多肽类蝎昆虫神经毒素胃毒型杀虫剂的研究

Scorpion anti-insect toxins with three characteristics of high selectivity, high insecticidal activity and structural stability have been considered as one of the most potential biological pesticides. But to date, there is no proper administration method could achieve the rapid lethal effect as scorpions prey predator in the nature. By molecular design and site directed mutagenesis, we constructed and screened three high efficiency and low toxicity candidate mutants. Among which, rLqh αIT-1 showed good stomach toxicity and the correction mortality rate increased three times, when coupling with 1% Calcofluor.The fact that the successfully application of protein PEGylation in human drug design reaching an equal effect to injection administration offers scientific, supports for developing peptide stomach toxicity pesticides. In this project, borrowing ideas from the human protein drug oral administration, we will continue to screen high efficiency, non-toxicity Lqh α IT candidate mutants; to improve their protease tolerance and enhance their absorption by PEGylation; to appraise PEGylation leads with the compatibility use of 1% Calcofluor. Thus the stomach toxicity of rLqh αITs can be increased. Applying this method, it will be expected to discover a fast and efficient administration method for the application of peptide pesticides.

蝎昆虫神经毒素具有选择性强、活性高及结构稳定三大特点，被视为最具开发潜力的生物杀虫剂之一。但是目前尚没有合适的给药方式，能够达到自然界蝎子捕食猎物迅速致死的效果。本课题组通过分子设计和定点突变，构建并筛选得到3个高效、低毒候选突变体，其中rLqh αIT-1与1% Calcofluor配伍后，表现出良好的胃毒性，矫正死亡率提高了3倍。人类蛋白类药物的PEG修饰，成功实现了药物的口服和注射给药疗效无显著差异，这给多肽类杀虫分子胃毒剂的研究提供了科学依据。因此，本项目拟在前期工作基础上，借鉴人类口服蛋白类药物的PEG化技术，继续筛选高效、无毒的Lqh αIT候选突变体；对其进行PEG修饰，增强对蛋白酶的耐受性和透膜吸收能力；探究在1% Calcofluor配伍下，先导PEG化产物。从而增加蝎毒素rLqh αITs的胃毒作用。本项目有望为多肽类杀虫分子的有效应用寻找一条快捷有效的给药途径。

本项目设计并筛选Lqh αIT的特定位点突变体，获得的6个单、双突变体表现出比天然毒素增强的昆虫毒性和减弱的哺乳动物毒性，其中rLqh αIT-1在两个方面表现最好。确定了对rLqh αIT N端进行PEG修饰化学方案及分离纯化方案，经阳离子交换色谱和凝胶过滤色谱两次纯化后，修饰产物纯度达85%以上。PEG产物较未修饰的rLqh αIT-1在中肠肠液的稳定性有所提高。PEG-rLqh αIT-1在与1% Calcofluor 配伍时,实现了蝎昆虫毒素经口给药的胃毒活性，表现出了较强的杀虫作用（同等给药剂量时,48小时死亡率达43%）。但是尚未达到自然界蝎子捕食猎物时的迅速致死效果。于是我们进一步寻找能够进一步增强rLqh αIT经口给药效果的物质。我们发现3个具有3,4-亚甲二氧基，4个具有鬼臼毒素反式五元内酯环结构的新木脂素类物质与PEG-rLqh αIT-1和1% Calcofluor 配伍使用时能够使杀虫效果提高2-3倍，是非常有潜力的增效剂。以上工作实现了多肽类昆虫神经毒素经口给药的胃毒性。我们还将继续从天然植物次生代谢产物中寻找多肽类昆虫毒素的增效剂,以期达到用量微、起效快的自然界杀虫效果。