The Immunological Characterization of the Epimastigote Antigens of Trypanosoma Cruzi with Chronically Infected Mouse Sera

• Main Author: 程新民
• Other Authors: 趙大衛
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/60146625265737702459

碩士 === 國立中山大學 === 生命科學研究所 === 85 === Trypanosoma cruzi is a protozoan parasite that can cause Chagas' disease in the Central and South America. The disease was divided into two phases, acute and chronic. In the acute phase, a large number of bloodstream form (BSF) trypo-mastigotes appear in the infected host circulation. While the disease is usually asymptomatic and trypanosomes are hardly seen in the peripheral blood during the chronic phase. In this study, BALB/c mice were infected intraperitoneally with 1×106 BSF of T. cruzi. In the first week, the number of parasite was few (<1×104) in the host blood. In the 2nd and 3rd week, there were 105-107 BSF/ml in the blood of the infected mice. The numbers of parasites decreased in the 4th week and could not detect with hemacytometer in the 5th week. Epimastigotes were cultivated to the exponential growth phase in a liquid metacyclic stage culture medium and harvested for antigen preparation. Sera collected on the 28th day of infection recognized three antigens from epimastigotes of molecular weights of 90, 65, and 61kDa. Results obtained from lectin binding analysis showed that 90kDa polypeptide is an O-linked glycoprotein, whereas 65 and 61kDa polypeptides are not glycoproteins. Epimastigote antigens were purified with Sephacryl S-300 chromatography. The first peak presented high immune response with the sera of the infected mice on the 28th day. The Western blot of the fraction 22 showed only a 65kDa polypeptide (p65), which reacted with the chronic antisera, and the pI value was 6.62 with the phast IEF electrophoresis. The polypeptide was used to diagnose the chronic mouse's sera; the efficiency of the ELISA is twice to eightfold than the crude antigen of epimastigotes. The fraction 22 was highly specific and sensitive to react with the sera of the infected mice on the 28th day. The efficiency of complement cytolysis of immunized rabbit sera was 56 percent, and the efficiency of the control group was only 6 percent. Mice immunized with fraction 22 (700ng/mice) exhibited only 40% of immunized mice presented detectable parasites in blood after challenge. All the parasitemia decreased when compared to the control group. Survival of the immunized mice was 100% after challenged with 2.62×104 BSF.