【摘 要】
：
目的 克隆和原核表达脑多头蚴dUTPase基因并分析其酶学活性.方法 从羊脑多头蚴原头节提取总RNA,采用RT-PCR技术扩增脑多头蚴dUTPase基因,将其克隆入原核表达载体pET-32a(+)中,构建pET-32a-dUTPase表达载体,转化大肠杆菌BL21(DE3)后诱导表达,用SDS-PAGE和Western-blot检测表达产物,用免疫荧光组化法检测dUTPase基因在多头带绦虫成虫体
【机 构】
：
四川农业大学动物医学院动物寄生虫病研究中心,成都 611130;成都农业科技职业学院,成都 611130
【出 处】
：
中国畜牧兽医学会兽医寄生虫学分会第十三次学术研讨会
论文部分内容阅读
目的 克隆和原核表达脑多头蚴dUTPase基因并分析其酶学活性.方法 从羊脑多头蚴原头节提取总RNA,采用RT-PCR技术扩增脑多头蚴dUTPase基因,将其克隆入原核表达载体pET-32a(+)中,构建pET-32a-dUTPase表达载体,转化大肠杆菌BL21(DE3)后诱导表达,用SDS-PAGE和Western-blot检测表达产物,用免疫荧光组化法检测dUTPase基因在多头带绦虫成虫体内的表达分布情况,并对dUTPase重组蛋白进行酶学活性分析.结果 基因序列分析结果表明脑多头蚴dUTPase基因与猪囊尾蚴、豆状囊尾蚴和亚洲带绦虫dUTPase基因的核苷酸序列相似性分别为100％、96％和99％；纯化后的原核表达产物中dUTPase蛋白的含量为0.907 mg.mL-1.免疫荧光染色结果显示dUTPase蛋白大量分布于多头带绦虫成虫的体壁处；纯化的脑多头蚴dUTPase的比活力为986.29U.mg-1,酶学活性试验证实dUTPase蛋白能特异性降解dUTP,同时EDTA可以抑制dUTPase的活性,而Mg2+可增强dUTPase的活性.结论 本研究成功表达了脑多头蚴dUTPase基因并鉴定了它的酶学活性,为进一步研究dUTPase在脑多头蚴中的生物学功能奠定了基础.
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