Construction and identification of recombinant plasmid containing SAG1 and MIC3 from Toxoplasma gondii

Objective To construct and identify an eukaryotic expression recombinant plasmid pcDNA3.1-SAG1-MIC3 for the further development of multiantigenic vaccine against toxoplasmosis.Methods Two gene fragments were amplified by PCR with primers that were designed according to the published gene sequence of SAG1;MIC3 from Toxoplasma gondii RH strain,identified and then subcloned into pMD18-T simple vector,then subcloned into pcDNA3.1(-) to generate eukaryotic expression plasmid pcDNA3.1-SAG1-MIC3.Ampicilin-resistant transformants were selected and identified by PCR,enzyme digestion and DNA sequencing analysis.The recombinant plasmid was transfected into Hela cells by LipofectamineTM 2000.Then,detect the expression by RT-PCR.Results Enzyme digestion and PCR analysis of the gene SAG1 and MIC3 T-A clone showed the length of fragment was about 933 bp and 789 bp,which fits the expected result.Enzyme digestion of pcNDA3.1-SAG1-MIC3 showed the length of fragment was 1 722 bp,corresponding to the length of SAG1-MIC3.The sequence analysis demonstrated that the sequence identities were 100% between recombinant SAG1-MIC3 gene and that from GenBank.RT-PCR analysis of cells transfected gene fragment display positive bands.Conclusion The recombinant eukaryotic expression plasmid of pcDNA3.1-SAG1-MIC3 has been successfully obtained,building a foundation for the further studies on the vaccine development for T.gondii infection.