Clonging,Expression And Identification The Microneme Protein 3 (MIC3) Gene Of GJS Strain Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a wide range of hosts, including human and domesticated animals throughout the world.So the toxoplasmsis can harm to people's health and cause considerable economic loss in the farming industry. The development of an effective vaccine against Toxoplasma gondii would be of great value to both human and veterinary medicine., due to microneme 3(MIC3) antigen is secreted by the microneme of tachyzoites and bradyzoites. They are highly conservative and immunogenic, were selected as antigens for diagnosis and vaccination totoxoplasmosis.In the present study, a pair of primers was designed based on the MIC3 gene sequences of RH strain Toxoplasma gondii in GenBank. The MIC3 gene was amplified by PCR from genomic DNA of GJS strain Toxoplasma gondii, and cloned into pMD-18T vector to construct the recombinant plasmid pMD-MIC3. The result showed that the identities of the cloned MIC3 gene with the data published in GenBank in nucleotide and amino acid sequence were 99.7% and 99.2%, respectively. After then, Primers without signal peptide were designed according to the ORF sequence of the MIC3 gene, and the pMD-MIC3 recombinant plasmid used as template in the PCR reaction, the amplified products were ligated to the pET-30a vector, and transformed into BL21(DE3) host cells. Expression of the recombinant plasmid was induced with IPTG, and resulted in a high level of protein expression. The expressed products were inclusion body. SDS-PAGE experiments revealed that fusion protein's molecular weight is approximately 44kD. Western-blot analysis showed that the expressed recombinant protein could be recognized by pig anti-Tg. positive serum.The recombination fusion protein was purified by lysis, washing, denaturation and renaturation. The specific band of expression was excised from the gel and used to immunize mice .The antisera were collected from the immunized mice ,examined with Excreted-secreted antigen(ESA) and recomident antigent by ELISA respectively .The result show that the sensitivity and specificity of recomident antigent were more ideal. This also provid theory and experiment data to search for new recombiant antigent and produce multi-molecule vaccine.