Immunoprotection Of Subunit Vaccine And Nucleic Acid Vaccine Of Toxoplasma Gondii ADF Gene

Toxoplasma gondii is an obligate intracellular protozoan parasite, can infect all warm-blooded animals including human beings,and cause toxoplasmosis in people and animals It have serious threat to human health and livestock industry. At present, there are no ideal drugs for the treatment of toxoplasmosis. In view of the experience of vaccine applications in past, and the host can obtain a certain responsed infection immunity against T.gondii, more ideal vaccine candidate genes should be screen.By prepared recombinant vaccines and DNA vaccines of these genes can induce protective immunity response against T.gondii infection.This is the vision measure and the application direction to prevention and treatment of toxoplasmosis. 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 T.gondii ADF can screen ideal vaccine candidate gene for immunological control of Toxoplasmosis and provide experimental basis. In the present study,ADF recombinant protein was expressed. The ADF was localizated in T.gondii tachyzoites cytoplasm by immuno- fluorescence technique,and mainly below the plasma membrane.Meanwhile,the ADF gene was connected with the eukaryotic expression vector pVAX1 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 T.gondii after mices were inoculated with them.Cloning,expressing and localizing of ADF gene According to ADF sequences reported in Genebank and designed primers .Extracted the total RNA of T. gondii, 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 98% 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-T.gondii serum, has a good immune responsiveness.The localization of ADF in T.gondii 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 T.gondii 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 pVAX1 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 mice The mices were inoculated with purfied recombinant ADF protein by intraperitoneal once every two weeks.The responses of specific humoral and 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 mices were increased significantly compared with control groups(P< 0.01),and the different of CD4+/CD8+ ratio among them is not signification (P>0.05).Other immounoprotective index were detected after mices were challenged with tachyzoites.Compare with the control mice,the cysts of experimental mices in brain were reduced, and decreased cysts rate of the experiental group is 30%.The average survival days of experiment mices was increased,but not significant(P>0.05). The above results suggested that the recombinant ADF protein have partly immunoprotective effect on T.gondii infection in mice.The protective immune response induced by nucleic acid vaccine in mice. All the BALB/c mice were immunized with the vaccine plasmids by Intramuscular pathway. The responses of specific humoral and cell immunity were elevated by the immunological methods such as the specific antibodies responses and the ratio of CD4+/CD8+. The level of CD4+,CD8+ T lymphocyte and antibody titers of immunized mices were increased significantly compared with control groups(P<0.01),while the CD4+/CD8+ ratio among them is not signification(P>0.05).The mices were challenged with tachyzoites in two weeks after third immunition. The average survival days were longer than those of control groups,and the cysts number of experiment group was reduced, the decreased cysts rate is 42.8%.