【摘要】 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。更多还原

【Abstract】 Objective To construct a high-efficiency prokaryotic expression system for the MIC3 gene of Toxoplasma gondii in order to produce a greater yield and highly purified recombinant MIC3 protein and evaluate its potential for serodiagnosis of T. gondii infection. Methods A gene encoding mature MIC3 protein was amplified by PCR from genomic DNA. The PCR product was then inserted into a pGEX-4T-3 expression vector. Escherichia coli transformed with the recombinant plasmid pGEX-4T/MIC3 was induced with IPTG to express the recombinant MIC3 (rTgMIC3) as a fusion protein with glutathione S transferase. rTgMIC3 purified using glutathione sepharose 4B was analyzed by SDS-PAGE and Western blot analysis with antisera from mice infected with the T. gondii ME49 strain. Enzyme-linked immunosorbent assay (ELISA) with rTgMIC3 as an antigen was developed and used to detect specific antibodies in uninfected mice,mice infected with the T. gondii ME49 strain,and mice infected Neospora caninum,respectively. Results The sequence of the amplified MIC3 in this study was identical to the MIC3 sequence in GenBank (accession No. AF509564). The measured molecular mass of rTgMIC3 agreed with the theoretical molecular mass. The yield of purified rTgMIC3 was more than 5 mg per liter of culture medium. Sera from mice infected with the T. gondii ME49 strain had a strong positive reaction to rTgMIC3. Antiserum against rTgMIC3 recognized natural MIC3. ELISA with rTgMIC3 clearly distinguished sera infected with T. gondii from uninfected sera and sera infected with N. caninum. Conclusion Results suggested that rTgMIC3 is suitable for use in developing a serodiagnostic test for T. gondii infection.更多还原