Huang Xiaohong, Xuan Xuenan, Suzuki Hiroshi, Sugimoto Chihiro, Nagasawa Hideyuki, Fujisaki Kozo, Mikami Takeshi, Igarashi Ikuo
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Clin Diagn Lab Immunol. 2002 Nov;9(6):1343-7. doi: 10.1128/cdli.9.6.1343-1347.2002.
A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats.
构建了携带刚地弓形虫SAG2基因的杆状病毒，并在昆虫细胞中表达了重组SAG2蛋白（S-rSAG2）。感染刚地弓形虫的猫和猪的血清可识别S-rSAG2。用S-rSAG2免疫的小鼠产生了高滴度的特异性免疫球蛋白G2a（IgG2a）和IgG1抗体。在间接荧光抗体试验中，所有抗S-rSAG2的小鼠抗血清与天然寄生虫反应强烈，但抗大肠杆菌表达的rSAG2（E-rSAG2）的抗血清仅显示非常弱的反应，尽管在蛋白质印迹分析中对变性抗原刚地弓形虫裂解物的反应未发现明显差异。结果表明，S-rSAG2在抗原性和免疫原性方面均优于E-rSAG2。用S-rSAG2进行的酶联免疫吸附测定（ELISA）可清楚地区分30只无特定病原体猫和4只实验感染猫的血清。用ELISA检测了日本家猫的血清样本，并与乳胶凝集试验（LAT）和用E-rSAG2进行的ELISA进行了比较。在187份样本中，所有35份LAT阳性血清对S-rSAG2和E-rSAG2均有强烈反应。在152份LAT阴性血清中，18份在用S-rSAG2进行的ELISA中呈阳性，而在用E-rSAG2进行的ELISA中仅2份呈阳性。尽管三种方法之间存在显著相关性，但用S-rSAG2进行的ELISA比其他方法更敏感，这可能是因为S-rSAG2与天然抗原具有一些共同的构象结构。结果表明，S-rSAG2将是检测猫刚地弓形虫感染的有用试剂。
