Clonging, Expression And Identification Of The Surface Antigen 1 (SAG1) Gene Of GJS Strain Of Toxoplasma Gondii

Toxoplasma gondii is an obligatory intracellur parasite and courses human and animal toxoplamosis, which seriously threat to people, s health and the development of the farming industry. Toxoplasma gondii surface antigen 1 (SAG1) is the first cloned membrane protein in the surface of Toxoplasma gondii tachjyzoite, which has potential application in both diagnosis and immunazition.One pair of specific primers was designed based on SAG1 of Toxoplasma gondii RH strain in GenBank, and gene fragment was amplified from the genomic DNA of Toxoplasma gondii GJS strain by PCR and cloned into pMD-18T vector to construct the recombinant plasmid pMD-SAG1. Homology analysis showed that the ORF of nucleotide sequences and amino acid sequences of SAG1 shared 98.9%, 97.3% identity with those of the published T. gondii SAG1 sequence (S76248). Another pair of specific primers without signal pepide was designed from strong epitope of SAG1. SAG1 was subcloned into a prokaryotic expression vector pET-30a, and the recombinants were transformed into E.coli BL21 (DE3) for expression under induction of IPTG. The expressed product existed in a form of inclusion body and the fusion protein's molecular weight is approximately 35kD. SDS-PAGE and Wester-blot analysis showed that the recombinant protein could be recognized by the goat serum against Toxoplasma gondii. The purified SAG1 (rSAG1) was used to immunize mice and after seven weeks, mice were challenged with highly virulent tachyzoites via intraperitoneal injection. The antisera were collected from the immunization mice and specific antibody were detected with recombinant antigen ELISA.The SAG1 protein vaccine could induce high titers of anti-SAG1 antibodies in immunized mice. The protective trial proved that there was no significant difference between control group and experimental group though the survival time of mice from experimental group had been prolonged. In order to gain recombinant proteins with configuration to natural SAG1, one pair of specific primers was designed and SAG1 gene was amplified from the genomic DNA of Toxoplasma gondii GJS strain by PCR. The amplified product was cloned into pcDNA3.1 eukaryotic expression vector and transformed into E.coli DH5αand identified by PCR, restriction enzyme digestion and sequencing. Sequence analysis indicated that the fragments of SAG1 gene were 1008bp. The pcDNA-SAG1 recombinant plasmid successfully constructed, which has established the foundation to feather study DNA vaccine of Toxoplasma gondii.