采用噬菌體展示技術淘選河豚毒素模擬表位的研究
發(fā)布時間：2018-09-05 08:57
【摘要】： 河豚毒素(Tetrodotoxin, TTX)是一種毒性很強的非蛋白類神經毒素,該毒素作用于中樞及周圍神經系統,選擇性地與細胞膜表面的鈉離子通道上的蛋白質結合,從而阻斷鈉離子通道,直接影響神經肌肉興奮性的傳導,使得神經肌肉呈麻痹狀態(tài),甚至引起死亡。該毒素理化性質穩(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。目前國際上TTX標準品價格不菲且TTX屬于生物戰(zhàn)劑遭到國際禁運,此外TTX是劇毒物對實驗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,并對其性質進行研究鑒定,無論是用于無毒檢測方法的研究,還是做為藥物篩選的前提,都具有較大意義。 曾有學者使用抗抗河豚毒素單克隆抗體獨特型抗體(AID)替代河豚毒素用于ELISA檢測,但由于抗獨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不能替代結合態(tài)TTX包被酶標板用于ELISA,而且抗獨特型抗體的篩選相當繁瑣。噬菌體隨機肽庫技術的出現為這個問題的有效解決提供了新的手段。噬菌體隨機肽庫技術是以大量隨機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,并保持其相對的空間構象和生物活性。本研究通過以抗TTX的單抗為配基,從噬菌體隨機肽庫中篩選與之特異性結合的TTX模擬表位,以篩選出來的抗原表位合成多肽替代TTX毒素,并對多肽的性質進行初步鑒定。主要研究內容如下： 以抗TTX單克隆抗體作為靶分子,對融合表達在M13絲狀噬菌體衣殼蛋白表面的隨機七肽庫進行親和淘選,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；通過對陽性克隆進行DNA測序確定插入序列的氨基酸序列,同時經化學方法合成多肽,對模擬表位進行驗證。通過4輪淘選,獲得了7株能與抗TTX單克隆抗體特異性結合的噬菌體,采用間接競爭ELISA,篩選到了3株能抑制TTX的陽性克隆。其中以4號噬菌體建立的免疫檢測方法,線性范圍為1～20ng/ml (TTX毒素濃度),R2=0.9947,50%抑制率為4ng/ml,檢測下限(20%抑制率)為1ng/ml。使用該方法與常規(guī)ELISA檢測方法同時檢測樣品,結果基本無差異。經DNA測序分析,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DNA測序分析結果,合成多肽(HGPYRHP),并建立免疫檢測方法,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標準品與合成多肽建立ELISA分析方法的競爭曲線,通過參照標準曲線中相同結合率時TTX標準品與合成多肽的濃度,可得到TTX標準品與合成多肽的劑量關系,二者呈現良好的線性,其R2=0.9978,表明該多肽可用于替代TTX標準品用于TTX的ELISA檢測。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：2223808
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
【參考文獻】
相關期刊論文 前10條
1 宮慶禮,黃海龍,崔建洲;河豚毒素的色譜檢測[J];水產科學;2001年06期
2 劉相葉;鄧洪寬;吳秀萍;王學林;于申業(yè);于艷玲;劉明遠;;噬菌體展示技術及其應用[J];動物醫(yī)學進展;2008年01期
3 ;河豚魚中毒[J];國外醫(yī)學.護理學分冊;1995年03期
4 周曉翠;謝光洪;劉國文;李妍;王帥玉;王照鵬;孔濤;宋文學;王哲;;河豚毒素檢測方法的研究進展[J];中國畜牧獸醫(yī);2008年07期
5 段紅,李少林;噬菌體展示技術與腫瘤[J];國外醫(yī)學(腫瘤學分冊);2005年09期
6 吳惠蘭;;噬菌體展示技術及其在藥物開發(fā)中的應用[J];海峽藥學;2006年04期
7 王冠男;謝麗萍;張榮慶;;河豚毒素的分布及動物對它的拮抗作用[J];海洋通報;2007年01期
8 許亮亮;;河豚毒素的毒性控制和應用前景[J];化學教學;2008年08期
9 陳常英,丁曉琴,潘心富,李玉林;河豚毒素(TTX)及其衍生物的電子結構和構效關系及與石房蛤毒素(STX)的比較研究[J];化學學報;1998年02期
10 鄧尚貴,彭志英,楊萍,吳鐵,陳芳;河豚毒素研究進展[J];海洋科學;2002年10期
，本文編號：2223808
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2223808.html
最近更新
教材專著
