【作者】 李键；

【导师】 凌飞；

【作者基本信息】 西北农林科技大学 ， 水产养殖， 2020， 硕士

【摘要】 由多子小瓜虫（Ichthyophthirius multifiliis）（简称小瓜虫）寄生于淡水鱼体表和鳃引起的小瓜虫病是危害最大的鱼类寄生虫病之一。小瓜虫具有具有分布广、无宿主专一性等特点,不仅能造成养殖和观赏渔业巨大的经济损失,而且也能在自然水域中爆发,引起鱼类大面积死亡。目前化学药物是现阶段小瓜虫病防治的主要方法和有效策略。然而,自特效药物-孔雀石绿和汞盐被禁止使用后尚无特效药。在自然环境中容易降解、对环境友好和资源丰富等优点的天然产物是水产抗寄生虫药的重要来源。厚朴酚是从中草药厚朴（Magnolia officinalis）中发现的主要活性成分之一,由于其简单的化学结构,易于进行人工合成以及修饰改造,因此是进行药物开发的理想先导化合物。前期研究发现精氨酸酶（Arginase,ARG）是厚朴酚作用于小瓜虫的药物靶点。因此,本研究利用原核表达的小瓜虫精氨酸酶构建最佳酶活反应体系,并以该系统为基础,测定厚朴酚/和厚朴酚衍生物对精氨酸酶的体外抑制效果并研究其定量构效关系,从而揭示化合物结构和活性之间的定量变化规律,为后续设计合成抗小瓜虫药物提供依据和理论基础。取得的结果如下:1. 小瓜虫精氨酸酶的原核表达本研究对小瓜虫精氨酸酶序列进行碱基优化后,构建了重组表达质粒p ET-32a-ARG,在大肠杆菌BL21（DE3）中成功表达并纯化到可溶性重组蛋白ARG,分子量约为60 k Da。2. 小瓜虫精氨酸酶的酶活性反应体系的建立利用纯化的重组蛋白ARG建立了体外酶活性分析体系,酶活性分析结果表明重组蛋白ARG酶活性约为25.23 U/mg,在反应体系中加入1 m M Mn²⁺时,其酶活性显著提高,约为116.06 U/mg,最适反应p H为9.5,最适反应温度为42℃,Km值为0.158mol/L。低温4℃和高温70℃均能使ARG失去酶活性。厚朴酚和精氨酸酶特异性抑制剂NOHA对ARG均有抑制作用。3. 厚朴酚/和厚朴酚衍生物抑制精氨酸酶活性的构效关系研究测定了73个厚朴酚、和厚朴酚衍生物的抑制效果,其中有35个化合物对精氨酸酶存在抑制效果;选取20个化合物作为训练集和4个化合物作为测试集,以活性最好的化合物21（3,5’-dipropyl-[1,1’-biphenyl]-2’,4-diol）为模板分子,建立比较分子场分析法（Comparative molecular field analysis,Co MFA）模型。模型的交叉验证系数q²为0.595,相关系数平方r²为0.967;利用Co MFA模型进行预测,试验值与预测值的拟合图表明模型具有的统计学意义,预测能力较好。利用偏最小二乘法（PLS）对模型建立三维等势图,表明在厚朴酚与和厚朴酚的C-9和C-9’位置增加取代基的体积会增加活性,在C-2、C-3、C-4和C-5位置增加取代基的体积会降低活性;在C-9和C-3’位置增加取代基的体积的负电性会增加活性,在C-4位置增加取代基的正电性会增加活性。综上所述,在厚朴酚与和厚朴酚的在C-9和C-9’位增加取代基的体积有利于抑酶活性的提高,在C-3和C-4位置增加取代基的体积不利于抑酶活性的提高;在C-4位置增加取代基的正电性有利于抑酶活性的提高。本研究为后续设计合成抗小瓜虫药物提供了依据和理论基础。更多还原

