Prokaryotic Expression,purification And Polyclonal Antibody Preparation Of Canine FGF12

Fibroblast growth factor 12(FGF12)is a member of the fibroblast growth factor family,which is expressed in neurons,mast cells,cardiomyocytes and other cells,and is involved in epileptic encephalopathy,arrhythmia,atherosclerosis.FGF12 can be used as a diagnostic marker for pulmonary arterial hypertension(PAH),which is currently considered to be the third most common cardiovascular disease,.Symptoms of canine pulmonary arterial hypertension are similar to those in humans,which is a high incidence disease in canine.Diagnosis is difficult because of no characteristic symptoms or signs clearly distinguished from other diseases.Canis fibroblast growth factor 12(cFGF12)may play an important role in the diagnosis and treatment of canine PHA,which has no report yet.In this study,the structure of cFGF12 gene was analyzed and the phylogenetic tree was constructed by bioinformatics software.The prokaryotic expression vector p EASY-Blunt E1-cFGF12 was constructed to expression the soluble cFGF12 protein,and the induction conditions were optimized..The biological activity of cFGF12 protein purified by Ni-NAT affinity chromatography was verified by CCK8 analysis with NIH3T3 cells.A specific rabbit-derived cFGF12 polyclonal antibody was prepared by immunizing New Zealand white rabbits and purified by protein A.This study not only provides a direction for obtaining a large amount of soluble protein of cFGF12,but also opens up a broad market application prospect for the clinical diagnosis and treatment of canine PAH.The main findings are as followed:1.Bioinformatics analysis of cFGF12 showed that cFGF12 was a hydrophilic protein without signal peptide and transmembrane structure,the theoretical molecular mass was 20.424 KDa,and it has 8 N glycosylation sites and 26 phosphorylation sites,the subcellular localization was in the cytoplasm,and the secondary structure consists ofɑ-helix,β-turn and random coil.The result of phylogenetic tree analysis showed that cFGF12 was most closely related to hFGF12.2.In this study,the prokaryotic expression vector of pEASY-Blunt E1-cFGF12 was constructed.The results of solubility analysis showed that cFGF12 was a soluble protein with a molecular weight of 21 KDa.The optimal induction condition of this protein was to induce by 0.1m M IPTG at 37℃for 2 h.The cFGF12protein was purified by Ni-NAT affinity chromatography and characterized by Western Blot.CCK8 analysis proved that cFGF12 protein had the biological activity of promoting the proliferation of NIH3T3 cells.3.In this study,the purified cFGF12 protein was used as the antigen to immunize NewZealand white rabbits four times.When the antiserum titer reached 1:10~6determined by indirect ELISA,cFGF12 protein was purified by Protein A from the serum,and its purity was identified by SDS-PAGE.HEK293 cells transfected with eukaryotic plasmid p CMV13-His-cFGF12 were used to identify the specificity of polyclonal antibodies.Western Blot analysis confirmed that the cFGF12 polyclonal antibody can recognize cFGF12 protein with good specificity.