Construction of pVAX1-SAG1 and pVAX1-SAG4 eukaryotic plasmids of an antigen gene of Toxoplasma gondii

Objective To construct pVAX1-SAG1and pVAX1-SAG4eukaryotic expression plasmids of an antigen gene of Toxoplasma gondii. Methods A pair of specific primers was designed and synthesized in accordance with the SAG1and SAG4gene sequences of T.gondii(RH strain).These sequences contain the HindⅢ/BamH Ⅰand BamH Ⅰ/XbaⅠ endonuclease cleavage sites.The genes SAG1and SAG4were amplified with PCR using genomic DNA fromT.gondii as a template.The PCR products were cloned into a PEGM-T Easy vector and were identified by digestion with restriction enzymes and colony PCR;The recombinant plasmid PEGM-T Easy-SAG1and PEGM-T Easy-SAG4and pVAX1vectors were digested with HindⅢ/BamH Ⅰand BamH Ⅰ/XbaⅠ,and then the target fragments SAG1and SAG4with endonuclease cleavage sites were subcloned into a pVAX1vector and identified by digestion with restriction enzymes and colony PCR. Results The target DNA fragments SAG1and SAG4yielded by PCR were sequenced and the results were compared with the reported sequences of the SAG1and SAG4genes of T.gondii(RH strain).The rate of concordance was 100%.The recombinant plasmid PEGM-T Easy-SAG1and PEGM-T Easy-SAG4,pVAX1-SAG1and pVAX1-SAG4were found to contain the target gene fragments SAG1(722bp)and SAG4(511bp)according to digestion with restriction enzymes and colony PCR. Conclusion The eukaryotic expression plasmids pVAX1-SAG1and pVAX1-SAG4encoding the SAG1and SAG4proteins of T.gondii were constructed,laying the foundation for studies using vaccines from T.gondii genes.