【Abstract】 Ichthyophthiriasis is one of the most harmful parasitic diseases of fish,which is caused by Ichthyophthirius multifiliis parasitizing on the body surface and gills of freshwater fish.With the characteristics of wide distribution and host-free specificity,I.multifiliis can not only cause huge economic losses in breeding and ornamental fishery,but also cause large areas of fish death in natural waters.At present,chemical drugs are the main methods and effective strategies for the prevention and treatment of Ichthyophthiriasis.However,no specific drugs have been available since the specific drugs malachite green and mercury salt were banned.Natural products with abundant resources are easy to degrade in the natural environment and are friendly to the environment.They are the ideal source of anti-parasite drugs for safe and pollution-free aquatic products.Magnolol,one of the main active ingredients found in Magnolia officinalis,is an ideal lead compound for drug development because of its simple chemical structure and its ease of synthesis and modification.Previous studies found that arginase is the drug target of magnolol for the prevention and treatment of I.multifiliis.In this study,the optimal enzyme activity reaction system was constructed using prokaryotic expressed arginase,and based on this system,the in vitro inhibitory effect of magnolol and honokiol derivatives on arginase was determined.Study its quantitative structure-activity relationship,reveal the quantitative change between the structure and activity of the compound,provide a basis and theoretical basis for the subsequent design and synthesis of anti-I.multifiliis drugs,and also provide reference for the research and development of other aquatic drugs.The results obtained in this work were as follows:1. Prokaryotic expression of I.multifiliis arginaseIn this study,after optimizing the arginase sequence of I.multifiliis,a recombinant expression plasmid p ET32a-ARG was constructed,which was successfully expressed in E.coli BL21（DE3）and purified to a soluble recombinant protein ARG,with a molecular weight of about 60 k Da.2. Establishment of the reaction system of arginase from I.multifiliisAn in vitro enzyme activity analysis system was established using the purified ARG recombinant protein.The results of the enzyme activity analysis showed that the enzyme activity of the recombinant protein ARG was about 25.23 U/mg.The enzyme activity was significantly increased when 1 m M Mn²⁺was added to the reaction system,which was about116.06 U/mg.The optimum p H is 9.5,the optimum temperature is 42℃,and the Km value is0.158 mol/L.Low temperature 4℃and high temperature 70℃can make ARG lose enzyme activity.Both magnolol and arginase-specific inhibitor NOHA inhibited ARG.3. Structure-activity relationship of magnolol and honokiol derivatives on arginase inhibitionThe inhibitory effects of 73 magnolol and honokiol derivatives were measured,among which 35 compounds had inhibitory effects on arginase.Twenty compounds were selected as the training set and four compounds as the test set.The most active compound 21（3,5’-dipropyl-[1,1’-biphenyl]-2’,4-diol）was used as the template molecule to build a Comparative molecular field analysis（Co MFA）model.The cross-validation coefficient q²was 0.595,and the correlation coefficient r² was 0.967.The Co MFA model was used for prediction,and the fitting graph of the experimental value and the predicted value showed that the model had statistical significance and good prediction ability.Partial least squares（PLS）was used to construct a three dimensional isopositive diagram of the model,which indicated that increasing the volume of substituents at the positions of magnolol and honokiol C-9 and C-9’would increase the activity,while increasing the volume of substituents at the positions of C-2,C-3,C-4 and C-5 would decrease the activity.Increasing the volume of the substituents at C-9 and C-3’increases the activity,and increasing the positivity of the substituents at C-4 increases the activity.In summary,the results showed that the introduction of large groups at the C-9 and C-9’positions of magnolol and honokiol was beneficial to the improvement of inhibitory activity,while and the introduction of large groups at the C-3 and C-4 positions was detrimental to the improvement of inhibitory activity.The increase of electronegativity in the c-4 position is not conducive to the improvement of the enzyme activity.This study provides a basis and theoretical basis for the subsequent design and synthesis of anti-I.multifiliis drugs.更多还原