肉毒毒素与蛋白受体相互作用的分子基础及其应用的研究

发布时间：2018-01-04 07:30

本文关键词：肉毒毒素与蛋白受体相互作用的分子基础及其应用的研究　出处：《中国人民解放军军事医学科学院》2007年博士论文　论文类型：学位论文

【摘要】： 肉毒毒素(Clostridium botulinum neurotoxin,BoNT)通过与靶细胞表面受体特异结合，在受体介导的胞吞作用下内化进入细胞发挥强神经毒性作用。其中，肉毒毒素受体结合区与其靶细胞表面受体的特异结合是决定毒素分子毒性发生的关键起始步骤。研究表明，肉毒毒素Hc片段是其受体结合区(Receptor Binding Domain，RBD)，SynaptotagminsⅡ(sytⅡ)是新近研究发现的肉毒素素特异蛋白受体。目前对肉毒毒素与其受体相互作用的分子基础还缺乏足够的了解，寻找和鉴定在肉毒毒素与蛋白受体相互作用中发挥关键作用的结构基序将有助于揭示肉毒毒素的分子致病机制，为肉毒毒素的特异性防治研究提供新的思路。本论文针对肉毒毒素与靶细胞相互作用的关键环节，主要进行了三部分研究。具体研究结果如下：一、肉毒毒素受体结合区的主要抗原表位的研究针对肉毒毒素受体结合区，采用生物信息学对其抗原表位进行综合分析及预测，并通过计算机模拟技术设计合成模拟表位肽，对其免疫学效应进行评价，同时利用噬菌体展示技术筛选针对模拟表位肽的特异结合短肽，评价噬菌体展示肽封闭特定抗原表位的毒素毒性中和能力，结果发现A、B型肉毒毒素受体结合区抗原表位的分布存在血清型的差异性，A型肉毒毒素受体结合区抗原表位分布较为宽泛，存在分散的多个优势抗原表位，主要位于片段1110-1172及1260-1295，实验结果还显示不同表位间存在协同保护作用；而B型肉毒毒素抗原表位的分布更为集中，主要位于受体结合区末端约四分之一的区域(1183-1291)。抗原表位的确定为肉毒毒素的防治提供明确的药物靶标，同时表位的分布特征也为不同血清型肉毒毒素抗毒素的研究策略提供了实验依据。二、肉毒毒素与特异蛋白受体sytⅡ相互作用结合位点的鉴定针对新近发现的肉毒毒素蛋白受体sytⅡ，首先进行重组制备获得可溶性受体sytⅡ-LD，分析其与不同血清型肉毒毒素及其Hc片段的结合活性，结果显示，可溶性受体可与B型肉毒毒素及其Hc片段特异结合，而与其他血清型没有结合，，表明可溶性受体与B型肉毒毒素Hc片段的结合是特异的。应用Biacore SPR技术对可溶性受体与重组BHc蛋白的特异结合活性进行动态分析，测定亲和力为2.99×10~(-7)M。进一步对可溶性受体与B型肉毒毒素优势模拟表位肽的相互作用进行分析，结果发现，重组功能受体能与P7、P8模拟表位肽特异结合，提示它们可能共同构成毒素的受体结合位点(1235-1291)。同时根据SytⅡ氨基端泡内区氨基酸序列，化学合成了两段重叠受体肽，通过与肉毒毒素重组BHc的特异识别结合，初步确定SytⅡ-LD的毒素特异结合域为G37-K50。三、功能蛋白受体的肉毒毒素毒性拮抗效应研究应用肉毒毒素敏感细胞及小鼠模型，分析肉毒毒素特异蛋白受体的毒素拮抗效应，免疫荧光结果显示，可溶性受体sytⅡ-LD能与细胞膜受体竞争结合肉毒毒素，减少结合到SytⅡ~+ PC12细胞上的毒素。动物保护实验结果显示，可溶性受体sytⅡ-LD融合蛋白具有剂量依赖的毒素抑制作用，浓度达到270μg／mL时，小鼠存活率为25％。双受体是否存在协同作用?结果显示，单独使用神经节苷脂未发现明显的保护作用，但可以显著增强蛋白受体SytⅡ-LD与毒素的相互作用，提高蛋白受体SytⅡ-LD的毒素拮抗效应，同样剂量条件下小鼠存活率达到60％；在此基础上，利用linker序列将肉毒毒素受体片段与抗B型肉毒毒素ScFv连接，构建ScFv-SytⅡ-LD双功能分子，所构建的双功能分子能够明显抑制毒素毒性，其抑制活性比单独可溶性受体sytⅡ-LD有明显提高，双功能活性增强了母本可溶性受体或抗体的毒素特异靶向性，提高毒素毒性拮抗效应，有效延长了小鼠发病和死亡的时间。以上研究为进一步加深对肉毒毒素受体结合区分子结构的认识，探讨肉毒毒素与宿主细胞受体相互作用的分子致病机制奠定了良好的基础，同时为肉毒毒素的特异性防治研究提供了新的思路以及作用明确的药物靶标。
[Abstract]:Botulinum toxin (Clostridium botulinum, neurotoxin, BoNT) by binding to specific cell surface receptor, in receptor-mediated endocytosis internalized into cells exert neurotoxic effects. Among them, botulinum toxin receptor binding region and target specific cell surface receptor binding is a key initial step decision. Toxin molecular toxicity study show that botulinum toxin Hc fragment is the receptor binding region (Receptor Binding, Domain, RBD), Synaptotagmins II (syt II) is a recently discovered botulinum toxin specific protein receptor. There is a lack of adequate understanding of the molecular basis of botulinum toxin receptor interaction, molecular mechanism and structure identification based search play a key role in botulinum toxin and protein receptor interactions in order to help reveal the botulinum toxin, the prevention and control of the specificity of botulinum toxin research to provide a new This paper focuses on three key parts of the interaction between botulinum toxin and target cells.
The results of the study are as follows:
A study of the major epitopes of the binding region of botulinum toxin receptor
The botulinum toxin receptor binding region, using bioinformatics of its epitope was analyzed and predicted by using the computer simulation technology, design and synthesis of mimotope peptide, evaluate its immunological effect, at the same time using phage display specific mimotopes of binding peptides, phage display peptide closed evaluation cytotoxin specific epitope neutralization ability, the results showed that A, the difference of botulinum toxin type B receptor binding region epitope existing serotypes of botulinum toxin type A receptor binding epitope distribution is relatively broad, dispersed multiple epitope fragment and 1110-1172 mainly located in the 1260-1295, the experimental results also showed that different epitopes has synergetic protective effect; and the distribution of botulinum toxin type B antigen epitope is more concentrated, mainly located in the receptor binding region at the end of about four points One area (1183-1291). The determination of antigen epitopes provides a clear target for the prevention and treatment of botulinum toxin. Meanwhile, the distribution characteristics of epitopes provide experimental evidence for different botulinum toxin antitoxin research strategies.
