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目的原核表达刚地弓形虫(Toxoplasma gondii)微线蛋白16(micronemal protein 16,Tg MIC16)的3个基因片段,并分析3个重组蛋白的免疫反应性。方法参照Gen Bank中Tg MIC16基因序列,根据功能活性区将其分为3个片段(M16D1、M16D2、M16D3),分别设计引物,以刚地弓形虫RH株速殖子RNA为模板,通过RT-PCR扩增,获得预期的3个DNA片段,双酶切后连接至原核表达载体p ET-32a(+),将重组质粒转化入大肠埃希菌(Escherichia coli)TOP10,经PCR、双酶切筛选阳性质粒,测序确认后转化至E.coli Rosetta,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表达产物。重组蛋白经镍柱纯化后,分别以抗组氨酸(His)单抗和弓形虫感染兔血清为一抗,Western blotting分析其免疫反应性。结果 Tg MIC16 3个基因片段的RT-PCR扩增产物分别为1 806、1 290、855 bp。双酶切和测序结果显示,3个Tg MIC16片段的重组表达质粒构建成功。SDS-PAGE分析结果显示,3个重组蛋白成功表达,且均为以包涵体的形式表达,相对分子质量(M_r)分别为88 000、68 000、52 000。Western blotting分析结果显示,3个纯化的表达蛋白均能被抗His单抗和弓形虫感染兔血清识别。结论成功构建并表达Tg MIC16的3个功能活性区,且3个重组蛋白均表现出良好的免疫反应性。
Objective To express three gene fragments of Toxoplasma gondii microneglial protein 16 (Tg MIC16) in prokaryotic cells and analyze the immunoreactivity of the three recombinant proteins. Methods According to the sequence of Tg MIC16 gene in GenBank, three fragments (M16D1, M16D2 and M16D3) were designed according to the functional domain. The primers were designed respectively. The tachyzoites of T. gondii RH strain were used as template, PCR amplification, obtained the expected three DNA fragments, double digested and then connected to the prokaryotic expression vector p ET-32a (+), the recombinant plasmid was transformed into Escherichia coli TOP10, by PCR, double digestion Positive plasmids were screened and confirmed by sequencing. The recombinant plasmids were transformed into E.coli Rosetta and induced by IPTG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) to detect the expression product. The purified recombinant protein was purified by nickel column, the anti-histidine (His) monoclonal antibody and Toxoplasma gondii sera were used as the primary antibody, and the immunoreactivity was analyzed by Western blotting. Results RT-PCR products of Tg MIC16 gene fragments were 1 806, 1 290 and 855 bp, respectively. Double enzyme digestion and sequencing showed that the three Tg MIC16 fragments were successfully constructed. SDS-PAGE analysis showed that the three recombinant proteins were successfully expressed, and all of them were expressed as inclusion bodies. The relative molecular mass (M_r) were 88 000, 68 000 and 52 000, respectively. The results of Western blotting showed that all the three expressed proteins could be recognized by anti-His monoclonal and Toxoplasma gondii-infected rabbit sera. Conclusion Three functional active regions of Tg MIC16 were successfully constructed and expressed, and all three recombinant proteins showed good immunoreactivity.