Immunoprotection Of Subunit Vaccine And Nucleic Acid Vaccine Of E.maxima ADF Gene

Chicken coccidiosis is induced by Eimeria an intestinal intracellular parasites. Coccidiosis distribute around the world, which is one of the major diseases damaged the poultry widely, can cause serious economic losses. At present, there are no effectual drugs for the treatment of coccidiosis. In view of the vaccine applications in past,and the infected host can be obtained a certain immunity response against E. maxima, more ideal vaccine candidate genes should be screen.By prepared recombinant vaccines and DNA vaccines of these genes can induce protective immunity response against E.maxima infection.This is the vision measure and the application direction to prevention and treatment of coccidiosis. Actin-depolymerizing factor (ADF) has been confirmed widespread in a variety of organisms, and have the capable of regulating polymerization and depolymerization of actin.Thus,the research of E.maxima ADF can screen ideal vaccine candidate gene for immunological control of E.maxima and provide experimental basis. In the present study about ADF recombinant protein was expressed. The ADF was localizated in E.maxima tachyzoites cytoplasm by immuno-fluorescence technique.and mainly below the plasma membrane.Meanwhile.the ADF gene was connected with the eukaryotic expression vector pVAXl and constructed nucleic acid vaccine. The recombinant protein and the nucleic acid vaccine were used to immunize animals and both could induce specific immune response, and have some protection against E.maxima after chicken were inoculated with them.Cloning and expressing of ADF gene According to ADF sequences reported in Genebank and designed a pair primers.Extracted the total RNA of coccidiosis, and amplified open reading frame of ADF by RT-PCR. Purified the ADF fragment and ligated with the pMD18-T vector, the positive plasmids was sequenced after double restriction enzyme digestion. The results indicated ORF was 365bp and the nucleotide sequence shared 99% sequence homology with original sequence compared by BLAST. The positive plasmid and pET-28a (+) expression vector were connected by T4 ligase and transformed into Rosetta (DE3).The bacteria which identified correct was induced experssion with IPTG.The molecular weight of the recombinant fusion protein was 17KDa analyzed by SDS-PAGE. The recombinant protein was highly expressed at 37℃under the condition of 1mM IPTG for 5h.The protein mainly in soluble form, and can specific identified by Western blot with anti-E.maxima serum, has a good immune responsiveness.The localization of ADF in E.maxima s were determined by immuno-fluorescence antibody technique with the ADF gene polyclonal antibody. The result showed that ADF is scattered throughout the tacyzoite cytoplasm and mainly below the plasma membrane.This result proved ADF indeed exist in tachyzoite and established foundations for the further study of E.maxima ADF gene.Construction and expression of eukaryotic expression vectors. The recombiant plasmid pVAX1-ADF was contructed by ligated the ADF gene with eukaryotic expression vector pVAXl and expressed in Hela cell strain. The recombinant plasmid was transient transfected into Hela cells.The specific recombiant proteins were detected in Hela cells by indirect immunofluorescence assay in 48h after transfection. The expression of proteins was confirmed by SDS-PAGE.and has a good immune responsiveness by western blot identification.The protective immune response induced by recombinant ADF antigen in chicken The chickens were inoculated with purfied recombinant ADF protein by intraperitoneal once every two weeks.The responses of cell immunity were elevated by the immunological methods such as the specific antibodies responses and the ratio of CD4+/CD8+.The specific immune responses were strengthened with the increase of immunization times.The level of CD4+and CD8+T lymphocyte of immunized chickens were increased significantly compared with control groups(P< 0.01),and the different of CD4+/CD8+ ratio among them is not signification (P>0.01).Other immounoprotective index were detected after chickens were challenged with tachyzoites.Compare with the control chicken.the cysts of experimental chicken in brain were reduced, and decreased cysts rate of the experiental group is 30%.The average survival day of experiment chickens was increased.but not significant(P>0.01). The above results suggested that the recombinant ADF protein have partly immunoprotective effect on E.maxima infection in chicken.The protective immune response induced by nucleic acid vaccine in chicken. DNA vaccine immune chickens, then could induce a higher cellular immune responses. Experimental group chicks CD4+, CDS+T lymphocytes were increased compared with the control group, but the CD4+/CD8+T lymphocyte numbers compared with the control group was not significant (P> 0.05). After oral inoculating E.maxima oocysts, oocysts were counted per gram of faeces (OPG), small intestinal disease score, weight measurement, the results show that recombinant plasmid can improve chicken weight gain and intestinal lesion score. The recombinant plasmid group anticoccidial index is 178.2,and it compared with the control group was significant difference (P<0.05).