三种截短的泡球蚴Em18基因原核表达质粒的构建、表达及初步鉴定

发布时间：2018-04-23 23:05

本文选题：泡球蚴 + Em18重组蛋白　；参考：《新疆医科大学》2006年硕士论文

【摘要】：目的：构建三种截短的泡球蚴18(Em18)基因的原核表达质粒，获得高效表达、有生物活性的三种重组蛋白并对其进行初步鉴定。方法：DNAman软件设计引物，，PCR法扩增并构建三种截短的pET41a-Em18.1，Em18.2，Em18.3原核表达质粒，测序鉴定插入序列正确性。IPTG诱导、表达和纯化rEm18.1，rEm18.2，rEm18.3，-GST重组蛋白，运用Western免疫印迹法和ELISA方法对其进行初步鉴定。结果：成功构建三种截短的pET41a-Em18.1，Em18.2，Em18.3，SDS-PAGE检测表明rEm18.(1，．2，．3)-GST重组蛋白得到成功表达，在相对分子量为41kDa，45.5kDa，45.5kDa处有表达条带。Western blot结果显示三种重组蛋白均能被AE病人血清识别。对56例多房棘球蚴病(AE)、88例细粒棘球蚴病(CE)、24例肝硬化、24例其他病、6例肺吸虫病、6例肝吸虫病、6例血吸虫病、6例囊虫病、24名健康人共236份血清进行ELISA检测，结果显示rEm18.1-GST、rEm18.2-GST对AE血清诊断的敏感性分别为19.6％、73.2％，特异性分别为98.9％、98.3％，阳性预测值分别为84.6％、93.2％，阴性预测值分别为79.8％、92.2％。结论：成功构建了三种截短的pET41a-Em18.1，Em18.2，Em18.3原核表达质粒，表达的三种重组蛋白中rEm18.2-GST ELISA结果显示Em18抗原的优势抗原表位可能位于Em18抗原的第81位-120位氨基酸中，为Em18抗原表位分析奠定了基础。
[Abstract]:Aim: to construct the prokaryotic expression plasmids of three truncated Elastoc 18 (E18) genes and to obtain three recombinant proteins with high expression and bioactivity. Methods three kinds of truncated prokaryotic expression plasmids pET41a-Em18.1Em18.2Em18.2Em18.3 were amplified and constructed by PCR with specific primers designed by the software: DNAman software. The inserted sequences were confirmed by sequencing and induced by IPTG. The recombinant proteins were expressed and purified. The recombinant proteins were preliminarily identified by Western Western blot and ELISA. Results: three kinds of truncated pET41a-Em18.1- Em18.2Em18.2- Em18.3- blot were successfully constructed. The results of SDS-PAGE showed that the recombinant proteins were successfully expressed and expressed at the relative molecular weight of 45.5kDa of 45.5kDa. The results of Western blot showed that the three recombinant proteins could be recognized by the serum of AE patients. A total of 236 serum samples from 56 patients with multilocular echinococcosis, 88 patients with echinococcosis, 24 patients with liver cirrhosis, 24 patients with liver clonorchiasis, 6 patients with paragonimiasis, 6 patients with schistosomiasis and 6 patients with cysticercosis were detected by ELISA. The results showed that the sensitivity of rEm18.1-GST-rEm18.2-GST to the diagnosis of AE serum was 19.6 and 73.2, the specificity was 98.9 and 98.3, the positive predictive value was 84.6and the positive predictive value was 99.82.The negative predictive value was 79.8 and 92.2respectively.The results showed that the sensitivity of rEm18.1-GST-rEm18.2-GST to the diagnosis of AE serum was 19.6and the specificity was 98.93.2, respectively. Conclusion: three truncated prokaryotic expression plasmids pET41a-Em18.1Em18.2Em18.2Em18.3 were successfully constructed. The results of rEm18.2-GST ELISA showed that the dominant epitopes of Em18 antigen might be located in the 81-120 amino acids of Em18 antigen. The results laid a foundation for the epitope analysis of Em18 antigen.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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