 wistar rats (wt 100±10g,4 w old) were randomly divided into 4 groups,5 each.GroupⅠwere given a free choice feeding and an injection with PBS; GroupⅡwere also given a free choice feeding and an injection with recombinant bovine resistin (1:1); GroupⅢfasted for 2d and were injected with PBS; GroupⅣfasted for 2d and were injected with recombinant bovine resistin (1:1).All of the 4 groups were injected at the begin of the fasting of groupⅢand groupⅣ(1.5mg each rat,an interval of 12h,4 times of injection).At the last injection(48h),blood samples were collected(marked as 0 min).Then blood samples were collected at15min,30min,60min,120min,180min after the first sampling and tested for glucose and insulin levels.The results shown recombinant bovine resistin could obviously increase glucose and insulin levels of both fasting rats and free-feeding rats,which confirmed the biological activity of recombinant bovine resistin.We cultured hepatocytes on cell culture plate and added recombinant bovine resistin when hepatocytes were growing well with 6 gradients (0,10,50,100,300,500ng/L)and 6 replicates.Cultured for 12h then rinsed with PBS, and then conduct ultrasonic and supernatant. Using Coomassie brilliant blue staining method supernatant protein content of bovine resistin. Remove 100μl supernatant of each tube and adding substrate buffer, then 37℃water bath 8min. Adding 1.5mmol/L of oxaloacetate 100μl, then reading the absorbance of 340nm in the microplate reader. Calculating the specific activity of enzyme PEPCK according to protein content and absorbance. The results showed that the activity of enzyme PEPCK was enhanced with recombinant bovine resistin concentrations.10ng/ L or more recombinant bovine resistin could increase PEPCK activity, and proved that once again recombinant bovine resistin has obvious biological activity.In summary, this study has successfully cloned cattle resistance gene, successfully constructed a bovine resistin gene eukaryotic expression plasmid, the expression product (recombinant bovine resistin protein) has significant biological activity.