多房棘球蚴myophilin、LDH蛋白的免疫原性与循环核酸检测研究

发布时间：2018-02-23 18:38

本文关键词： 多方棘球蚴亲肌肉蛋白乳酸脱氢酶免疫原性循环核酸检测　出处：《青海大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.克隆表达多房棘球蚴亲肌肉蛋白(myophilin)和乳酸脱氢酶(LDH)基因,鉴定myophilin和LDH重组蛋白的免疫原性,为基于myophilin和LDH蛋白的多房棘球蚴感染的免疫诊断研究奠定理论基础。2.检测泡型包虫病患者血清中特异的多房棘球蚴miRNA和DNA分子,将其作为虫体感染的生物标志物,为基于循环核酸的多房棘球蚴感染的检测方法研究提供理论依据。方法:1.通过PCR技术克隆myophilin和LDH基因,将其连接入pET15b载体中,构建重组表达质粒,再转化至E.coli Rosetta(DE3)感受态细胞进行诱导表达。通过SDS-PAGE检测重组蛋白的表达形式,Ni-IDA树脂亲和层析纯化重组蛋白。通过Western blotting法鉴定重组蛋白的免疫原性,ELISA法检测泡型包虫病患者(57例)、囊型包虫病患者(33例)和正常人(50例)血清,初步评价重组蛋白的免疫诊断效果。通过DNAstar软件预测重组蛋白的B、T细胞抗原表位。2.通过高通量测序技术分析泡型包虫病患者血清miRNA表达情况,从中确定血清中特异的多房棘球蚴mi RNA分子。通过PCR技术检测泡型包虫病患者血清中的多房棘球蚴DNA,确定血清中特异的多房棘球蚴DNA序列。结果:1.成功克隆了myophilin和LDH基因并表达纯化出相应的重组蛋白。Western blotting结果显示,myophilin和LDH重组蛋白可被泡型和囊型包虫病患者血清识别,而不被正常人血清识别。ELISA结果显示,myophilin重组蛋白对泡型和囊型包虫病患者血清的诊断敏感性分别为80.70%和78.79%;LDH重组蛋白对泡型和囊型包虫病患者血清的诊断敏感性分别为84.21%和84.85%。DNAstar软件预测显示,myophilin蛋白可能有8个B细胞抗原表位,分别为15-29、41-48、72-77、110-115、130-136、145-161、169-177和181-188位氨基酸;6个T细胞抗原表位,分别为46-53、59-75、82-96、100-108、117-123和155-163位氨基酸。LDH蛋白可能有4个B细胞抗原表位,分别为52-57、81-87、97-102和221-227位氨基酸;3个T细胞抗原表位,分别为39-45、76-86和225-235位氨基酸。2.高通量测序筛选出3个可能的多房棘球蚴miRNA分子,分别为emu-miR-124a、emu-miR-87和emu-miR-9,但与人的miRBase数据库比对显示同源性均为100%,不具有特异性。目前使用PCR技术筛选了48对引物,尚未检测出血清中特异的多房棘球蚴DNA序列。结论:1.多房棘球蚴myophilin和LDH重组蛋白具有较高的免疫原性,对包虫病具有较好的免疫诊断价值。2.尚未检测到泡型包虫病患者血清中特异的多房棘球蚴miRNA和DNA分子。
[Abstract]:Objective to clone and express myophilin) and lactate dehydrogenase (LDH) genes from echinococcus multilocularis and to identify the immunogenicity of recombinant myophilin and LDH proteins. To lay a theoretical foundation for the immunological diagnosis of echinococcus multilocularis infection based on myophilin and LDH protein. 2. To detect the specific miRNA and DNA molecules in serum of patients with alveolar hydatid disease, and to use them as biomarkers of the infection of echinococcus multilocularis. Methods: 1. Myophilin and LDH genes were cloned by PCR technique and ligated into pET15b vector to construct recombinant expression plasmid. The recombinant protein was purified by SDS-PAGE and purified by Ni-IDA resin affinity chromatography. The immunogenicity of the recombinant protein was identified by Western blotting. The immunogenicity of the recombinant protein was detected by Elisa. There were 57 cases of disease, 33 cases of cystic hydatid disease and 50 cases of normal controls. DNAstar software was used to predict the antigen epitopes of BT cells of recombinant proteins. The expression of miRNA in serum of patients with hydatid disease was analyzed by high-throughput sequencing. PCR technique was used to detect the multilocular echinococcal DNA in the sera of patients with hydatid disease, and the specific DNA sequence of the multilocularis was determined. Results: 1. Myophilin was cloned successfully. Western blotting showed that myophilin and LDH recombinant proteins could be recognized by the sera of patients with vesicular and cystic hydatidosis. The results of Elisa showed that the sensitivity of myophilin recombinant protein to the diagnosis of vesicular and cystic hydatidosis patients was 80.70% and 78.79 respectively, and the sensitivity of the recombinant protein to diagnosis of vesicular and cystic hydatidosis was 80.70% and 78.79 respectively. The prediction of 84.21% and 84.85. DNAstar software showed that there might be 8 B cell antigen epitopes in the myophilin protein. The amino acids at the 110-115N 130-136C145-161C 169-177 and 181-188, the T cell epitopes at 46-53r-59-79-79-7108107-123 and 155-163, respectively, may have four B cell epitopes, which are 52-57-81-81-87-102 and 221-227 amino acids, respectively, and the three T cell epitopes may have four B cell epitopes, the three T cell epitopes, the three T cell epitopes, and the three T cell epitopes, the three T cell epitopes, the 3 T cell epitopes, the 3 T cell epitopes, the 3 T cell epitopes, the 3 T cell epitopes, the 3 T cell epitopes, the 3 T cell epitopes, the 3 T cell epitopes, The amino acids at position 76-86 and 225-235 of Echinococcus multilocularis were identified by high throughput sequencing. They are emu-miR-124aemu-miR-87 and emu-miR-9, respectively, but the homology with human miRBase database is 100, which is not specific. At present, 48 pairs of primers were screened by PCR technique. The specific DNA sequence of Echinococcus multilocularis has not been detected in serum. Conclusion: 1. The recombinant proteins of myophilin and LDH have high immunogenicity. The specific miRNA and DNA molecules of hydatid hydatid were not detected in the sera of patients with hydatid disease.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R532.32

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