Ab Preparation By Chicken PPARγ And C/EBPa Genes Immunization And Their Function

Evidences from mammals indicated that adipocyte differentiation was a complex process regulated by transcription factors coded by some genes, such as PPARs (peroxisome proliferator-activated receptors) and C/EBPs (CCAAT/ enhance binding proteins) gene family. Among these transcription factors, PPARγand C/EBPαgenes play important roles in adipocyte differentiation.In the current study, the cDNA sequence of chicken PPARγand C/EBPαgene was amplified according to chicken PPARγand C/EBPαgene sequence, and then prokaryotic and eukaryon expression vectors of the two genes were constructed, respectively. The pcDNA3/C/EBPαwas injected to mice by plasmid and so antiserum was prepared; the fusion protein GST-PPAR and GST-C/EBP were used to immunize broilers for analysis the levels of antibody of PPARγand CEBPαand the effects on abdominal fat contents in broiler.The results showed that pGEX-4T-1/ PPARγ,pcDNA3/ PPARγ,pGEX-4T-1/ C/EBPα,pcDNA3/ C/EBPαwere constructed successfully the high titer and specifically antiserum were prepared;The active immunization results showed that there were high levels of antibody against PPARγand C/EBPαin serum of broiler, and the broilers of PPARγgroup had significant lower abdominal fat contents than control group.1. The construction of expression vector of PPARγand C/EBPαGenes from chickenTo construct the prokaryotic and eukaryotic expression vector and prepare the antiserum of peroxisome proliferator-activated receptor-gamma (PPARγ) gene and CCAAT/enhancer binding protein alpha(C/EBPα) in chicken. The sequence of PPARγand C/EBPαgene were amplified by reverse transcriptase-PCR (RT-PCR) from the total RNA of chicken fatty acid and insert into pGEX-4T-1 vector and pcDNA3 vector. 2. The preparation of PPARγand C/EBPαGenes antiserumTo further study the function of PPARγand CEBPα, PPARγand CEBPαwere cloned into pcDNA3 to form pcDNA3- PPARγand pcDNA3-CEBPαDNA vaccines, and then vaccinate mice by two DNA delivery ways. ELISA and Western blot showed that antibody obtained from the serum of mice by injecting recombinant pcDNA3- PPARγand pcDNA3-CEBPαwere high effective and strong specific. The recombinant pcDNA3-PPARγand pcDNA3-CEBPαin the current study would provide a powerful tool for overexpression research of chicken PPARγgene in cell level;3. PPARγand C/EBPαfusion protein and their effects on Abdominal fat in BroilerFusion protein GST-PPARγand GST-C/EBPαwere expressed in E.coli. BL21(DE3) which has prokaryotic expression plasmids pGEX-4T-1/PPARγand pGEX-4T-1/C/EBPαwith IPTG induction. The expressed fusion proteins were purified by Glutathione Sepharose 4B affinity chromatography, and then were used to immunize broilers for analysis the levels of antibody of PPARγand CEBPαand the effects on abdominal fat contents in broiler. SDS-PAGE showed that the GST-PPARγfusion protein formed mostly inclusion body in E.coli. And its molecular weight was about 80 kDa, and the GST- C/EBPαfusion protein was expressed in E.coli. with mostly soluble-form protein and its molecular weight was about 62 kDa. Inactive immunization results showed that there were high levels of antibody against PPARγand C/EBPα.in serum of broiler, and the broilers of PPARγgroup had significant lower abdominal fat contents than control group.In this study, these results were provided as the experimental materials for the PPARγand C/EBPαfunction analysis.