本文選題：多房棘球蚴病　切入點(diǎn)：Em18基因　出處：《新疆醫(yī)科大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:克隆泡球蚴18(Em18)抗原基因,獲得高效表達(dá)、有生物活性的Em18重組蛋白,對(duì)其用于兩型包蟲病的免疫診斷特性進(jìn)行研究。方法:DNAman軟件設(shè)計(jì)引物。分別經(jīng)PCR篩選,從已構(gòu)建的新疆株泡球蚴cDNA文庫(kù)中克隆出Em18抗原基因;及Trizol試劑提取泡球蚴原頭節(jié)總RNA,RT-PCR反轉(zhuǎn)錄克隆出Em18抗原基因,并構(gòu)建pMD18-T/Em18質(zhì)粒,測(cè)序確定序列。構(gòu)建pET41a-Em18原核表達(dá)質(zhì)粒,測(cè)序鑒定插入序列正確性。IPTG誘導(dǎo)表達(dá)rEm18-GST重組蛋白和GST重組蛋白,谷光甘肽-Sepharose 4B親合層析柱純化,SDS-PAGE法和Western Blot法分析鑒定。ELISA法及Western Blot法檢測(cè)523份血清(其中59份AE病人血清,240份CE病人血清),確定Em18重組蛋白對(duì)于兩型包蟲病的免疫診斷特性。結(jié)果:測(cè)序顯示Em18基因長(zhǎng)度為486bp,編碼161個(gè)氨基酸,為一新序列,被GenBank收錄(AY513691)。成功構(gòu)建了pET41a-Em18原核表達(dá)質(zhì)粒,經(jīng)IPTG誘導(dǎo),SDS-PAGE檢測(cè)表明rEm18-GST重組蛋白得到成功表達(dá),在相對(duì)分子量(KDa)為50處有表達(dá)條帶;Western Blot分析顯示rEm18-GST重組蛋白能被泡型棘球蚴病人陽(yáng)性血清識(shí)別,具有良好的抗原性。523份血清經(jīng)rEm18-GST重組蛋白檢測(cè),ELISA法顯示其敏感性為91.52%,特異性為94.61%;Western Blot法檢測(cè)顯示敏感性為93.22%,特異性為94.82%。結(jié)論:成功克隆Em18抗原基因,表達(dá)的rEm18-GST重組蛋白對(duì)兩型包蟲病具有較高的免疫鑒別特性和免疫診斷價(jià)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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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本文編號(hào)：1558129