泡球蚴Em18抗原表位分析的初步研究

发布时间：2018-05-04 08:07

本文选题：噬菌体7肽库 + Em18　；参考：《新疆医科大学》2007年硕士论文

【摘要】： 目的:应用噬菌体肽库技术和分子生物学方法,分析和确定泡球蚴Em18抗原表位。方法:DNAman软件设计引物,PCR法扩增并构建截短的PET41a-Em18.4(81aa-160aa),经诱导、表达和纯化后,Western Blot及ELISA方×法对其进行鉴定。用纯化后的rEm18-GST免疫新西兰白兔,获得抗rEm18-GST的多克隆抗体,用加入GST抗原的镍-螯合物亲和层析树脂(His·Bind Resin)对此抗体进一步纯化,以获得去除GST抗体的抗Em18多克隆抗体,ELISA法确定效价。以之作为靶分子,应用噬菌体随机7肽库进行筛选。经过5轮的淘筛和富集,随机挑取46个阳性噬菌斑扩增,凝胶电泳分析后,提取DNA测序,结果经BLAST软件分析,并与Em18进行同源性比较。结果:成功构建了PET41a-Em18.4原核表达质粒,rEm18.4-GST重组蛋白得到成功表达,SDS-PAGE显示相对分子量为41KDa,Western blot和ELISA结果显示rEm18.4-GST重组蛋白未能被AE病人阳性血清识别。获得去除抗GST的抗Em18多克隆抗体,ELISA确定效价为1:5 1200,Western Blot显示该抗体不与GST发生交叉反应。使用噬菌体7肽库经过5轮筛选后,噬菌体富集率达到10~3倍,46个噬菌体克隆DNA序列结果经Blast软件分析后,发现为9个不同的序列,与Em18核苷酸同源性比较后未发现有同源性。结论:Em18的抗原表位可能不位于81—160aa部分,所筛选的Em18抗原噬菌体7肽是否是Em18抗原的模拟表位,有待进一步验证。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】