Expression Of SAG1 In E.coli And Development Of An Indirect ELISA For Detection Of Anti-T.gondii Antibodies

Toxoplasma gondii(T. gondii), an obligate intracellular protozoan parasite, is able to infect all species of mammals(including human) and birds. It is an opportunistic protozoan and responsible for toxoplasmosis. The hosts usually have no symptom with a normal immune system. In the hosts who are immunocompromised (e.g, neoplastic disease, organ transplantation, and AIDS patients), the tachyzoites of T.gondii may frequently cause a serious symptoms and subsequent complications, even death sometimes.SAG1 protein, a major surface antigen of T. gondii, is a highly immunogenic protein in diagnosis and vaccine. Only a little can it be derived from T. gondii however. When we need large amounts of pured SAG1, genetic engineering technique is chosen a lot to experess the target protein in vitro.According to the SAG1 gene sequence of T. gondii published on GenBank, a pair of primers was designed to amplify a part of gene of SAG1 by PCR technique. The PCR product was purified and cloned into pMD18-T vector. When the target DNA sequence was testified, it was subcloned into prokaryotic expressing vector pET-28a(+) and transfected into E.coli strain BL21. Induced by IPTG, the transformant express the recombinant protein. The recombinant protein after purified was analyzed by SDS-PAGE and Western-blotting. The result revealed that the protein had a molecular weight between 26kD and 35kD, which could be recognized specifically by anti-T.gondii antibody. With a good immunogenicity, the recombinant protein can be used as an antigen in ELISA to detect anti-T.gondii antibody in sera of rat. Using the recombinant protein as coated antigen, an indirect ELISA for detection of anti-T.gondii antibody was established. A series of experiments was taken to optimize the ELISA. The concentration of coated antigen, the dilution of serum and second antibody, the standard of determining as positive sample were defined. The results of specificity test, sensitivity test, stability test and contrast test suggested that the diagnosis method was specific and sensitive. There was an application prospect for the T. gondii ELISA.