【摘要】： 河豚毒素(Tetrodotoxin, TTX)是一種毒性很強(qiáng)的非蛋白類神經(jīng)毒素,該毒素作用于中樞及周圍神經(jīng)系統(tǒng),選擇性地與細(xì)胞膜表面的鈉離子通道上的蛋白質(zhì)結(jié)合,從而阻斷鈉離子通道,直接影響神經(jīng)肌肉興奮性的傳導(dǎo),使得神經(jīng)肌肉呈麻痹狀態(tài),甚至引起死亡。該毒素理化性質(zhì)穩(wěn)定,能耐高溫及日光暴曬,家庭的烹飪過程幾乎不破壞其毒性。河豚毒素在臨床上可用做止痛劑、麻醉劑和降壓藥等等,但需慎重把握其用量。目前國(guó)際上TTX標(biāo)準(zhǔn)品價(jià)格不菲且TTX屬于生物戰(zhàn)劑遭到國(guó)際禁運(yùn),此外TTX是劇毒物對(duì)實(shí)驗(yàn)操作人員也存在安全隱患。因而如果能制備一種河豚毒素的替代物,并對(duì)其性質(zhì)進(jìn)行研究鑒定,無論是用于無毒檢測(cè)方法的研究,還是做為藥物篩選的前提,都具有較大意義。 曾有學(xué)者使用抗抗河豚毒素單克隆抗體獨(dú)特型抗體(AID)替代河豚毒素用于ELISA檢測(cè),但由于抗獨(dú)特型抗體與抗體之間的親和力通常高于抗體與毒素之間的親和力,因此制備的AID不能替代結(jié)合態(tài)TTX包被酶標(biāo)板用于ELISA,而且抗獨(dú)特型抗體的篩選相當(dāng)繁瑣。噬菌體隨機(jī)肽庫技術(shù)的出現(xiàn)為這個(gè)問題的有效解決提供了新的手段。噬菌體隨機(jī)肽庫技術(shù)是以大量隨機(jī)編碼的多肽序列插入噬菌體載體,形成噬菌體展示文庫,每個(gè)噬菌體粒子表面只展示一種序列的外源肽鏈,并保持其相對(duì)的空間構(gòu)象和生物活性。本研究通過以抗TTX的單抗為配基,從噬菌體隨機(jī)肽庫中篩選與之特異性結(jié)合的TTX模擬表位,以篩選出來的抗原表位合成多肽替代TTX毒素,并對(duì)多肽的性質(zhì)進(jìn)行初步鑒定。主要研究?jī)?nèi)容如下： 以抗TTX單克隆抗體作為靶分子,對(duì)融合表達(dá)在M13絲狀噬菌體衣殼蛋白表面的隨機(jī)七肽庫進(jìn)行親和淘選,通過ELISA方法篩選和鑒別具有模擬表位的陽性噬菌體克隆,并用陽性克隆建立ELISA分析方法；通過對(duì)陽性克隆進(jìn)行DNA測(cè)序確定插入序列的氨基酸序列,同時(shí)經(jīng)化學(xué)方法合成多肽,對(duì)模擬表位進(jìn)行驗(yàn)證。通過4輪淘選,獲得了7株能與抗TTX單克隆抗體特異性結(jié)合的噬菌體,采用間接競(jìng)爭(zhēng)ELISA,篩選到了3株能抑制TTX的陽性克隆。其中以4號(hào)噬菌體建立的免疫檢測(cè)方法,線性范圍為1～20ng/ml (TTX毒素濃度),R2=0.9947,50%抑制率為4ng/ml,檢測(cè)下限(20%抑制率)為1ng/ml。使用該方法與常規(guī)ELISA檢測(cè)方法同時(shí)檢測(cè)樣品,結(jié)果基本無差異。經(jīng)DNA測(cè)序分析,模擬表位肽的共有序列是組氨酸-甘氨酸-脯氨酸-酪氨酸-精氨酸-組氨酸-脯氨酸。 根據(jù)DNA測(cè)序分析結(jié)果,合成多肽(HGPYRHP),并建立免疫檢測(cè)方法,其線性范圍為1-20μg/ml, R2= 0.9995,20%抑制率為1.2μg/ml,50%抑制率6.3μg/ml。分別以不同濃度的TTX標(biāo)準(zhǔn)品與合成多肽建立ELISA分析方法的競(jìng)爭(zhēng)曲線,通過參照標(biāo)準(zhǔn)曲線中相同結(jié)合率時(shí)TTX標(biāo)準(zhǔn)品與合成多肽的濃度,可得到TTX標(biāo)準(zhǔn)品與合成多肽的劑量關(guān)系,二者呈現(xiàn)良好的線性,其R2=0.9978,表明該多肽可用于替代TTX標(biāo)準(zhǔn)品用于TTX的ELISA檢測(cè)。
[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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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