Construction and expression-in-BHK-21-cells of eukaryotic expression plasmid with MIC3 gene of Toxoplasma gondii GJS strain.

The gene encoding microneme protein 3(MIC3) was amplified by PCR from the genomic DNA of Toxoplasma gondii GJS strain and cloned into the vector pMD18-T.After identification by PCR,restriction digestion and sequencing,the positive plasmid was digested and the MIC3 gene was subcloned into an eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid pcDNA3-MIC3 was identified by PCR,restriction enzyme digestion and sequencing analysis.Then,the pcDNA3-MIC3 plasmid was transfected into BHK-21 cells mediated by Lipofectamine 2000.An indirect immunofluorescence test showed that the recombinant plasmid was expressed in BHK-21 cells.Western-blot analysis showed that bands approximately 39.2 ku in size in BHK-21 cells lysate and supernatant of the culture medium were recognized by the goat-serum against T.gondii,indicating that the MIC3 protein expressed transiently in the cells had antigenicity.