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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。
OBJECTIVE: To screen phage display phage display random peptide library phage mimic shellfish toxoid GTX2, 3 antigen epitope, preliminary identification of synthetic specificity GTX2, 3 epitope peptide. Methods: The anti-paralytic shellfish toxin GTX2,3 monoclonal antibody (mAb) was used as a target. The phage random 12-peptide library was screened by immunoaffinity and identified by sandwich ELISA. The positive phage clones Specificity. Positive clones were subjected to DNA sequencing and deduced amino acid sequences exhibited by phage. Competitive ELISA identifies the specificity of synthetic peptides that mimic the GTX2,3 epitope. Results: After 3 rounds of screening, 20 positive phage clones with high affinity to the target were obtained. Sequence analysis showed that DXLXPP is a conserved sequence (X is any amino acid). Competitive ELISA test showed that paralytic shellfish toxin GTX2,3 can inhibit the positive phage cloning phage2 and anti-GTX2,3 mAb binding. Short peptides synthesized based on the positive sequence can inhibit the binding of phage2 to anti-GTX2,3 mAb (IC50 = 13 μg / mL). Conclusion: The mimotopes of paralytic shellfish toxoid GTX2,3 were successfully obtained by phage display peptide screening. The results showed that the short peptides synthesized based on this method could accurately and specifically mimic the epitope of GTX2,3.