抗日本血吸虫14-3-3蛋白单克隆抗体5C6抗原识别表位的鉴定  

Epitope identification of monoclonal antibody 5C6 against 14-3-3 protein of Schistosoma japonicum

作　　者：钱春艳[1] 宋丽君[1] 余传信[1] 殷旭仁[1] 王玠[1] 张伟[1] 金一[1] 柯雪丹[1] 

机构地区：[1]江苏省血吸虫病防治研究所、卫生部寄生虫病预防与控制技术重点实验室,无锡214064

出　　处：《中国血吸虫病防治杂志》2012年第1期45-49,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家重大科技专项(2008ZX10004-11);国家自然科学基金(30972581);江苏省自然科学基金(BK2008110);江苏省科技厅公益专项(BM2007704);江苏省卫生厅科技项目(H201066);江苏省卫生厅预防医学项目(YZ201030)

摘　　要：目的采用噬菌体展示肽技术鉴定抗日本血吸虫14-3-3蛋白单克隆抗体5C6的抗原识别表位。方法将纯化的抗日本血吸虫14-3-3蛋白单克隆抗体5C6作为固相配基筛选噬菌体随机展示12肽库,按照吸附-洗脱-扩增的淘洗过程进行3轮生物淘洗,富集特异性结合的噬菌体颗粒。随机挑取第3轮洗脱的独立噬菌体克隆,分别依次进行噬菌体扩增、基因组DNA抽提和DNA序列分析。选择外源性插入DNA序列完全相同或高度相似的同源性噬菌体,采用ELISA及免疫印迹分析对其免疫反应性作进一步鉴定,以确定单克隆抗体5C6的抗原识别表位。结果第3轮淘洗物中33个独立噬菌体克隆的DNA测序结果显示,其中25个噬菌体编码外源性插入肽的核酸序列均为5′-CCACCTAGTAGCAGACCGATTCT-CAGTCGAAGGAAA-3′,其对应的演绎氨基酸序列为PPSSRPILSRRK,该肽与日本血吸虫信号蛋白14-3-3及自然界其他已知蛋白的氨基酸序列均无同源性。免疫印迹试验证实此短肽可被自然感染的日本血吸虫病人血清所识别,而不与健康人血清反应。结论本研究成功鉴定了抗日本血吸虫14-3-3蛋白单克隆抗体5C6所识别的模拟抗原表位,并证明该表位为空间构像表位。Objective To identify the epitope of monoclonal antibody （McAb） 5C6 against 14-3-3 protein of Schistosoma japonicum by phage display peptide library. Methods The phage display 12-peptide library was screened with purified McAb 5C6 against 14-3-3 protein of S. japonicum three rounds of bio-panning ＂adsorption-elution-amplification＂ to enrich the specific binding phages. The single phage clones selected randomly were amplified, their genomic DNA were extracted and sequenced. The immune response characterization of phages with the same or high homologous foreign inserted DNA sequence was identified by ELISA and Western blotting for further defining the epitope recognized by McAb 5C6. Results A total of 33 single phage clones were selected and sequenced. Among them, 25 shared the same foreign inserted DNA sequence of 5′-CCACCTAGTAGCAGACCGATTCTCAGTCGAAGGAAA-3′, encoding a deduced peptide PPSSRPILSRRK. This peptide was not homologue to Sj14-3-3 protein or any other known native protein in the world. The results of Western blotting showed that this peptide could be recognized by the sera of patients with schistosomiasis, but not by those of healthy persons. Conclusion The mimic antigen epitope of McAb 5C6 against 14-3-3 protein of S. japonicum, which is a conformational epitope, has been defined successfully in this study.

关 键 词：日本血吸虫 信号传导蛋白14-3-3 单克隆抗体 噬菌体随机展示肽库 模拟表位 生物淘洗 

分 类 号：R383.24[医药卫生—医学寄生虫学]