本文選題：噬菌體7肽庫 + Em18��； 參考：《新疆醫(yī)科大學》2007年碩士論文

【摘要】： 目的:應用噬菌體肽庫技術和分子生物學方法,分析和確定泡球蚴Em18抗原表位。方法:DNAman軟件設計引物,PCR法擴增并構建截短的PET41a-Em18.4(81aa-160aa),經誘導、表達和純化后,Western Blot及ELISA方×法對其進行鑒定。用純化后的rEm18-GST免疫新西蘭白兔,獲得抗rEm18-GST的多克隆抗體,用加入GST抗原的鎳-螯合物親和層析樹脂(His·Bind Resin)對此抗體進一步純化,以獲得去除GST抗體的抗Em18多克隆抗體,ELISA法確定效價。以之作為靶分子,應用噬菌體隨機7肽庫進行篩選。經過5輪的淘篩和富集,隨機挑取46個陽性噬菌斑擴增,凝膠電泳分析后,提取DNA測序,結果經BLAST軟件分析,并與Em18進行同源性比較。結果:成功構建了PET41a-Em18.4原核表達質粒,rEm18.4-GST重組蛋白得到成功表達,SDS-PAGE顯示相對分子量為41KDa,Western blot和ELISA結果顯示rEm18.4-GST重組蛋白未能被AE病人陽性血清識別。獲得去除抗GST的抗Em18多克隆抗體,ELISA確定效價為1:5 1200,Western Blot顯示該抗體不與GST發(fā)生交叉反應。使用噬菌體7肽庫經過5輪篩選后,噬菌體富集率達到10~3倍,46個噬菌體克隆DNA序列結果經Blast軟件分析后,發(fā)現(xiàn)為9個不同的序列,與Em18核苷酸同源性比較后未發(fā)現(xiàn)有同源性。結論:Em18的抗原表位可能不位于81—160aa部分,所篩選的Em18抗原噬菌體7肽是否是Em18抗原的模擬表位,有待進一步驗證。
[Abstract]:Objective: to analyze and identify Em18 epitopes of hydatid alveolar hydatid by phage peptide library technique and molecular biological methods. Methods the truncated PET41a-Em18.4AA-160aA was amplified and constructed by PCR with primers designed by the software: DNAman. After induction, expression and purification, it was identified by Western Blot and ELISA method. The polyclonal antibody against rEm18-GST was obtained by immunizing New Zealand white rabbits with purified rEm18-GST. The antibody was further purified by means of the Ni-chelate affinity chromatography resin his Bind. The anti-Em18 polyclonal antibody was obtained to determine the titer of anti-Em18 antibody by Elisa. The phage random 7 peptide library was used to screen the target molecule. After five rounds of screening and enrichment, 46 positive plaque samples were randomly selected for amplification. After gel electrophoresis, DNA was extracted and sequenced. The results were analyzed by BLAST software and compared with Em18 homology. Results: the PET41a-Em18.4 prokaryotic expression plasmid rEm18.4-GST recombinant protein was successfully constructed. The SDS-PAGE showed that the relative molecular weight of the recombinant protein was 41 K Dawei Western blot and the ELISA result showed that the rEm18.4-GST recombinant protein could not be recognized by the positive serum of AE patients. The anti Em18 polyclonal antibody to remove GST was obtained by Elisa and the titer of the antibody was confirmed to be 1:5 1200 Em18 Blot, which showed that the antibody did not cross with GST. After 5 rounds of screening, the phage enrichment rate of phage 7 peptide library was 103 times. The results of 46 phage clone DNA sequences were analyzed by Blast software and 9 different sequences were found, but no homology was found after comparing with Em18 nucleotide homology. Conclusion the antigenic epitopes of 10% Em18 may not be located in the 81-160aa part. Whether the Em18 antigen phage 7 peptide is the mimic epitope of Em18 antigen needs further verification.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

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