| The obligate intracelllar protozoan parasite Toxoplasma gondii (T. gondii)is worldwide spread, and is responsible for toxoplasmosis and able to infect all species of mammals (inclde human) and birds. Nearly one-third of the human popluation is exposed to the threat of this parasite,It is a severe zoonosis and genetic disorders disease. It can case considerable economic loss on the people health and farming losses. Therefore, the effective prevention and timely diagnosis of toxoplasmosis is particlarly important. At present, the effective protection of gene of Toxoplasma gondii screening work is studying at home and abroad. While the dense granle protein is a kind of important secretion of metabolic antigen of Toxoplasma gondii, it has strong Original reaction and immunogenicity in human and animal experimental infection. So far, it have been reported to have10different tachyzoites dense granle protein (GRA1-GRA10). In the diagnosis of toxoplasmosis and immune prevention has potential value. Where GRA3can stimulate the body to generate humoral immunity,The antigen have potential application value for diagnosis of toxoplasmosis and as a vaccine candidate molecular.This study aims is main in constructing GRA3prokaryotic expression vector, And use the expression of recombinant GRA3protein to prepare GRA3monoclonal antibody, and it is going to identify and applicate GRA3monoclonal antibody. The antigen has potential application value for diagnosis of toxoplasmosis and as a vaccine candidate molecularThis study according to the GenBank AF414079published in Toxoplasma gondii GRA3gene seqences. The687bp RH strain GRA3gene of Toxoplasma gondii were cloned by polymerase chain reaction (PCR), On the gene seqence analysis, the result shows that the gene fragment is GRA3gene of toxoplasma; And it is Succeed in construct the prokaryotic expression vector pGEX-GRA3; induced by IPTG,they were expressed in Ecoli BL21, by means of SDS polyacrylamide gel electrophoresis display,that obtained the moleclar weight of50ku fusion protein GST-GRA3, it is most of the soluble protein. Western-Blotting immunoblotting analysis, fusion protein reacted with Toxoplasma gondii positive serum specific.The results show the protein has some antigenicity and specificity.For the further study of recombinant proteins is as novel diagnostic antigen of possibility. This study is the efficient expression of recombinant GRA3protein immunized BALB/c mice, sing cell fusion technology, after indirect ELISA method screened and limited dilution, obtained a hybridoma cell strain5E4. After identification, the McAb subtype of type is IgGl. ELISA and Western blotting test results show that,5E4MAb can distingished Toxoplasma gondii GRA3protein,.It is suggested5E4McAb recognition epitopes may be located in the GRA3protein conserved region.And the Rabbit anti-Toxoplasma gondii IgG is as coated antibody against recombinant GRA3protein, monoclonal antibody is as the detection of antibodies, Antibody sandwich ELISA method was Successed in establishing of the detection of Toxoplasma gondii, the method was for detection of Toxoplasma gondii in sensitivity that is to0.154g/ml. By the method to a pig farm in Yanbian152pigs were detected, the positive rate is13.2%.The study cloned and expresed the GRA3gene of Toxoplasma gondii and prepared of recombinant GRA3protein McAb, that established a monoclonal antibody sandwich ELISA method. For clinical research of Toxoplasma gondii diagnostic reagents, reagent treatment laid the foundation, at the same time for detection of Toxoplasma gondii and the regional epidemiological investigation provided a simple and rapid diagnosis method. |
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