【作者】 王维；

【导师】 蒋作君； 沈继龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2002， 硕士

【摘要】 弓形虫(Toxoplasma gondii)是严重危害人类健康的机会致病性寄生虫。目前，弓形虫病研究的两大热点仍然是寻找敏感性高、特异性强的诊断抗原和有效的候选疫苗分子，基因重组技术为该研究提供了可行之路。已知生物体进化过程中，14-3-3家族是一组真核细胞表达的保守蛋白，细胞内五十多种信号蛋白是其配体，通过结合激酶、磷酸酶、跨膜受体等多种配体成为信号转导和细胞周期调控介质。本课题从弓形虫分裂增殖生命活动重要环节——细胞信号转导途径寻找重组诊断和候选疫苗分子。根据GenBank收录的弓形虫Beverley株猫肠上皮细胞期信号蛋白14-3-3 mRNA序列，设计并合成引物，RT-PCR，扩增出RH株速殖子14-3-3(Toxo14-3-3)基因，克隆入pGEM-T载体，经酶切和PCR法鉴定及DNA测序证实；Toxo14-3-3具有一个长度为798bp的完整开放阅读框(ORF)，编码265个氨基酸，理论分子量31KDa，与GenBank收录(编号为ABO12775)的弓形虫14-3-3基因完全一致；将目的基因亚克隆入双表达质粒pBK-CMV、转化宿主菌E.coliXL-1 blue，获得pBK-CMV/Toxo14-3-3阳性重组子，IPTG诱导目的蛋白表达，表达产物经SDS-PAGE显示在31Kda—43KDa之间有一条明显的蛋白表达条带，理论推测为Toxo 14-3-3蛋白(31 KDa)和β-半乳糖苷酶(3 KDa)融合蛋白，但在凝胶中的实际迁移率慢于理论预期值(34 KDa)，分子量超出约5KDa，Western-blot法鉴定该蛋白表达条带能被兔抗鼠14-3-3ε多克隆抗体识别，证实为Toxo14-3-3融合蛋白。更多还原

【Abstract】 Toxoplasma gondii(T. gondii) is a harmful opportunistic protozoa. However. So far no practical antigens has been obtained. Currently the two issues of T. gondii are still in the search of the antigens of diagnosis with high sensitivity and specificity and of candidated molecular vaccine. The recombinant DNA technology is an available approach to screen the recombinant proteins. As known, an challenged field is the cell signal transduction between host immune system and parasites. So we manage to develop the signal transduction 14-3-3 protein which control some basic biochemical metabolism of T. gondii. 14-3-3 is an evolutionarily conserved protein expressed in all eukaryotic cells including plants, yeasts, protozoa, helminth, insects and mammalian animals. 14-3-3 proteins exhibit a remarkable degree of sequence conservation among species and share some essential biochemical properties. 14-3-3 proteins bind multitude of functionally diverse signaling protein, including kinases. phosphatases. transrnembrane receptors and become signal transduction and cell cycle regulation f mediator. Therefore, a pair of primers were designed and synthesized based on the . mRNA sequence cloned from the enteroepithelial stage of T. gondii (Beverley strain). Toxo 14-3-3 gene(RH strain) was amplified by RT-PCR. the PCR products were cloned into pGEM-T vector and the inserted DNA fragements w-ere confirmed by restriction -endonuciease digestion and PCR followed by DNA sequencing. A complete openreading frame(ORF) of 798bp was verified, which encodes 265 amino acid with theorial molecular weights of 31KDa and is identical to Toxol4-3-3 gene submitted to GenBank(Accession No ABO 12775). Then, the Toxo14-3-3 DNA fragments were subcloned into pBK-CMV expressing vector and transformed the recombinant plasmid into E.coli XL-1 blue. The positive recombinant pBK-CMV/Toxo 14-3-3 was induced by IPTG and the cell h sates were analysed with SDS-PAGE. which showed an obvious band of fusion protein between 31KDa?3KDa. The migration of the expression, however, is 5KDa slower than expected. The fusion protein can be recognized by anti-14-3-3 ?immunogen.更多还原