Cloning, Expression And Polyclonal Antibody Preparation Of C-reactive Protein Gene From Epinephelus Coioides

Based on the Smart cDNA libraries which constructed with head kidney of Epinephelus coioids, we cloned CRP gene. The cDNA length of CRP gene is 1001 bp,and includes a 148bp 5'-UTR, a 178bp 3'-UTR, a 675 bp ORF and encoding 225 amino acids. Sequence analysis reveals that EC-CRP gene shares a highest amino acids identity of 42.58% with Salmo salar. The CRP gene was cloned into the plasmid pET-21a(+) and expressed in Escherichia coli. The CRP gene was efficiently expressed in host bacterium E. coli BL21(DE3) following induction with IPTG. The expressed protease purified by Ni-NTA affinity chromatography. The SDS-PAGE analysis showed the recombinant EC-CRP was expressed as inclusion body,and the molecular weight was 25kDa. The results are important for the future study on molecular structure, function and diagnosis of disease-related genes of E. coioides.After injection of Vibrio alginolyticus, We found the concentration of CRP significant increasing in liver, head kidney, spleen, kidney, intestine, heart, gill and thymus of E. coioides by Real-time PCR. The highest concentration of CRP was in head kidney and liver second, following in kidney, spleen, intestines, heart and gills, and the lowest in thymus. As infection time increasing, the peak of expressed CRP was different in the organization at different times. The concentration of CRP was 1.5, 2, 5, 50, 1.5 and 20 times of the normal level in the gill at 3h, thymus at 6h, spleen at 12h, head kidney at 24h, intestinal at 24h and liver at 48h, respectively. The changes of expressed CRP were studied when E. coioids was exposed in the water containing CuSO4(2ppm), CdCl2(20ppm), phenol (10ppm), respectively. The maximum levels of CRP were15, 30 and 50 times higher than normal level at 6h. The results indicated that E. coioids was different in the detoxification extent under the different pollution stress. The amount of expressed CRP level associated with the exposure time of the natural pollutants. The results suggested that the natural pollution can be used as pollution biomarkers.The natural CRP had been purified by calcium dependent affinity chromatography with a phosphorylcholine (PC)–Sepharose column in the sera of E. coioides. SDS-PAGE analysis revealed that the protein contained only one kind of subunit and the molecular weight was 26kDa. In this study, rabbit antiserum against natural CRP and fusion CRP were prepared. Antigen-antibody reaction just occured between rabbit antiserum against natural CRP and the serum of E. coioides. rabbit antiserum against natural CRP couldn't both react with the serum of Lutjanus sanguineus. rabbit The rabbit antiserum against fusion CRP had no reaction with the sera of E. coioides and nature CRP.