ABSTRACT
To obtain the functional fusion protein of rhoptry protein 2, compound rhoptry protein2 and surface antigen 1 of Toxoplasma gondii. the ROP2 and P30 genes from genomic DNA of T.gondii RH strain were amplified by PCR, and were inserted into pMD18-T cloning vector. Then the ROP2 fragment was subcloned to pET-30a(+) plasmid digested by EcoRⅠand Hind Ⅲ to construct plasmid pET-ROP2. Furthermore,the P30 fragment was subcloned into pET-ROP2 digested by BglⅡand EcoRⅠto create plasmid pET-ROP2-P30, the resulting recombinant plasmids , transformed into E.coli BL21 (DE3), were induced with IPTG. and the proteins identified by SDS-PAGE were further purified and refolded. The biological activity was analyzed by Western blot with specific antibody. It was found that the sizes of ROP2 and ROP2-P30 were 1212 and 1896bp with corresponding molecular weight 50- kDa and 75-kDa, respectively. The recombinant protein ROP2 (50-kDa) could specifically react with rabbit-polyclonal antiserum, and complex fusion protein ROP2-P30 (75- kDa) could react with P30 monoclonal antibody.
ABSTRACT
Objective To study the protective effect of ROP2 nuclei acid vaccine in mice.Methods Forty-two BALB/c mice were divided into three groups.Each mouse in experiment group was injected with 50 ?g recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris.In control groups,each mouse was injected with 50 ?g blank plasmid pc-DNA3 and with 50 ?l PBS respectively.All mice were immunized for three times with an interval of three weeks.The volume was doubled for the final injection in the two plasmid groups.Blood,spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+,CD8+ T cells and cytokines 2 weeks after the final immunization.The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation.Results The vaccine induced strong cellular and humoral immune response.The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro.The lymphocyte phenotype was analyzed.CD4+ T cells proliferated sharply(69.5?3.4)%,and the ratio of CD4+/CD8+ increased considerably by(4.69?1.32)%(P