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目的构建弓形虫微线体蛋白3(MIC3)原核表达体系,制备高纯度MIC3蛋白,评价其在血清学诊断方面的应用价值。方法应用PCR技术扩增去除信号肽序列的MIC3基因,将其插入原核表达载体pGEX-4T-3中,转化入大肠埃希菌DH5α中并克隆,用IPTG诱导表达与谷胱甘肽S转移酶融合的MIC3蛋白(rTgMIC3),用谷胱甘肽琼脂糖凝胶4B纯化rTgMIC3,用SDS-PAGE和Western blot鉴定rTgMIC3纯度及抗原性。以纯化的rTgMIC3为抗原,用ELISA检测人工感染刚地弓形虫ME49株的BALB/c小鼠血清IgG抗体,并观察其消长动态。结果扩增的MIC3基因与GenBank中相应基因序列(AF509564)的相似性为100%;表达的MIC3融合蛋白分子质量为61.2ku,与理论预测值一致,每升菌液能纯化出5mg的rTgMIC3。Western blot显示,rTgMIC3能被弓形虫ME49株感染鼠血清识别;ELISA显示,感染弓形虫ME49株鼠血清能与rTgMIC3起阳性反应。结论本实验成功实现了MIC3基因在大肠埃希菌中的高效表达,以rTgMIC3为抗原建立的ELISA具有较高的敏感性和特异性,为开发弓形虫感染快速免疫诊断方法及弓形虫侵入宿主细胞的分子机制的研究奠定了基础。
Objective To construct the prokaryotic expression system of MIC3 in Toxoplasma gondii and to prepare high purity MIC3 protein and to evaluate its value in serodiagnosis. Methods The MIC3 gene was amplified by PCR and inserted into prokaryotic expression vector pGEX-4T-3. The recombinant plasmid was transformed into Escherichia coli DH5α and cloned into E.coli DH5α. The MIC3 gene was induced to express with glutathione S transferase The fusion protein MIC3 (rTgMIC3) was purified by glutathione sepharose 4B, and the purity and antigenicity of rTgMIC3 were identified by SDS-PAGE and Western blot. Using purified rTgMIC3 as antigen, the serum IgG of BALB / c mice infected with Toxoplasma gondii ME49 artificially infected with IgG was detected by ELISA, and its growth and development was observed. Results The similarity of the amplified MIC3 gene to the corresponding gene sequence in GenBank (AF509564) was 100%. The molecular weight of the expressed MIC3 fusion protein was 61.2ku. Consistent with the theoretical predictions, 5 mg of rTgMIC3 was purified per liter of bacterial culture. Western blot showed that rTgMIC3 could be recognized by the serum of mice infected with Toxoplasma gondii ME49 strain. ELISA showed that the sera from ME49 infected mice could positively react with rTgMIC3. CONCLUSIONS: The high expression of MIC3 gene in Escherichia coli was successfully achieved in this experiment. The ELISA with rTgMIC3 as antigen has high sensitivity and specificity. In order to develop a rapid immunoassay for the detection of Toxoplasma gondii infection and invasion of host cells by Toxoplasma gondii The molecular mechanism of the study laid the foundation.