Cloning And Expression Of The P30 Gene In The NT Strain Of Toxoplasma Gondii And Establishment Of ELISA Diagnosis Method For Toxoplasmosis

The obligate intracellular protozoan parasite Toxoplasma gondii(T.gondii) is worldwide spread, and is responsible for toxoplasmosis and able to infect all species of mammals (including human) and birds. In hosts who are severely immunocompromised(e.g, neoplastic disease, organ transplantation, and AIDS patients), the tachyzoites of T.gondii may frequently cause a dissrmination and subsequent complications, sometimes become fatal. In addition, at the veterinary level, toxoplasmosis is one of the main cause of fetal abortion, stillbirth neonatal mortality in domestic animals, and cause considerable economic loss in the farming industy. SAG1 is the major surface antigen of the tachyzoite. It could induce IgG, IgM, IgA antibodies and CD8+T cell to kill Toxoplasma godii. It has been shown that P30, the major surface antigen of Toxoplasma gondii is a highly immunogenic protein and an appropriate antigen for development into a caccine. The gene encoding surface antigen 1(P30)of Toxoplasma gondii was cloned into the plasmid pET-32a(+) and subsequently expressed in Escherichia coli(E.coli) BL21(DE3) as a glutathi-one-s-transferase fusion protein. The recombinant SAG1(rSAG1) was evaluated in an enzyme-linked immunosorbent assay(ELISA) for serological diagnosis of toxoplasmosis. It is very important to establish an effective and convenient method to diagnose toxoplasmosis.According to P30 gene sequence of toxoplasma, two pairs of primers were designed and synthesized, which were used to amplify the part different fragments of P30 gene from the genomic DNA of NT strain of T.gondii with PCR, and then the corresponding T/A clones were constructed with the part fragments. PCR amplification and enzyme digestion after identifying the two fragments, then the fragments were sequenced and subcloned into pET-32(a)+ plasmid, then transformed into susceptible E.coli DH5αto express. The positive clones were selectec from the ampicillin positive LB plate after identification by PCR and enzyme digestion. The expression of the target genes was induced with 1mmol/L IPTG after all the recombinant plasmids were identified and transformed into the competent cells of the host cell E.coli BL21 (DE3). The expression for the fusion proteins were analyzed by SDS-PAGE and Western-blotting. Results showed that the recombinant vectors were successfully constructed and the relative molecular mass of the fusion proteins were measured to be 48.3kDa. The purified fusion proteins were obtained by Ni-sepharose affinity chromatography, which could be applied to learn its immunogenicity.The gene encoding surface antigen 1(SAG1) of Toxoplasma gondii was cloned into the plasmid pET-32a(+) and subsequently expressed in Escherichia coli(E.coli) BL21(DE30) as a glutathi-one-s-transferase fusion protein. The recombinant SAG1(rSAG1) was refolded using 8M urea solution followed purifying by affinity chromatography with Ni2+-NTA and thereafter evaluated in an enzyme-linked immunosorbent assay(ELISA) for serological diagnosis of toxoplasmosis. The positive and negative of Toxoplasma gondii sera were used for developing an indirect ELISA for P30 antibody detection. The suitable concentration of antigen was determined by square matrix titration,and the specificity and repetitiveness of this method were determined. 96-well plates were coated with 2.75μg/mL commercially synthesized P30 protein, optimal blocked with 5% skimmed milk at 37℃for 2h, optimal serum samples were diluted by 1:600 and incubated at 37℃for 1h, optimal HRP-conjugated secondary antibodies were diluted by 1:2000 and incubated at 37℃for 1h, and the chromogen, TMB was added and after 15min incubation at 37℃, the reaction was stopped by 2M H2SO4. It was not occurred cross reaction with the positive serum of the HCV,PRRSV and PCV.Serum samples from pig farms were tested by IHA and ELISA at the same time, the coincidence rate is up to 90.17%,the difference between the two methods is not remarkable. These results indicate that the ELISA has high specificity and good repetitiveness. This method offered a sort of technique instrument carried through the toxoplasmosis diagnosis and the serological survey, moreover settled prophase the basic work for the developing and exploiture of reagent box.