本文選題：泡球蚴　切入點：Em18　出處：《新疆醫(yī)科大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的:應(yīng)用噬菌體12肽庫篩選泡球蚴Em18抗原的模擬表位,為研究和開發(fā)特異、靈敏的包蟲病診斷抗原提供實驗依據(jù)。方法:IPTG誘導、表達和純化重組蛋白Em18-GST,SDS-PAGE電泳進行初步鑒定。將純化后的rEm18-GST蛋白作為免疫原,免疫兔獲得抗rEm18-GST的多克隆抗體,ELISA檢測其效價。將抗體用鎳.螫合物親和層析樹脂His Bind Ni Resin(His柱)進一步純化,Western blot及ELISA進行鑒定。以Em18抗體作為靶分子,篩選噬菌體隨機12肽庫。隨機挑取19個噬菌斑進行擴增,提取單鏈DNA,凝膠電泳鑒定并送測序,分析其獲得的DNA序列及編碼的氨基酸序列,并與Em18進行同源性比較。結(jié)果:rEm18-GST重組蛋白得到成功表達,在相對分子量為50kDa處有表達條帶。獲得非特異性雜帶少純度高的rEm18-GST重組蛋白。經(jīng)5次免疫獲得兔抗tEm18-GST抗體,ELISA檢測抗體最終效價為1:51200。Western blot及ELISA鑒定rEm18-GST抗體去除了與GST的交叉反應(yīng),保留有抗Em18抗體。經(jīng)5輪肽庫篩選,所獲得的噬菌體克隆有較高程度的富集,第5輪篩選回收的噬菌體比第1輪高出約1000倍。19個克隆測序后發(fā)現(xiàn)主要有四個不同序列。其插入片段的DNA序列分別為:序列1:GTTGCGGCTTCTCCTAAGTGGACTAATCTTGAGTGG(f1-f4,f10)序列2:CATTCGAAGTGGCATATTCCGTCTGAGTGGCATGTG(f5-f9)序列3:CAGCATGGGCATAAGATTTGGTCGCCGAGTTATGTT(f11-f12,f14)序列4:CATCATTGGAGTTATTTTTATATTTTGGAGAGTCCG(f13,f15-f19),將獲得的四個序列與Em18比較,發(fā)現(xiàn)沒有同源性。結(jié)論:獲得了去除與GST交叉反應(yīng)的抗Em18多克隆抗體:篩選獲得的噬菌體12肽,與Em18比較無同源性,是否為Em18抗原的模擬表位還需要進一步研究。
[Abstract]:Objective: to screen mimic epitopes of hydatid Em18 antigen using phage 12 peptide library in order to provide experimental basis for the study and development of specific and sensitive antigen for diagnosis of hydatidosis. The recombinant protein Em18-GST-SDS-PAGE was identified by SDS-PAGE. The purified rEm18-GST protein was used as immunogen. The polyclonal antibody against rEm18-GST was obtained from immunized rabbits to detect its titer by Elisa. The antibody was further purified by nickel and chelate affinity chromatography resin His Bind Ni Resin(His column. The Em18 antibody was used as the target molecule. The phage random 12 peptide library was screened. 19 phage spots were randomly selected for amplification, single strand DNA was extracted, identified by gel electrophoresis and sequenced. The DNA sequence and amino acid sequence were analyzed. The homology of the recombinant protein was compared with that of Em18. The rEm18-GST recombinant protein with less specific heterologous bands and high purity was obtained at the relative molecular weight of 50 kDa. The final titer of rabbit anti tEm18-GST antibody detected by Elisa was 1: 51200.Western blot and ELISA confirmed that the cross reaction with GST was removed. After five rounds of peptide library screening, the phage clones were highly enriched. The number of bacteriophages recovered in the fifth round was about 1 000 times higher than that in the first round. After 19 clones were sequenced, there were mainly four different sequences. The DNA sequences of the inserted fragments were as follows: sequence 1: GTTGCGGCTTCTCTTCCTAAGTGTACTTTATTATTATTATTATTATTATTATTGTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATATTTATTATTATTGTATTATTGTATTATTGTATTATTGTATTATTGTATTATTGTATTATTATF19f19f19f1), and the four sequences were compared with the sequences of Em18, and the four sequences were compared with the sequences of GCAGCATGCATAAGAGTAGTATTATTTTATTT Tf14. the sequence of GTG13f19f19f1915 was compared with that of Em18. Conclusion: the anti Em18 polyclonal antibody to remove the cross reaction with GST was obtained. The phage 12 peptide obtained by screening has no homology with Em18. It is necessary to further study whether it is a mimic epitope of Em18 antigen.
【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

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