基于新靶点的三种抗菌肽一级结构解析、可溶性表达以及作用机制研究

Three antimicrobial peptides were purified from scorpion toxin and their partial amino acid sequence were resolved, using the Edman method. Their molecular mass were determined by biological mass spectrometry. Furthermore, the cDNA and their gene sequences were obtained by biotechnology. The result of this experiment is to resolve the primary structures of three antimicrobial peptides from scorpion. Three antimicrobial peptides were successfully expressed in E. coli in soluble form and the recombinant antimicrobial peptides were prepared. Then three standard strains from the Gram-positive bacteria, Gram-negative bacteria and fungi respectively were selected to verify the antibacterial activity of the recombinant peptides. Three active peptides and the scorpion analgesic-antimicrobial peptides preobtained were studied with patch clam，fluorescent reagents combined with fluorescence plate reader technology, their effect and the extent on sodium channels of three strains' protoplasts were investigated. We also investigated the influence that the varying concentrations of sodium ions in bacteria effect on the membrane potential and the concentration of calcium ions as well as the relationship between them. The effect of antimicrobial peptides on sodium channels of human, was explored preliminarily. Depending on the bioinformatic techniques, their potential active sites in the interaction between antimicrobial peptides and sodium channels of bacterial was explored preliminarily by molecular docking. Our research was not only for the development of the antibacterial drug with originally intellectual property rights and a new target, as well as the in-depth development of new drugs, but also laid a solid material foundation and theoretical basis for the evaluation system of the sodium channel which the antimicrobial drug targets for. We will try to explore new ideas in order to flourish the traditional Chinese pharmaceutical treasure.

从全蝎毒纯化获得三种抗菌肽，利用Edman法解析部分氨基酸序列、生物质谱检测分子量、生物技术获得cDNA、基因及其序列实验结果，解析三种蝎抗菌肽一级结构。实现三种抗菌肽基因在大肠杆菌的可溶性表达并制备获得重组抗菌肽。选择G+菌、G-菌和真菌三种标准株，验证重组肽的抗菌活性。利用膜片钳、荧光试剂结合荧光酶标仪技术，研究上述三种活性肽以及前期获得的蝎镇痛抗菌活性肽，对三种菌株原生质体的钠离子通道作用及程度，菌体内钠离子浓度变化对膜电位、钙离子浓度的影响以及关系。初步探讨抗菌肽对人钠离子通道的作用，利用生物信息学技术，通过抗菌肽与细菌钠离子通道的分子对接，初步探讨抗菌肽、细菌钠通道相互作用的各自潜在活性位点。研究工作，不仅为拥有原创性自主知识产权的新靶点抗菌新药研发及其深层次新药研发，也为基于钠通道抗菌药物作用靶点评价体系奠定坚实物质基础和理论依据，为发扬光大祖国传统医药宝库,尝试、开拓新思路。

由于抗生素的不合理用药、乃至滥用，使得细菌耐药问题日趋严重，目前已出现了能对抗所有抗生素的耐药菌。如何解决细菌耐药性，是当前抗细菌性感染和细菌性疾病药物研发的重点和热点。对此解决策略有多种，其中新靶点、新机制、新结构抗菌候选药物是重中之重。中医利用全蝎在抗各种细菌性感染和治疗细菌性疾病的临床应用显现出令人振奋的疗效，但有关蝎以及蝎毒中抗菌活性成分的结构，国内外专利及文献报道极少。. 本课题从蝎毒中分离纯化三种抗菌活性肽并解析其一级结构；实现三种抗菌肽在大肠杆菌的可溶性表达并制备获得重组抗菌肽；在 G+菌、G-菌标准株，验证重组肽的抗菌活性；利用膜片钳、荧光试剂结合荧光酶标仪术，探讨上述三种重组活性肽对菌体钠离子通道作用及程度，菌体内钠离子浓度变化对膜电位、钙离子浓度的影响以及关系。初步探讨抗菌肽对人钠离子通道的作用以及抗菌肽SYPU-32的活性区域及其氨基酸残基的性质与其活性作用强度与特异性关系。. 新型三种抗菌活性肽一级结构解析结果表明：抗菌肽均为单一肽链，由64-66 个氨基酸残基组成的碱性多肽，含有四对二硫键，属于蝎长链毒素家族。通过融合表达模式，三种抗菌肽在大肠杆菌细胞中均得以表达，可溶性表达水平在25%——86%之间，纯化的融合表达产物经酶切割（效率在90%以上）去除融合蛋白后，纯化获得重组抗菌肽，重组活性肽对G+菌、G-菌和真菌均具有抑制其生长作用。新型三种抗菌活性肽作用机制结果表明：两种抗菌肽的作用机制属于经典抗菌肽作用范畴，即通过破坏细胞壁与细胞膜结构，使其胞内生物大分子——酶泄漏而导致细菌死亡；抗菌肽SYPU-32（已经申请了发明专利）对人电压门控钠离子通道具有作用，但并不是通过作用菌体细胞离子通道产生抗菌作用，而是通过改变菌体细胞内钠离子浓度而产生作用。初步确定抗菌肽-SYPU2作用的潜在靶点——钠离子转运系统。. 课题研究，为拥有原创性自主知识产权的新靶点、新机制抗菌新药研发及其深层次新药研发，也为基于钠离子转运体的抗菌药物作用靶点及其评价体系，奠定了坚实的物质基础和理论依据。