Construction And Expression Of The Eukaryotic Expressed Plasmid Of MIC3 From GJS Strain Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects all warm- blooded animals including humans. Toxoplasmosis causes serious public health problems and is of great economic importance worldwide. The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite which is expressed in tachyzoites, bradyzoites and sporozoites. MIC3 has two potential valuble applycation for general research, including diagnosis and immunity.One pair of primers was designed based on the MIC3 gene sequences of RH strain Toxoplasma gondii in GenBank. The gene fragment encoding MIC3 was amplified by PCR from the genomic DNA of Toxoplasma gondii GJS strain and cloned into the vector pMD18-T. After PCR, restrictive digestion and sequencing identification, the positive plasmid was digested and subcloned into an eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression recombinant plasmid pcDNA3-MIC3 was identified by PCR, restriction enzyme digestion and sequencing analysis. Then, the pcDNA3-MIC3 plamid was transfected into BHK-21 cells mediated by Lipofectamine 2000. Indirect immunofluorescence showed that the recombinant plamid was expressed in BHK-21 cells. Western-blot analysis showed that a band approximately 39.2 ku in size in BHK-21 cells lysate and supernatant of the culture medium were recognized by the goat serum against Toxoplasma gondii, indicating that the MIC3 protein expressed transiently in the cells had antigenicity. After mice were injected with the recombinant plasmid pcDNA3-MIC3, the sera antibody were detected by indirect ELISA and survival time of mice after challenge with tachyzoites of Toxoplasma gondii GJS strain were observed. The results showed that the recombinant plasmid pcDNA3-MIC3 was successfully constructed and the immunized mice showed higher specific antibody level (P<0.01) than control. The survival time of vaccined mice was longer than that of control groups. It indicated that the nucleic acid vaccine had good immunity and could induce the immunoprotection in mice.