采用噬菌体展示技术淘选河豚毒素模拟表位的研究

发布时间：2018-09-05 08:57

【摘要】： 河豚毒素(Tetrodotoxin, TTX)是一种毒性很强的非蛋白类神经毒素,该毒素作用于中枢及周围神经系统,选择性地与细胞膜表面的钠离子通道上的蛋白质结合,从而阻断钠离子通道,直接影响神经肌肉兴奋性的传导,使得神经肌肉呈麻痹状态,甚至引起死亡。该毒素理化性质稳定,能耐高温及日光暴晒,家庭的烹饪过程几乎不破坏其毒性。河豚毒素在临床上可用做止痛剂、麻醉剂和降压药等等,但需慎重把握其用量。目前国际上TTX标准品价格不菲且TTX属于生物战剂遭到国际禁运,此外TTX是剧毒物对实验操作人员也存在安全隐患。因而如果能制备一种河豚毒素的替代物,并对其性质进行研究鉴定,无论是用于无毒检测方法的研究,还是做为药物筛选的前提,都具有较大意义。曾有学者使用抗抗河豚毒素单克隆抗体独特型抗体(AID)替代河豚毒素用于ELISA检测,但由于抗独特型抗体与抗体之间的亲和力通常高于抗体与毒素之间的亲和力,因此制备的AID不能替代结合态TTX包被酶标板用于ELISA,而且抗独特型抗体的筛选相当繁琐。噬菌体随机肽库技术的出现为这个问题的有效解决提供了新的手段。噬菌体随机肽库技术是以大量随机编码的多肽序列插入噬菌体载体,形成噬菌体展示文库,每个噬菌体粒子表面只展示一种序列的外源肽链,并保持其相对的空间构象和生物活性。本研究通过以抗TTX的单抗为配基,从噬菌体随机肽库中筛选与之特异性结合的TTX模拟表位,以筛选出来的抗原表位合成多肽替代TTX毒素,并对多肽的性质进行初步鉴定。主要研究内容如下：以抗TTX单克隆抗体作为靶分子,对融合表达在M13丝状噬菌体衣壳蛋白表面的随机七肽库进行亲和淘选,通过ELISA方法筛选和鉴别具有模拟表位的阳性噬菌体克隆,并用阳性克隆建立ELISA分析方法；通过对阳性克隆进行DNA测序确定插入序列的氨基酸序列,同时经化学方法合成多肽,对模拟表位进行验证。通过4轮淘选,获得了7株能与抗TTX单克隆抗体特异性结合的噬菌体,采用间接竞争ELISA,筛选到了3株能抑制TTX的阳性克隆。其中以4号噬菌体建立的免疫检测方法,线性范围为1～20ng/ml (TTX毒素浓度),R2=0.9947,50%抑制率为4ng/ml,检测下限(20%抑制率)为1ng/ml。使用该方法与常规ELISA检测方法同时检测样品,结果基本无差异。经DNA测序分析,模拟表位肽的共有序列是组氨酸-甘氨酸-脯氨酸-酪氨酸-精氨酸-组氨酸-脯氨酸。根据DNA测序分析结果,合成多肽(HGPYRHP),并建立免疫检测方法,其线性范围为1-20μg/ml, R2= 0.9995,20%抑制率为1.2μg/ml,50%抑制率6.3μg/ml。分别以不同浓度的TTX标准品与合成多肽建立ELISA分析方法的竞争曲线,通过参照标准曲线中相同结合率时TTX标准品与合成多肽的浓度,可得到TTX标准品与合成多肽的剂量关系,二者呈现良好的线性,其R2=0.9978,表明该多肽可用于替代TTX标准品用于TTX的ELISA检测。
[Abstract]:Tetrodotoxin (TTX) is a highly toxic non-protein neurotoxin. It acts on the central and peripheral nervous system and selectively binds to the protein on the sodium channel on the cell membrane surface, thus blocking the sodium channel, directly affecting the conduction of neuromuscular excitability and paralysing the neuromuscles. Tetrodotoxin can be used clinically as analgesics, anesthetics and antihypertensive drugs, but its dosage should be carefully controlled. At present, the international standard TTX is expensive and TTX belongs to biological warfare agent is international. In addition, TTX is a highly toxic substance and has potential safety hazards for laboratory operators. Therefore, if a substitute for tetrodotoxin can be prepared and its properties can be studied and identified, it is of great significance not only for the study of non-toxic detection methods, but also for the premise of drug screening.
Anti-tetrodotoxin monoclonal antibody idiotypic antibody (AID) has been used to replace tetrodotoxin in ELISA, but the affinity between anti-idiotypic antibody and antibody is usually higher than that between antibody and toxin, so the prepared AID can not replace the binding TTX coated enzyme tag for ELISA, and anti-idiotypic antibody. The emergence of phage random peptide library technology provides a new way to solve this problem. Phage random peptide library technology inserts a large number of randomly encoded peptide sequences into phage carriers to form phage display libraries. Each phage particle displays only one sequence of exogenous peptide chains on its surface and preserves them. In this study, TTX mimic epitopes were screened from phage random peptide libraries using anti-TTX monoclonal antibodies as ligands. The antigen epitopes synthesized by the mimic epitopes were used to replace TTX toxins and the properties of the peptides were preliminarily identified.
Using anti-TTX monoclonal antibody as target molecule, the random heptapeptide library fused to the capsid protein of M13 filamentous phage was screened and identified by ELISA. The positive phage clones with mimic epitopes were identified by ELISA, and the ELISA analysis method was established by positive clones. The amino acid sequence of the sequence was synthesized by chemical method and the mimic epitope was validated. Seven phages specifically binding to anti-TTX monoclonal antibody were obtained by four rounds of panning. Three positive clones were screened by indirect competitive ELISA. The immunoassay method established by phage 4 was linear. The inhibition rate of R2=0.9947,50% was 4ng/ml and the detection limit was 1ng/ml. There was no difference between this method and conventional ELISA. The common sequence of mimic epitope peptides was histidine-glycine-proline-tyrosine-arginine-histidine by DNA sequencing analysis. Proline.
According to the results of DNA sequencing analysis, synthetic peptides (HGPYRHP) were synthesized and an immunoassay method was established. The linear range was 1-20 ug/ml, R2=0.9995, the inhibition rate of 20% was 1.2 ug/ml, and the inhibition rate of 50% was 6.3 ug/ml. The dose relationship between TTX standard and synthetic polypeptide was obtained at the concentration of TTX standard and synthetic polypeptide, and the two showed good linearity, R2=0.9978, indicating that the polypeptide could be used as a substitute for TTX standard for ELISA detection of TTX.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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