创伤弧菌溶细胞素抗原表位预测及其噬菌体展示肽的构建

Epitope prediction of Vibrio vulnificus cytolysin and construction of phage display peptides

目的预测创伤弧菌溶细胞素(vibrio vulnificus cytolysin)的抗原表位,为研制多抗原肽疫苗(MAP)奠定基础。方法采用生物信息学技术对Vvc的T细胞和B细胞表位进行预测及分析。选取Vvc主要T细胞和B细胞联合表位肽构建噬菌体展示系统。PEG/NaCl沉淀法提纯重组噬菌体,SDS-PAGE鉴定目的重组PIII蛋白(rPIII)。Ni-NTA亲和层析法纯化的重组蛋白rVvc常规皮下免疫法制备兔抗血清。rVvc抗血清为一抗,采用Western blot对上述表位肽进行鉴定和筛选。结果预测的4个主要表位肽Vvha1、Vvah2、Vvha3和Vvha4在M13噬菌体中获得成功表达。采用rVvc抗血清为一抗,Western blot对上述表位肽进行鉴定和筛选:Vvha2、Vvha4反应强度高于Vvha1和Vvha3。结论本实验成功地构建Vvc主要T细胞和B细胞联合表位肽噬菌体展示系统。

To predict the antigenic epitopes in Vibrio vulnificus cytolysin（Vvc） for the development of multiple antigenic peptide（MAP）vaccine,the epitopes on T and B cells of Vvc were predicted and analyzed by using bioinformatic techniques and its major combined epitope peptides on T and B cells were selected to construct the phage display system. The PEG/NaCl precipitation method and SDS-PAGE were used to extract the recombinant phage and to identify the target recombinant PIII（rPIII） respectively. Meanwhile,the recombinant protein of Vvc（rVvc） purified with Ni-NTA chromatography was applied to immunize rabbits subcutaneously to prepare the rabbit anti-Vvc serum. By using the rabbit anti-Vvc serum as the first antibody, Western blot assay was established to identify and to screen the epitope peptides, It was found that the 4 major predicted epitope peptides, Vvhal,Vvha2,Vvha3 and Vvha4 were successively expressed in their recombinant M13 phages, in which the reaction intensities of Vvha2 and Vvha4 as demonstrated in Western blot assay were remarkably stronger than those of Vvhal and Vvha3. It is evident that the phage display system of the major combined epitope peptides on T and B cells of Vibrio vulnificus cytolysin were successively constructed in the present study.