Cloning, Expression, Purification And Identification Of Bradyzoite-specific Antigens BSR4of Toxoplasma Gondii

Objective：To clone gene fragment of a bradyzoite-specific antigen BSR4from the totalDNA of Toxoplasma gondii PRU strain, and construct the recombinant plasmid ofpET28a(+)-BSR4. Analyze the homology of BSR4among T.gondii PRU strains andME49strains, and predict its bioinformatics features. To express and purify the BSR4protein from pET28a (+)-BSR4, analyze its immunoreactivity and bioactivity fordiagnosis, vaccines and pathogenesis of toxoplasmsis.Method: The Kunming mice were intraperitoneally inoculated with T.gondii PRUstrain. The brain tissue homogenate was collected, and the cyst was purified withLydroxypropylmethyl Cellulose. Genomic DNA of T.gondii PRU strain was extracted,a pair of specific primers with Restriction Enzyme cutting site of NcoI and HindⅢwere designed from DNA sequence of T.gondii ME49strain bradyzoite surface antigenBSR4and used to amplify the BSR4DNA by PCR. After purification, the PCRfragment was cloned into vector pET28a(+) with T4ligase in order to constructerecombinant cloning plasmid pET28a(+)-BSR4. The recombinant plasmid wasidentified by double digested and sequenced. The physical and chemical nature,homology, secondary structure and epitope location of recombinant BSR4werepredicted by using bioinformatics. The recombinant plasmid pET28a (+)-BSR4wastransformed in E.coli BL21, and then the single colony was picked, amplified andinduced by IPTG. The expression product was analyzed by SDS-PAGE after destroyedby ultrasonic wave. The inclusion body was purified by His Trap Ni affinitychromatography after lysised by8M urea, and then analyzed by SDS-PAGE andWestern-Blottting. The protein concentration was determined by Bradford assay. Thesplenocytes of T.gondii PRU strains infected and non-infected mice were separated byLydroxypropylmethyl Cellulose. The splenocytes were stimulated with purified antigens. After culture for48h, the3H-TdR was added. The lymphocyte proliferationwas measured by using3H-TdR incorporation assay.Results: The product of target gene amplified by PCR was1194bp. The sequencingresult showed that target fragment was BSR4gene. The BLAST analysis showed thatthe encoding sequences of PRU strain BSR4gene was100%homology with that ofME49strain. The double digested reaction and sequencing analysis demonstrated thatthe BSR4gene had been properly connected into the recombinant plasmid pET28a (+)-BSR4. The predicted BSR4protein molecular mass is42344.75，which can form twofunctional domains, with a signal peptide located before40amino acids. TheN-terminal of BSR4was a signal peptide, and the C-terminal was a hydrophobic regionwhich suggested that it is a GPI-anchored surface protein. There were18potentialantigenic epitopes and2coserved domains in BSR4. After IPTG induction，therecombinant BSR4was expressed in an inclusion body form in E.coli BL21at37℃.After lysised with urea, the protein was purified with His Trap Ni. The purified proteinwas solubility. A specific protein band at45kDa position was detected byWestern-Blotting, which can specifically react with T.gondii positive serum from mice.Lymphocyte proliferation assay showed that SI was higher in infected T.gondii PRUstrains than that in non-infected T.gondii PRU strains.Conclusion: The sequence of BSR4was successfully cloned from T.gondii PRU strain.Recombinant plasmid pET28a (+)-BSR4was successfully constructed. Therecombinant BSR4was expressed highly in an inclusion body form in E.coli BL21, anddisplayed specific immunoreactivity. This antigen can effectively stimulate memorylymphocyte proliferations in vitro. Recombinant BSR4may be used as a potentialvaccine candidate of T.gondii and a novel diagnostic antigen to detect chronic T.gondiiinfection.