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目的:筛选鼠疫耶尔森菌F1抗原的中和性表位,构建基于鞭毛蛋白佐剂的重组表位疫苗。方法:利用鼠疫菌F1抗原的中和抗体F2H5筛选噬菌体随机12肽库,对得到的阳性克隆采用ELISA进行特异性鉴定,采用竞争抑制ELISA确定具有竞争性的噬菌体单克隆并对其DNA测序,重组表达并纯化获得肽序列与截短型鞭毛蛋白Fli Cdel的融合蛋白,并通过Western印迹和ELISA鉴定重组蛋白与F2H5的结合。结果:获得了2株能够与F1抗原竞争结合F2H5的噬菌体单克隆12-1和12-14,其中12-14的竞争能力较强;通过序列比对,并没有发现这2株噬菌体克隆的插入肽序列与F1抗原序列存在一致性,但这2个插入肽序列与Fli Cdel的重组蛋白在Western印迹和ELISA结果中均显示出能够被抗F1的单克隆抗体识别。结论:F1中和性抗体筛选出的肽序列与截短的鞭毛蛋白融合表达后能够被F2H5特异性识别,为进一步对重组表位抗原进行免疫保护评价奠定了基础。
Objective: To screen neutralizing epitopes of Yersinia pestis F1 antigen and construct a recombinant epitope vaccine based on flagellin adjuvant. Methods: The phage random 12 - peptide library was screened by F2H5, a neutralizing antibody against the Y. pestis F1 antigen. The positive clones were identified by ELISA. Competition - restricted ELISA was used to determine competitive phage clones. The DNA was sequenced and recombined The fusion protein of the peptide sequence and the truncated flagellin Fli Cdel was expressed and purified, and the binding of the recombinant protein to F2H5 was confirmed by Western blotting and ELISA. Results: Two phage clones 12-1 and 12-14 were obtained that were able to compete with F1 antigen for binding to F2H5, of which 12-14 were more competitive. No insertion of the two phage clones was found by sequence alignment The peptide sequence is identical to the F1 antigen sequence, but both of the inserted peptide sequences and the Fli Cdel recombinant protein have been shown to be recognized by anti-F1 monoclonal antibodies both in Western blot and ELISA. CONCLUSION: The fusion protein of F1 neutralizing antibody can be specifically recognized by F2H5 after fusion with truncated flagellin, which lays a foundation for further immunoprotection evaluation of recombinant epitope antigen.