O1群小川型霍乱弧菌单克隆抗体识别表位模拟肽的筛选鉴定

Screening and identification of eiptope mimic peptide of monoclonal antibodies against vibrio cholerae O1 serotype Ogawa

摘要:

【摘 要】目的：以纯化的有高度特异性的IXiao3G6单抗为靶分子，对噬菌体随机7肽库进行淘筛，以期筛选到与O1群小川型霍乱弧菌单抗有高亲和力的模拟肽，为基于表位的霍乱弧菌模拟多肽疫苗的研制奠定基础。方法：运用噬菌体随机7肽库技术，以纯化的IXiao3G6单抗为靶蛋白，进行噬菌体随机肽库 3 轮筛选，以双抗夹心ELISA和竞争抑制实验分析获得的噬菌体短肽与筛选配基的结合能力及特异性，并进行阳性克隆序列测定和同源性分析。结果：3轮筛选后，阳性噬菌体得到有效富集，夹心ELISA检测随机挑取的25个噬菌体克隆，有22个噬菌体克隆为阳性，测序表明其中20个阳性克隆的氨基酸序列完全一致，均为APAIPAS。对该序列同源性分析，未发现有相同的已知序列。竞争抑制实验表明，O1群小川型霍乱弧菌脂多糖（lipopo-lysaccharide，LPS）能很好地抑制阳性克隆与IXiao3G6 McAb的结合，抑制程度随LPS浓度的升高而增加，而对照细菌的LPS则不具有相应的抑制作用，进一步提示噬菌体克隆展示肽模拟了O1群小川型霍乱弧菌LPS抗原表位。结论：筛选到模拟O1群小川型霍乱弧菌LPS的抗原表位的模拟肽，为基于表位的霍乱弧菌模拟多肽疫苗的开发与设计提供了实验依据。

Abstract:

Objective：To obtain the mimic peptide highly binding to cholerae O1 serotype Ogawa by using purified IXiao3G6 McAb to biospan in phage display random 7 peptides library and to establish the basement for further research of the mimic peptide vaccine of vibrio cholerae. Methods：Purified IXiao3G6 McAb was used to biospan in phage display random 7 peptides library and mimic epitopes were acquired after 3 rounds of biospanning. ‘Double-antibodysandwich’ ELISA and specific inhibition test were used to analyze the combining ability and specificity of the obtained phage short peptide and the IXiao3G6 McAb. DNA sequence analysis was used to an－alyze the sequence of the positive clones and the related bioinformatic tools were used to analyze the alignments. Results：After three rounds of biospanning，the positive phages were enriched effectively. Twenty-two phage clones were positive of the 25 phage clones se－lected randomly by ‘double-antibodysandwich’ ELISA. DNA sequence analysis showed that the amino acid sequences of 20 positive clones were identical completely，which was identified as APAIPAS. Alignment analysis showed no homologous known sequence. Spe－cific inhibition test showed that the lipopolysaccharide（LPS） of cholerae O1 serotype Ogawa could inhibit the binding of the positive clones with IXiao3G6 McAb and the inhibitory intensity was enhanced by increment of the content of LPS，while the LPS of control bacteria did not show the corresponding inhibitory effect. These results further demonstrated phage display peptide could be mimetic to the antigen epitope of the LPS of cholerae O1 serotype Ogawa. Conclusions：Mimic peptide mimesis to the antigen epitope of the LPS of cholerae O1 serotype Ogawa serves as the basement for development of the mimic peptide vaccine of vibrio cholerae.