环形泰勒虫spm-N蛋白的表达及间接ELISA诊断方法的建立

发布时间：2019-03-15 18:47

【摘要】：为深入研究环形泰勒虫(Theileria annulata)spm-N基因的功能,以建立一种方便快速检测环形泰勒虫病的诊断方法,本论文对环形泰勒虫spm-N基因进行了扩增,并将目的基因与原核表达载体pET30a(+)和真核表达载体PGBKT7进行连接,首先用原核表达质粒转化BL21(DE3)感受态细胞,经IPTG诱导表达重组蛋白,获得了以分泌型表达的重组蛋白,以此作为抗原,建立了诊断环形泰勒虫病的间接ELISA诊断方法,该方法与中华泰勒虫(T.sinensis),瑟氏泰勒虫(T.sergenti),牛巴贝斯虫(Babesia bovis)等梨形虫的阳性血清均不发生交叉反应,表明具有良好的敏感性和特异性,可以用于环形泰勒虫病的诊断和流行病学调查;然后又用真核表达质粒转化酵母Y187感受态细胞,检测其在酵母菌株中的自激活性、对酵母生长的影响及其在酵母细胞中的表达情况。结果显示,重组质粒在酵母细胞内成功表达,且对酵母细胞的生长没有影响,无毒害作用,没有自激活活性,可被用于筛选与环形泰勒虫spm-N蛋白相互作用的物质。
[Abstract]:In order to study the function of (Theileria annulata) spm-N gene and to establish a convenient and rapid diagnostic method for the detection of Thaleria circularis, the spm-N gene was amplified in this paper, and the results were as follows: (1) in this paper, the spm-N gene of Taylor ringworm was amplified by PCR. The target gene was ligated with prokaryotic expression vector pET30a () and eukaryotic expression vector PGBKT7. First, the prokaryotic expression plasmid was transformed into BL21 (DE3) competent cells. The recombinant protein was induced by IPTG to express the recombinant protein, and the recombinant protein expressed in secretory form was obtained. Based on this antigen, an indirect ELISA diagnostic method was established for the diagnosis of circular Taylor disease. This method was associated with T.sinensis, T.sergenti, and Tylutella sinensi, and was used as antigen for the diagnosis of Thalerellosis annularis. None of the positive sera of Bovine Babesia (Babesia bovis) and other piriform worms showed cross-reaction, which indicated that they had good sensitivity and specificity, and could be used in the diagnosis and epidemiological investigation of Thalerellosis circialis. Then Eukaryotic expression plasmid was used to transform yeast Y187 competent cells to detect its self-stimulating activity in yeast strain, its effect on yeast growth and its expression in yeast cells. The results showed that the recombinant plasmid was successfully expressed in yeast cells and had no effect on the growth of yeast cells, no toxic effect and no self-activating activity. The recombinant plasmid could be used to screen the substances interacting with the spm-N protein of Taylor ringworm.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

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