Preliminary Research On Biological Features Of Recombinant EG95-(C3d)3 Protein

Echinococcosis (Hydatidosis) caused by infection with larval stage of Echinococcus granulosus(Eg), affects both humans and domestic animals, and is one of the five major parasitic diseases which is prevented and controlled by Ministry of Health Planning. The drugs using and cyst removal treatment of hydatid disease is not satisfactory, so the disease is now mainly focusing on prevention. Because of efficiency and specificity characteristics, genetically engineered vaccines become the most rational way to prevent the disease.In recent years, the research found that genes of EG95 as immune antigen produced anti-infection was significantly higher than the capacity of several other antigens. And it can produce specific antibodies in vivo duration of up to 1 year. The study also found that while the production of the antibodies can generate by genetic ewes lamb. And there is research that the adjuvant can enhance protective effect of the EG95 antigen in the body. Complement C3d molecule as a novel molecular adjuvant can increase the immune response. The data show that series of 3 copies of C3d has better immune enhancement. However, the expression of the immunity protein is often low, insoluble which restricts the production and development of vaccine.Based on the above background, we expressed the EG95-(C3d)3 gene by baculovirus expression system, and also prepared Echinococcus granulosus EG95 gene monoclonal antibody. Using of monoclonal antibodies specific antigen recognition, we compared the protein conformation and the antigen recognition sites of prokaryotic and baculovirus expression of the EG95-(C3d)3 gene.In order to obtain the target gene EG95-(C3d)3, we connected the EG95 gene and 3 copies of C3d gene. Using the transfer vector pFastBac-HTa transfer EG95-(C3d)3 into the Bac-to-Bac baculovirus expression system, and expressed the recombination protein by sf9. The results of Western blot showed that this EG95-(C3d)3 protein could response to Echinococcus granulosus-specific positive serum, indicating that EG95-(C3d)3 protein has immune activity.Using in vitro fusion, the spleen cells of immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Hybridoma cells were screened by Enzyme-linked-immunosorbent assay (ELISA). All the 12 monoclonal antibodies were tested to be IgG1 subtype. Western-blot analysis showed that all strains of MAbs against Echinococcus granulosus EG95 protein were specific reaction.Comparing the conformation and the antigen recognition sites of prokaryotic and baculovirus expression of the EG95-(C3d)3 gene, we found that the conformation of baculovirus expression had some different with the prokaryotic expression. Monoclonal antibody only recognized the conformation of baculovirus expressed protein.In general, we acquired a new antigen of Echinococcus granulosus. These research laid the foundation for the comparison of the Eukaryotic and the prokaryotic expression of the EG95-(C3d)3 gene.