【作者】 都建；

【导师】 沈继龙； 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2004， 硕士

【摘要】 目的 体外扩增弓形虫RH株信号转导蛋白14-3-3（Toxo14-3-3）和膜表面主要抗原SAG1编码基因，克隆至原核表达载体pET28a，并表达出Toxo14-3-3和SAG1重组蛋白。利用此重组蛋白，探讨rToxo14-3-3和rSAG1用于诊断弓形虫感染的价值。方法收集、纯化弓形虫RH株速殖子，提取总RNA，逆转录生成cDNA，然后以其为模板扩增出Toxo14-3-3、SAG1编码基因，将扩增产物克隆入原核表达载体pET28a。在原核细胞表达Toxo14-3-3、SAG1重组蛋白。重组子双酶切、PCR和测序鉴定，转化大肠杆菌BL21．并以IPTG诱导表达。制备、纯化rToxo-14-3-3和rSAG1蛋白抗原，利用纯化的rToxo-14-3-3和rSAG1抗原，间接ELISA法探讨其诊断弓形虫病的价值。结果RT-PCR扩增出一具有803bp完整开放读码框的Toxo-14-3-3的cDNA片段和1011bp完整开放读码框的SAG1的cDNA片段，成功地将其克隆入pET28a；将pET28a／Toxo14-3-3和pET28a／SAGl分别做EcoRI、Xho Ⅰ和EcoRI、Hind Ⅲ双酶切，分别获得了一个大小与其相应PCR扩增产物一致的插入片段；用IPTG诱导重组质粒转化的大肠杆菌BL₂₁（DE₃），分别得到了30.7kDa和34.6kDa的重组蛋白；rToxo-14-3-3的融合蛋白可被弓形虫速殖子感染的兔血清、弓形虫缓殖子感染的鼠血清、抗14-3-3epsilon多克隆抗体、弓形虫感染阳性病人血清识别；rSAG1的融合蛋白可被弓形虫速殖子感染的兔血清识别。间接ELISA和Western-blot结果显示，rToxo-14-3-3作为抗原检测急、慢性弓形虫感染可获得很高的敏感性和特异性，具有实用价值。结论成功地从弓形虫RH株基因组DNA中获取了14．3．3基因和SAGl基因，构建了pET28a／Tox014—3．3和pET28a／SAGl重组质粒，并获得了高效表达，间接ELISA及Western-blot表明，rToxo．14．3．3在急慢性弓形虫感染诊断中具有实用价值。本研究为弓形虫病的免疫学诊断和蛋白质疫苗的研究创造了条件。更多还原

【Abstract】 Objective: To generate the signaling protein 14-3-3 of T. gondii (Toxol4-3-3) andsurface antigen SAG1 following inserts ligation, cell transformation, and induced expression for quick and accurate immunodiagnosis of toxoplasmosis. Methods: The specific primers were designed and synthesized. The cDNA fragment encoding T. gondii signaling protein 14-3-3 and surface antigen SAG1 were amplified by RT-PCR from RNA of RH strain tachyzoites. The PCR products were cloned into pGEM-T vector and recombinants were screened by PCR, enzyme digestion and sequencing. Then the 14-3-3 gene and SAG1 were subcloned into pET28a expression vector, and-E. coli BL21(DE3) was used for transformation. The expression of Toxo 14-3-3 and SAG1were induced by IPTG and identified by Western blot. In order to evaluate rToxo14-3-3 and rSAGl, we use them to detect positive sera from Toxoplasma-infected cases. Results: The size of pGEM-T,Toxo14-3-3, pET28a, Toxol4-3-3 digested with EcoR I and Xho I were all the same in length as the Toxol4-3-3-PCR product; pGEM-T ,SAGl, pET28a,SAGl digested with EcoR I and Hind III were all the same in length as the SAG1-PCR product . DNA sequencing indicated the Toxol4-3-3 gene had an open reading frame of 803bp, which encodes 266 amino acid with a molecular standard of 30.7 kDa identical to Toxo 14-3-3 gene submitted to GenBank (ABO 12775); SAGl has an open reading frame of 1011bp, which encodes 336 amino acid with a molecular standard of 34.81 kDa identical to SAGl gene submitted to GenBank (S76248). Conclusion: Toxo 14-3 -3 and SAGl gene were successfully cloned-and expressed in E.coli BL21.The fusion proteins have been confirmed by serai recognition of rabbit infected with tachyzoites. After simple purification procedure, the expressed product was identified by western blot. These results of serai detection test indicate that the ELISA using rToxo 14-3-3 can be useful for effective and reliable serodiagnosis of toxoplasmosis.更多还原