应用噬菌体展示技术筛选兔出血症病毒抗原模拟表位

摘要

以抗兔出血症病毒(RHDV)的单克隆抗体A3c作为靶物质,应用噬菌体展示技术筛选RHDV抗原表位.将纯化的单抗A3c包被固相载体,经3轮亲和筛选后,挑取25株噬菌体单克隆并扩增,用EIISA测定后,提取阳性克隆单链DNA并测序,用阳性噬菌体克隆免疫小鼠制备高免血清,检测筛选抗原表位的免疫原性.结果表明:3轮亲和筛选后,特异性噬菌体克隆得到了有效富集,25株噬菌体单克隆中有19株为阳性克隆；测序结果表明,获得了与抗原高度同源的序列GTDDMDPGTTAA,即抗原的模拟表位,其中,氨基酸基序DXXDP为表位中的核心氨基酸；制备的小鼠高免血清与抗原具有较好的反应性,阳性噬菌体克隆与兔RHDV高免血清也具有较好的反应性.因此,该表位具有良好的免疫原性和反应原性.该研究为RHDV抗原表位的研究和新型疫苗的探索积累了资料.%The monoclonal antibody (McAb) A3c against rabbit hemorrhagic disease virus had been used as solid-phase selective molecule to screen the epitope of RHDV by phage display technology. McAb A3c was coated on the solid-phase and then three rounds of biopanning were carried out; 25 phages were selected and amplified to be identified by ELISA, and the positives were sequenced; the serum of mice immunized three times was prepared to determine the immunogenic-ity of the epitope. The results showed that the specific phages were enriched effectively after three rounds of biopanning; 19 of 25 phages were positive. The sequence analysis of the positive clones showed that highly homogenous sequence (GTDDMDPGTTAA) comparing to antigen was got, which is the antigen mimotopes of RHDV. The sequence DXXDP was the core amino acids in the epitope. In addition, the serum of mice reacted with antigen well and so did positive phages with hyper-immune serum of rabbit against RHDV, which indicated that the epo'tipe had good immunogenicity and reactionogenicity. This study provide theoretical basis for studying the epitopernof RHDV and new vaccines.