Two, identification of binding sites for the interaction of botulinum toxin and specific protein receptor syt II
According to the newly discovered botulinum toxin syt protein receptor II, first prepared recombinant soluble receptor syt II -LD analysis of binding activity with different serotypes of botulinum toxin and its Hc fragment showed that soluble receptors can be combined with botulinum toxin B and Hc specific fragment, not combined with other serum according to type, soluble receptor and type B botulinum toxin Hc fragment is specific. The specific application of Biacore SPR technology of soluble recombinant BHc protein and receptor binding activity for dynamic analysis, determination of the affinity of 2.99 * 10~ (-7) M. of soluble receptor and botulinum toxin type B advantage to simulate the interaction between epitopes peptide analysis, results showed that the recombinant receptor function with P7, P8 epitope specific binding peptide, indicating that they may constitute the toxin receptor binding site (1235-1291). At the same time, according to the Syt - II The amino acid sequences in the end vesicle region were chemically synthesized, and the two overlapping receptor peptides were synthesized. By combining with the specific recognition of recombinant BHc of botulinum toxin, the toxin specific binding domain of Syt II -LD was preliminarily determined to be G37-K50..
Three, study on the toxic antagonism of botulinum toxin of functional protein receptor
The use of botulinum toxin sensitive cells and mice model, analysis of toxin antagonistic effect of botulinum toxin specific protein receptor, immunofluorescence results showed that the soluble receptor syt II -LD and cell membrane receptor competitive binding of botulinum toxin, reduce the binding to Syt II + PC12 cells of toxins. Animal experimental results showed that the soluble receptor syt II -LD fusion protein has a dose-dependent inhibitory effect of melittin, the concentration reached 270 g / mL, the survival rate of mice was 25%. double receptor whether there is a synergistic effect? Results show that ganglioside used alone without obvious protective effect, but can significantly enhance the interaction between -LD and Syt II receptor protein toxins, improve antagonistic effect of Syt toxin protein receptor II -LD, the same dose conditions, the survival rate of mice reached 60%; on this basis, using linker sequence of botulinum toxin receptor fragment with anti type B meat Toxin ScFv connection, construct ScFv-Syt II -LD bifunctional molecule, bifunctional molecule constructed can inhibit the toxicity, the inhibitory activity was obviously higher than single soluble receptor syt II -LD bifunctional activity enhanced toxin specific targeting maternal antibody or soluble receptor to enhance toxicity, antagonistic effect, prolong the mice of morbidity and mortality.
The above research to further deepen the understanding of the molecular structure of binding region of botulinum toxin receptor, has laid a good foundation to explore the molecular mechanism of botulinum toxin and host cell receptor interaction, and provides ideas and drug clear new target of specific prevention of botulinum toxin.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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