【作者】 王俨；

【导师】 丁剑冰；

【作者基本信息】 新疆医科大学 ， 免疫学， 2005， 硕士

【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,有望更多还原

【Abstract】 Objective: To clone Em 18 antigen gene and express recombinant Em18 antigen. Serological evaluate ReEm 18 antigen for diagnostic differentiation between alveolar echinococcosis (AE) and cystic echinococcosis (CE). Methods: The primers of Em18 were designed by DNAman biosoftware .The Em 18 antigen gene was amplified by PT-PCR from total RNA of Echinococcus multilocularis, and by TD-PCR from Em cDNA library for construction of plasmid pMD18/T-Em18. The Em18 antigen gene was identified by sequencing. The prokaryotic expression plasmid pET41a-Em18 was constructed and identified by sequencing. rEm18-GST fusion protein and GST were epressed by induction with IPTG and were detected by SDS-PAGE Western-Blot. rEm18-GST fusion protein was affinity purified against the GST tag. A total of 523 human serum samples were evaluated for the diagnostic sensitivity and specificity with the fusion protein by immunoblotting (IB) and enzyme-linked immunosorbent assay (ELISA), respectively (59 serum samples were collected from patients with AE, 240 serum samplesfrom patients with CE) . Results: DNA sequence analysis of Eml8 gene fragment indicated that the length of Em 18 was 486bp, code 161aa and was accepeted by Genbank as a new sequence (AY513691). The pET4la-Em 18 positive clone was the exact recombinant plasmid and rEml8-GST recombinant protein by induction with IPTG was espressed as a band of 50KDa by SDS-PAGE detection. The result of Western-Blot showed that rEml8-GST antigen has better antigenicity. 523 sera samples were detected by rEml8-GST antigen. The overall sensitivity was 91.52%/93.22% and the overall specificity was 94.61 %/94.82 by ELISA and Western Blotting, respectively. Conclusion: Eml8 antigen gene was cloned successfully. The combined levels of sensitivity and specificity achieved rather high with the rEml8-GST fusion protein for AE diagnosis, rEml8 has considerable advantage for serodiagnosis between AE and CE and possible development of a diagnosis kit.更多还原