Prokaryotic Expression Of Gene Fragments Of AjC3 And Preparation Of Polyclonal Antibody From Apostichopus Japonicus

The complement component C3(AjC3) of Apostichopu japonicus plays an important role in the natural immune system. AjC3 is highest content in composition of complement system. The crack products of AjC3 have the function of regulating immune system. AjC3 can clear the target cells by forming membrane attact complex. The methods of detecting AjC3 of Apostichopu japonicus is not effective at present. The objective of this study was to clone the genes of AjC3, express and purify recombinant protein in prokaryotic cells and prepare the polyclonal antibody of AjC3 to lay the foundation for further research on immunologic mechanism of Apostichopus japonicus.The partial genes of AjC3 was cloned by PCR. The prokaryotic expression recombinant plasmid was constructed. The recombinant plasmid was transformed into the transetta(DE3) and induced to express at different temperatures, IPTG concentrations and time. The forms of recombinant protein was analyzed after ultrasonic decomposition by SDS-PAGE. The expression products were purified by Ni-NTA. The recombinant protein as antigen was employed to immunize mice to prepare the polyclonal antibody of AjC3. The title was determined by indirect ELISA. The specificity of polyclonal antibody and the distribution of AjC3 in different tissues were determined by Western blot.The length of PCR product was 1185bp(1d), 873bp(2d), 555bp(3d), respectively. DNA sequencing suggested that the prokaryotic expression recombinant plasmid pGS-21a-1d,pGS-21a-2d,pGS-21a-3d was successfully construced. pGS-21a-1d not expressed protein, the optimal expression condition of pGS-21a-2d and pGS-21a-3d were 0.2mmol/L IPTG for 6h at 30℃. The recombinant protein pGS-21a-2d and pGS-21a-3d were expressed in the form of inclusion body by SDS-PAGE.The purity of recombinant protein reached 90% and the molecular weight were 67KDa(pGS-21a-2d)and 56KDa(pGS-21a-3d). The title were 1:400(pGS-21a-2d) and 1:25600(pGS-21a-3d) by indirect ELISA. Western blot detection suggested that the polyclonal antibody had high specificity, and the AjC3 protein expressed in coelomic fluid and body wall, but the intestinal, breathing tree, tetacle did not express.The prokaryotic expression recombinant plasmid was construced; The recombinant protein was successfully supurified; The polyclonal antibody of AjC3 with high title and specifity was successfully preparated; The distribution of AjC3 in different tissues were investigated. it provides a detecion tool for further researches on AjC3.