Cloning And Expression Of SAG5B And SAG1 Of Toxoplasma Gondii And Their Immunoprotective Role In Mice Against T.gondii

Objective: To clone and express sag1 and sag5b from the genome of Toxoplasma gondii RH strain and observe their immunoprotectivity to mice against T.gondii infection. To compare the difference between the genome of RH strian and Praugniud strian of T.gondii on sag5b by PCR reaction. Methods: RH strain tachyzoites,by laboratory mouse passage,were harvested from ascites of mice and genome DNA was prepared. PCR reaction, using the genome of RH strian of T.gondii and brian tissue contianing cysts of Praugniud strian of T.gondii, was taken.The crude antigen was prepared with tachyzoites isolated from the peritoneal exudates of infected mice. Newzealand rabbits were immunized with both recombinant and crude antigens to gain the multi-antibody sera. Fragments of sag5b and sag1 were generated by PCR amplification from genome of T.gondii. The PCR products were ligated to vectors of pGEM-T. The recombinants were confirmed by EcoRI/XhoI and EcoRI/HindIII digestion, PCR,and DNA sequencing and were cloned into expression vector of pET28a. The recombinants were transformed into E.coli BL21(DE3). Fusion expressions were induced by isopro-pyl-beta-D-thiogactoside(IPTG) followed by identification with Western blotting and purified through the collumn of affinity chromatography with Ni2+(nickel sulfate,NiSO4). The concentration of recombinant proteins were tested by improved Comass brilliant blue. The Balb/c mice were immuned with both rSAG5B and rSAG1 and crude antigen through subcutaneous inoculation, respectively with two weeks of interval shots. And the mice inoculated with Freund's Adjuvant Complete were taken as control. One month after last immunization, the tachyzoites were injected peritonealy into mice to observe the immunoprotectivity of the rSAG5B. Results: T.gondii SAG5B encoding gene with a molecular size of 1104bp was obtained, which was completely homologous to the sequence previously reported(Accession No AF013968). PCR reaction,taking the genome of RH strain of T.gondii and brian tissue containing cysts of Praugniud strian of T.gondii as template,showed a difference between the two strains. The expressions were confirmed by SDS-PAGE and Western blotting. The fusion expressions were induced by IPTG, and purified through affinity chromatography. The comparative study showed that rSAG5B could protect mice against T.gondii infection as effectively as SAG1 ( P30) and crude antigen (P>0.05).All of them showed a significant difference compared to the control mice. Conclusion: sag5b may act as a genetic marker to differentiate virulent from avirulent strains of T.gondii.The recombinant SAG1 and SAG5B of T.gondii have generated immunoprotectivity against T.gondii infection. This study has laied a foundation for the study of valid vaccine against T.gondii infection.