麻痹性贝类毒素GTX2,3模拟表位的初步研究

A Paralytic shellfish poisoning GTX2,3 mimic peptide isolated from phage display:characterization and potential applications

目的:利用噬菌体展示随机肽库筛选可模拟麻痹性贝类毒素GTX2,3抗原表位的噬菌体,初步鉴定人工合成的GTX2,3抗原表位肽的特异性。方法:以抗麻痹性贝类毒素GTX2,3单克隆抗体(mAb)为靶分子,对噬菌体随机12肽库进行3轮免疫亲和筛选,以夹心ELISA方法鉴定噬菌体克隆,竞争ELISA鉴定阳性噬菌体克隆的特异性。对阳性克隆进行DNA测序并推导噬菌体所展示的氨基酸序列。竞争ELISA鉴定模拟GTX2,3表位的合成肽的特异性。结果:经过3轮筛选获得20株能与靶分子高亲和力结合的阳性噬菌体克隆。序列分析表明DXLXPP为保守序列(X为任意氨基酸)。竞争ELISA检测表明,麻痹性贝类毒素GTX2,3可抑制阳性噬菌体克隆phage2与抗GTX2,3mAb结合。根据阳性序列合成的短肽可以抑制phage2与抗GTX2,3mAb的结合(IC50=13μg/mL)。结论:通过噬菌体肽库筛选技术,成功地获得麻痹性贝类毒素GTX2,3的模拟表位,并初步证实以此为基础合成的短肽能够准确和特异的模拟GTX2,3的表位。

AIM：To screen peptide mimics of PSP （Paralytic shellfish poisoning） GTX2,3 from a random 12-mer phage display peptide library and to identify and characterize the specificity and accuracy of the peptide mimotope of GTX2,3. METHOTHS： The monoclonal antibody against GTX2,3 （mAb E9F10） was used as a target to screen the 12-mer phage display peptide ibrary and the specificity of phage clones were identified by sandwich ELISA and blocking assay. The peptide sequences of positive phage clones were determined and analyzed by DNA sequencing. The affinity and specificity of synthetic peptide were identified by a competitive ELISA. RESULTS： The 20 clones were idenfified to be specific reactivity with the mAb E9F10. Amino acid sequence analysis revealed seven different types of mimotope sequence, most of which contained a common motif DXLXPP（X presents random acid amino）, X was random amino acid. The Phage No.2（phage 2）, the clone with mimotope sequence WPSLDXLXPPSY showed the strongest binding, and inhibited the reactivity of the mAb with GTX2,3. Another ELISA result showed that synthetic peptide-1 （SP-1） which contain mimotope amino acid sequence was able to inhibit the mAb-GTX2,3 interaction. CONXLUSION： The mimotope peptide of GTX2,3 is obtained by using the phage display technology. The results also showed that SP-1 bind to mAb E9F10 at the same site as GTX2,3.