The entire coding sequences of the T. gondii SAG4 gene were amplified by PCR from genomic DNA of T. gondii strain RH with synthetic primers. Trans TagTM High FidelityDNA Polymerase (TransGen, Beijing, China) was used in PCR amplification.
SAG4: Forward primer: 5′-GGGGTACC ATGACGAAAAATAAAATT-3′ Reverse primer: 5′-CGGGATCC TTACATTGATATCAACA-3′ (introduced KpnI and BamH I recognition sites, respectively, are underlined).
The PCR production of SAG4 gene was cloned into the pEASY-T1 Vector (TransGen, Beijing, China), digested with the appropriate restriction enzyme (KpnI and BamH I), and purified from agarose gels. SAG4 gene fragment was inserted into the eukaryocyte vector pEGFP-C1, generating pSAG4. The recombinant plasmids were then propagated in Escherichia coli DH5α and confirmed by restriction analysis and sequencing. Endotoxin-free plasmid DNA was isolated using a Plasmid Purification Kit (TianGen, Beijing, China). The concentrations of the purified plasmids were detected by spectrophotometer at 260 and 280 nm, and the 260:280 ultraviolet absorption ratio was between 1.8 and 2.0. All the plasmids were diluted into 1 mg/ml by sterile endotoxin-free PBS and stored at -20°C before use.
SAG4: Forward primer: 5′-GG
The PCR production of SAG4 gene was cloned into the pEASY-T1 Vector (TransGen, Beijing, China), digested with the appropriate restriction enzyme (KpnI and BamH I), and purified from agarose gels. SAG4 gene fragment was inserted into the eukaryocyte vector pEGFP-C1, generating pSAG4. The recombinant plasmids were then propagated in Escherichia coli DH5α and confirmed by restriction analysis and sequencing. Endotoxin-free plasmid DNA was isolated using a Plasmid Purification Kit (TianGen, Beijing, China). The concentrations of the purified plasmids were detected by spectrophotometer at 260 and 280 nm, and the 260:280 ultraviolet absorption ratio was between 1.8 and 2.0. All the plasmids were diluted into 1 mg/ml by sterile endotoxin-free PBS and stored at -20°C before use.