T. Gondii Nucleic Acid Vaccine Of Pcdna <sub> 3 </ Sub> / The Gra <sub> 1 </ Sub> Research

Aim: Constructing a recombinant plasmid encoding GRA1 protein of Toxoplasma gondii and observing the protective immunity induced by recombinant plasmid DNA vaccination.Methods: 1. Toxoplasma gondii cDNA library was screened with 2 monoclonal antibodies and sera from rabbits, which were immunized by T.gondii RH strain tachyzoit antigens. The sequences of positive clones were analyzed. 2.The sequence encoding GRA1 was amplified by PCR. The amplified product and eukaryotic expression vector pcDNA3 were digested with EcoR I and Xho I respectively. GRA1 gene-coding region was directly inserted into the poly-linker of pcDNA3. The recombinant plasmid was characterized by agarose gel electrophoresis of its PCR products, restriction enzyme digestion and DNA sequencing analysis. 3. The naked recombinant plasmid pcDNA3/GRAl was injected into BALB/c mice intramuscularly. The specific antibodies were analyzed by ELISA and the DNA of GRAl gene-coding region from different tissues were detected by PCR. The expression of GRAl in muscle tissues was also analyzed with immunofluorescene. The vaccinated mice were challenged with RH strain tachyzoites and the mice's survival time was observed.Results: 1. Twelve positive clones were obtained by immunoscreening T.gondii cDNA library. The sizes of the inserted cDNA fragments were 450bp~2400bp. After searching all gene databases and analyzing putative protein sequences with expert protein analysis system, 3 novel genes were identified and named as Toxo-W5, Toxo-W2 and Toxo-W4 (GenBank accession number: AY223538, AY238892, AY208994). 2. The recombinant plasmid pcDNA3/GRAl that expresses the full length of GRAl was constructed successfully. 3. The specific antibodies in sera from vaccinated mice were detected by ELISA. The GRAl gene-coding region was amplified from different tissues of immunized mice using as tissue DNA templates. The muscle tissues from vaccinated mice reacted positively with the immunized rabbit serum. Theaverage survival time of vaccinated mice was significantly longer than that of control groups after challenged with T.gondii RH strain tachyzoites.Conclusion: 3 novel genes were isolated from T.gondii cDNA library. The mammalian expression vector pcDNA3/GRAl that expresses the full length of GRA1 was constructed successfully. The preliminary data suggest that immunization of mice with the naked pcDNA3/GRAl DNA can induce specific immune responses and a partial protective immunity against the challenge of T.gondii RH tachyzoites.