泡球蚴Em18抗原噬菌体多肽筛选及其模拟表位分析的初步研究

发布时间：2018-03-12 08:05

本文选题：泡球蚴　切入点：Em18　出处：《新疆医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:应用噬菌体12肽库筛选泡球蚴Em18抗原的模拟表位,为研究和开发特异、灵敏的包虫病诊断抗原提供实验依据。方法:IPTG诱导、表达和纯化重组蛋白Em18-GST,SDS-PAGE电泳进行初步鉴定。将纯化后的rEm18-GST蛋白作为免疫原,免疫兔获得抗rEm18-GST的多克隆抗体,ELISA检测其效价。将抗体用镍.螫合物亲和层析树脂His Bind Ni Resin(His柱)进一步纯化,Western blot及ELISA进行鉴定。以Em18抗体作为靶分子,筛选噬菌体随机12肽库。随机挑取19个噬菌斑进行扩增,提取单链DNA,凝胶电泳鉴定并送测序,分析其获得的DNA序列及编码的氨基酸序列,并与Em18进行同源性比较。结果:rEm18-GST重组蛋白得到成功表达,在相对分子量为50kDa处有表达条带。获得非特异性杂带少纯度高的rEm18-GST重组蛋白。经5次免疫获得兔抗tEm18-GST抗体,ELISA检测抗体最终效价为1:51200。Western blot及ELISA鉴定rEm18-GST抗体去除了与GST的交叉反应,保留有抗Em18抗体。经5轮肽库筛选,所获得的噬菌体克隆有较高程度的富集,第5轮筛选回收的噬菌体比第1轮高出约1000倍。19个克隆测序后发现主要有四个不同序列。其插入片段的DNA序列分别为: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),将获得的四个序列与Em18比较,发现没有同源性。结论:获得了去除与GST交叉反应的抗Em18多克隆抗体:筛选获得的噬菌体12肽,与Em18比较无同源性,是否为Em18抗原的模拟表位还需要进一步研究。
[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.
【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

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