泡球蚴Em18抗原基因的克

发布时间：2018-03-02 21:28

本文选题：多房棘球蚴病　切入点：Em18基因　出处：《新疆医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:克隆泡球蚴18(Em18)抗原基因,获得高效表达、有生物活性的Em18重组蛋白,对其用于两型包虫病的免疫诊断特性进行研究。方法:DNAman软件设计引物。分别经PCR筛选,从已构建的新疆株泡球蚴cDNA文库中克隆出Em18抗原基因;及Trizol试剂提取泡球蚴原头节总RNA,RT-PCR反转录克隆出Em18抗原基因,并构建pMD18-T/Em18质粒,测序确定序列。构建pET41a-Em18原核表达质粒,测序鉴定插入序列正确性。IPTG诱导表达rEm18-GST重组蛋白和GST重组蛋白,谷光甘肽-Sepharose 4B亲合层析柱纯化,SDS-PAGE法和Western Blot法分析鉴定。ELISA法及Western Blot法检测523份血清(其中59份AE病人血清,240份CE病人血清),确定Em18重组蛋白对于两型包虫病的免疫诊断特性。结果:测序显示Em18基因长度为486bp,编码161个氨基酸,为一新序列,被GenBank收录(AY513691)。成功构建了pET41a-Em18原核表达质粒,经IPTG诱导,SDS-PAGE检测表明rEm18-GST重组蛋白得到成功表达,在相对分子量(KDa)为50处有表达条带;Western Blot分析显示rEm18-GST重组蛋白能被泡型棘球蚴病人阳性血清识别,具有良好的抗原性。523份血清经rEm18-GST重组蛋白检测,ELISA法显示其敏感性为91.52%,特异性为94.61%;Western Blot法检测显示敏感性为93.22%,特异性为94.82%。结论:成功克隆Em18抗原基因,表达的rEm18-GST重组蛋白对两型包虫病具有较高的免疫鉴别特性和免疫诊断价值,有望
[Abstract]:Objective: to clone the antigen gene of Elastococcus alveolaris 18 Em18 and obtain highly expressed and bioactive Em18 recombinant protein, and to study the immunological characteristics of the recombinant protein used in the diagnosis of hydatid disease. Methods: the primers were designed by the software of 10% DNAman and screened by PCR, respectively. The Em18 antigen gene was cloned from the cDNA library of Xinjiang alveolar hydatid strain, and the Em18 antigen gene was extracted by Trizol reagent. The Em18 antigen gene was cloned into pMD18-T/Em18 plasmid by reverse transcription RT-PCR. The pMD18-T/Em18 plasmid was sequenced and the expression plasmid of pET41a-Em18 was constructed. Sequencing confirmed the correctness of the insert sequence. IPTG induced the expression of rEm18-GST recombinant protein and GST recombinant protein. SDS-PAGE and Western Blot methods were used to analyze and identify 523 sera (59 AE patients, 240 CE patients), and to determine the immunogenicity of Em18 recombinant protein against two types of hydatid disease. Results: sequencing showed that the length of Em18 gene was 486 BP, encoding 161 amino acids. The prokaryotic expression plasmid AY513691was successfully constructed by GenBank. The IPTG induced SDS-PAGE analysis showed that the rEm18-GST recombinant protein was successfully expressed. Western Blot analysis showed that the recombinant protein of rEm18-GST could be recognized by positive serum of Echinococcus alveolar type patients at a relative molecular weight of 50. The sensitivity of Elisa was 91.52%, the specificity was 94.61%, the sensitivity was 93.2222 and the specificity was 94.82.Conclusion: Em18 antigen gene was cloned successfully. The expressed rEm18-GST recombinant protein has high immunological differential characteristics and immunological diagnostic value for the two types of hydatid disease, and it is expected to be helpful in the diagnosis of hydatid disease.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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