抗蛇毒蛋白PLIγ的分子改造及模拟肽设计

Snake venom phospholipase A2s exhibit a wide range of physiopathological effects, including haemorrhagin, neurotoxicty, cardiotoxicity, and anti-aggregation of platelet, etc., and is known as the major toxin. Therefore, inhibitors of PLA2 are able to diminish the mortality and morbidity of snake envenomation, and are considered as potential antivenom candidate besides antivenin. Natural PLA2 inhibitor (PLI) proteins are mainly found in snake species as product of co-evolution with venom. In our previous assays, we discovered a new gamma PLI from Sinonatrix annularis that strongely inhibited enzymatic and hemorrhagic acitivities of svPLA2. What's more, PLIγ were expressed in a variety of snake livers, but differed in acitivities. In order to investigate the structure-function relationship, we schemed to modify the PLIγ molecule of Sinonatrix annularis by DNA shuffling, error-prone PCR and site-specific mutagenesis. Phage display technique are going to be used to screen PLIγ mutations that with higher inhibition effeciency. Bioinformation analysis on these high acitivity PLIγs' secondary and 3D structure will be carried out for a further understanding of key amino acids and domains. Subsequently, mimic peptides design and validation will be conducted by employing molecular docking and assay. These experiments are benificial to drug discovery of antivenom and anti-inflammation diseases.

蛇毒PLA2家族具有出血毒、神经毒、心脏毒、抗血小板聚集等毒性，是蛇毒中的主要毒素。因此，PLA2抑制剂可以有效降低毒蛇咬伤的致死致残率，是除抗蛇毒血清之外的良好抗蛇伤药。天然的PLA2抑制蛋白主要来自蛇本身，它是与蛇毒协同进化的产物。通过筛选，我们前期在华游蛇中发现了一种可强烈抑制蛇毒PLA2酶活性和出血毒性的蛋白-PLIγ。我们还发现PLIγ在多种蛇肝脏均有表达，但活性差异较大。为了深入了解PLIγ结构与功能的关系，我们拟采用DNA改组、易错PCR和DNA定点突变等方法，对华游蛇PLIγ进行分子改造，利用噬菌体表面展示技术，筛选更强抑制活性的PLI γ突变体。我们还将通过生物信息学方法，分析高活性PLIγ的二级和空间结构特点，进一步明确PLIγ的关键氨基酸和结构域。基于这些位点和结构域，通过分子对接和活性实验，我们希望得到强活性PLIγ小分子模拟肽，为抗蛇毒和抗炎性疾病药物奠定基础。

本项目中，我们系统开展了12种蛇血清PLIγ活性测定，克隆获得9种PLIγ基因。建立了“蛇毒-sepharose 4B”亲和柱，实现了一步分离蛇血清PLIγ。根据活性筛选结果，选择华游蛇（saPLIγ）和王锦蛇PLIγ(ecPLIγ)为亲本，进行了PLIγ DNA改组。将突变体库PLIγ插入噬菌体质粒pCANTAB5e，以五步蛇毒为靶标，用噬菌体展示库淘选技术，获得了三种与蛇毒有高亲和力的嵌合PLIγ（cPLIγ）。测序和比对结果表明，三种cPLIγ主要是华游蛇和王锦蛇PLIγ在不同位点的序列拼接结果。构建了亲本和三种cPLIγ的pET28表达质粒，比较了亲本和cPLIγ蛋白的抗五步蛇毒出血毒活性。结果表明cPLIγ2活性高于亲本，在二级和三级结构方面与华游蛇PLIγ更相似，150–155位的α-螺旋可能与PLIγ活性有关。本研究中，我们系统阐述了蛇毒PLA2作为广谱抗蛇毒药靶点的科学基础和意义，并系统研究了华游蛇PLIγ和伐瑞拉迪（PLA2小分子抑制剂）广谱抗蛇毒出血毒、肌肉毒和系统毒活性，探讨了PLA2抑制剂对中毒肌肉再生和功能重建的影响。另外，我们采用低保真Taq酶，对华游蛇PLIγ进行易错PCR，构建了突变体库。采用定点突变方法，将华游蛇PLIγ的PGLPFSQLN区突变为赤练蛇型PGLPLSYPN。结果表明该区域三个氨基酸发生突变后，突变的PLIγ抗出血毒活性下降，表明该区域是PLIγ的活性区域。本研究为广谱抗蛇毒药物PLIγ的开发和活性改进提供了立论依据和实验基础。