Construction Of Gene Vaccine Of PVAX1-SAG1and PVAX1-SAG4from Toxoplasma Gondiiand Immune Protection Effect Of Animals

Objective To obtain Toxoplasma gondii SAG1/SAG4gene in vitro byPCR, and construct recombinant eukaryotic expressed plasmids pVAX1-SAG1/pVAX1-SAG4.Mice were immunized with recombinant eukaryotic expressedplasmids pVAX1-SAG1/pVAX1-SAG4respectively or mixing,and then themice were attacked by Toxoplasma gondii RH strain tachyzoites.animal serumantibodies and protective for infection of Toxoplasma gondii evaluate effects ofvaccines,lay the foundation for the application of toxoplasmosis DNA vaccine.Methods Genomic DNA of Toxoplasma gondii RH strain was extracted,and specific primers of SAG1/SAG4were designed according to the standardgene sequence of Toxoplasma gondii RH strain registered in GenBank,andamplified the SAG1/SAG4gene by PCR.The results of PCR were identified byagarose gel electrophoresis,DNA sequence analysis and BLAST.The PCRproduct of SAG1/SAG4was cloned into PEGM-T Easy vector in order toconstructed cloned plasmid PEGM-T Easy-SAG1/PEGM-T Easy-SAG4,whichidentified by colony PCR,restriction enzymes and sequencing.The targetfragment with restriction enzymes Hind Ⅲ/BamHⅠ and BamHⅠ/Xba Ⅰfrom PEGM-T Easy-SAG1/PEGM-T Easy-SAG4was subcloned into pVAX1.Therecombinant eukaryotic expression plasmids pVAX1-SAG1/pVAX1-SAG4wereconstructed and the positive clones were identified by colony PCR,restrictionenzyme and sequencing analysis.A large of recombinant eukaryotic expressionplasmids pVAX1-SAG1/pVAX1-SAG4were prepared for DNA vaccines.TheDNA vaccine pVAX1-SAG1/pVAX1-SAG4respective and mixed were injectedin hind limb muscle of Bala/c mice for three times,Each time interval2weeks.Serum from mice tail vein blood was separated to measure Toxoplasmaantibodies by ELISA.The mice were attacked with Toxoplasma gondii RH straintachyzoites in2weeks after the last immunization,observing incidence andmortality of mice.Results The SAG1/SAG4genes were amplified with the length of772bpand511bp.The BLAST analysis indicated that the two fragments correspondedwith Toxoplasma gondii RH strain SAG1and SAG4cDNA gene homology99%and100%respectively.Cloned plasmid PEGM-T Easy-SAG1/PEGM-TEasy-SAG4was identified by agarose gel electrophoresis displayed a specificband at800bp and500bp,and sequencing results also showed that SAG1/SAG4gene fragment had been correctly inserted downstream of cloning plasmid T7promoter,and contained Hind Ⅲ/BamH Ⅰa nd BamHⅠ/Xba Ⅰ restriction site.The recombinant eukaryotic expression plasmids pVAX1-SAG1/pVAX1-SAG4identified by colony PCR and double restriction enzyme digestion.There werespecific bands displayed at800bp and500bp by agarose gel electrophoresis.After the mice were immunized with pVAX1-SAG1/pVAX1-SAG4monovalentand multivalent combination DNA vaccines, serum antibody OD value of eachgroup were negative.Attacks experiment multivalent combination vaccinessurvived longer than the monovalent vaccine,the DNA vaccine pVAX1-SAG1 than the vaccine pVAX1-SAG4survive for a long time.Conclusions Successfully amplified SAG1/SAG3genes of Toxoplasmagondii RH strain by PCR,and constructed cloned plasmid PEGM-TEasy-SAG1/PEGM-T Easy-SAG4and recombinant eukaryotic expressionplasmid pVAX1-SAG1/pVAX1-SAG4.The DNA vaccines pVAX1-SAG1/pVAX1-SAG4could make animals induce protective immunity,immuneprotection pVAX1-SAG1monovalent DNA vaccines better thanpVAX1-SAG4,which consists of a mixture of both multivalent vaccine effect ismore than the respective monovalent vaccine better.