Wang L F, Du Plessis D H, White J R, Hyatt A D, Eaton B T
CSIRO Australian Animal Health Laboratory, Geelong, Victoria.
J Immunol Methods. 1995 Jan 13;178(1):1-12. doi: 10.1016/0022-1759(94)00235-o.
We describe the use of a gene-targeted random epitope library for the mapping of antigenic determinants. A DNA clone encoding the target antigen was digested randomly with DNase I to generate a population of DNA fragments of different sizes and sequences. After size fractionation, small DNA fragments (100-200 bp) were isolated and cloned into the phage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of the phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epitope libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Using this approach, we have mapped a monoclonal antibody (mAb)-defined epitope on the bluetongue virus outer capsid protein VP5. This epitope is not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although the example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy described should be equally applicable to genes of unknown sequence and for screening of epitopes using polyclonal antibodies. The approach can also be extended to identify immunodominant epitope from much more complex genome-targeted random epitope library for virus, bacteria and eukaryotic organisms. Other applications of recombinant phages expressing defined immunodominant epitopes include serodiagnosis and vaccine development.
我们描述了一种用于绘制抗原决定簇图谱的基因靶向随机表位文库的应用。用脱氧核糖核酸酶I对编码靶抗原的DNA克隆进行随机消化，以产生一系列大小和序列不同的DNA片段。经过大小分级后，分离出小的DNA片段（100 - 200碱基对）并克隆到噬菌体表达载体fUSE2中，形成一个表达文库，该文库在噬菌体基因III蛋白的N端以融合蛋白的形式展示随机多肽序列。这个文库被称为基因靶向随机表位文库，以区别于完全随机的合成表位文库，然后通过亲和选择筛选出被感兴趣的抗体特异性结合的重组噬菌体。利用这种方法，我们绘制了蓝舌病毒外膜蛋白VP5上一个单克隆抗体（mAb）定义的表位图谱。这个表位在完整病毒表面是不可接近的，但在病毒感染期间能被绵羊和牛的免疫系统识别。虽然这里给出的例子使用了已知序列的DNA片段，并且文库是针对mAb定义的表位进行筛选的，但所描述的策略同样适用于未知序列的基因，以及使用多克隆抗体筛选表位。该方法还可以扩展到从针对病毒、细菌和真核生物的更复杂的基因组靶向随机表位文库中鉴定免疫显性表位。表达确定的免疫显性表位的重组噬菌体的其他应用包括血清学诊断和疫苗开发。
