多头带绦虫dUTPase基因的原核表达、组织定位及酶学活性分析

发布时间：2018-03-20 10:57

本文选题：多头带绦虫　切入点：dUTPase基因　出处：《畜牧兽医学报》2017年12期 　论文类型：期刊论文

【摘要】：探讨多头带绦虫脱氧尿苷三磷酸酶(Tm-dUTPase)的基因特征,并初步分析Tm-dUTPase的酶学活性。通过原核表达,得到重组Tm-dUTPase蛋白,并对其进行Western blot分析、免疫荧光定位及酶学活性分析。序列分析结果显示,Tm-dUTPase基因ORF框为447bp,编码148个氨基酸,预测的蛋白质分子大小为16.39ku,等电点为6.263;与猪囊尾蚴和豆状囊尾蚴dUTPase基因的氨基酸序列相似性分别为97%和91%。原核表达的TmdUTPase为可溶性蛋白,纯化后的蛋白质质量浓度为0.907mg·mL~(-1),Western blot分析显示,该蛋白质能够被山羊脑多头蚴阳性血清所识别,具有较强的反应原性。免疫荧光定位显示Tm-dUTPase主要分布于多头带绦虫成虫的体内实质区,同时广泛分布于多头带绦虫幼虫(脑多头蚴)的头节和包囊囊壁外层。酶学活性试验测得重组Tm-dUTPase的比活力为986.29U·mg~(-1),EDTA能够抑制Tm-dUTPase活性,而金属离子Mg~(2+)、Zn~(2+)、Cu~(2+)能够增强Tm-dUTPase活性。上述结果为进一步研究Tm-dUTPase基因的生物学功能奠定了基础。
[Abstract]:To study the gene characteristics of deoxyuridine triphosphatase (Tm-dUTPase) of Taenia multiceps, and to analyze the enzymatic activity of Tm-dUTPase. The recombinant Tm-dUTPase protein was obtained by prokaryotic expression and analyzed by Western blot. The result of sequence analysis showed that the ORF frame of Tm-dUTPase gene was 447bp, encoding 148 amino acids. The predicted protein molecular size is 16.39kuU, the isoelectric point is 6.263, and the amino acid sequence similarity with dUTPase gene of cysticercus cellulosae and lentiform cysticercus is 97% and 91.The prokaryotic expression of TmdUTPase is a soluble protein. Western blot analysis of the purified protein with a concentration of 0.907mg 路mL ~ (-1) showed that the protein could be recognized by the positive sera of goat brain polycaria. Immunofluorescence localization showed that Tm-dUTPase was mainly distributed in the parenchymal region of Taenia multiceps. The specific activity of recombinant Tm-dUTPase was 986.29 U 路mg ~ (-1) U 路mg ~ (-1). The metal ion Mg~(2 can enhance the activity of Tm-dUTPase, which lays a foundation for the further study of the biological function of Tm-dUTPase gene.
【作者单位】：四川农业大学动物医学院;成都农业科技职业学院;攀枝花农林科学研究院;
【基金】：四川省科技支撑计划项目(2015NZ1504)
【分类号】：S852.734

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