【作者】 韩彤彤；

【导师】 肖向红；

【作者基本信息】 东北林业大学 ， 动物学， 2006， 硕士

【摘要】 两栖类皮肤抗菌肽是两栖类在进化过程中获得的一道抵御外源微生物入侵的屏障,其种类众多、功能多样,具有抗细菌、抗真菌、抗肿瘤等方面的性质和机制,特别是近几年来,研究发现两栖类抗菌肽对疱疹病毒、艾滋病毒等有抑制作用,且在抑杀病毒的同时对宿主细胞无伤害。在当今多种病毒性疾病的传播给全球经济造成了巨大损失,各种病毒的迅速变异的情况下,以及抗病毒药物疗效降低和较严重的毒副作用等,寻找安全、有效的抗病毒药物成为全球关注的焦点,而两栖类皮肤活性肽因其独特的抗微生物特点,有望成为开发新型抗病毒药物的主要途径之一。 本实验以东北林蛙(Rana dybowskii)作为两栖类的代表物种,从蛙皮肤分泌物中提取并纯化抗菌肽,通过以SAH水解酶活性抑制为评价指标的体外广谱抗病毒初筛模型检测皮肤抗菌肽的抗病毒活性,并以H9N2亚型禽流感病毒为试验对象,进一步检测蛙皮肤抗菌肽对有包膜病毒病毒的抑制作用。实验中的抗菌肽全部由本实验室提取。 结果表明:(1)从鲜牛肝脏中提取纯化的SAH水解酶,比活力为1.23U/mg,在1分钟内SAH水解酶催化30.7%的腺苷(Hcy)与高半胱氨酸(Ado)合成为S-腺苷-L高半胱氨酸(SAH);(2)向SAH水解酶反应体系加入皮肤抗菌肽后,1分钟内有44.7%的Hcy与Ado合成为SAH,说明东北林蛙皮肤活性肽对SAH水解酶有抑制作用,东北林蛙皮肤中含有广谱抗病毒作用的活性成分;(3)东北林蛙皮肤抗菌肽对H9N2病毒的杀灭作用较明显,与攻毒组比较,杀灭组、治疗组和预防组血凝效价差异极显著(P<0.01),杀灭组血凝效价(Log2)从(104±0.82)降低至(3±0.82),预防组血凝效价(Log2)降低至(5.5±0.50),治疗组血凝效价(Log2)降低至(5.3±0.58)。与治疗组和预防组相比较,杀灭组对H9N2病毒的抑制作用更明显。 结论:(1)本研究建立了SAH水解酶活性抑制的体外广谱抗病毒初筛模型,采用比色法从合成方向检测了SAH水解酶活性,该检测方法是本实验室首创,具有重复性好,操作简单,实验周期短,易于推广等特点;(2)东北林蛙皮肤抗菌肽对SAH水解酶有抑制作用,说明东北林蛙皮肤抗菌肽中含有广谱抗病毒成分;(3)东北林蛙皮肤抗菌肽对H9N2亚型禽流感病毒有抑制作用,说明东北林蛙皮肤抗菌肽对有包膜病毒有抑制作用。更多还原

【Abstract】 The skin antimicrobial peptides(AMPs) are the barrier for amphibians to defense invading of microorganism. They have many families and lots of functions, such as anti bacteria, anti fugin and anti tumor. Recently, it reported that AMPs have inactivate properties to herpes virus and HIV-1, and simultaneity no harm for host cells. Nowadays the spread of virus diseases depress the globle economy, to find safe, effective antiviral medicine became the focus. For the unique characters of amphibian skin peptides, it is hoped to be the important way of exploring new antibiotic.In this paper, we isolate and purified AMPs from northeast forest frogs(Rana dybowskii), choose the SAH hydrolase as the antiviral filtration model to determine the broad-spectrum antiviral properties, then choose the zoonosis virus—H9N2 avian influenza virus to investigate its inactive properties to enveloped virus.Result: (1) We extract and purified SAH hydrolas from bovine liver, the specific activity of the extract SAH hydrolase is 1.23U/mg, it can catalyse 30.7% Hcy to SAH within 1 minute;(2) When put the skin peptide of Northeast forest frog into the reaction system, there is 44.7% Hcy change into SAH within 1 minute, It indicate that the skin peptide of Northeast forest frogs regress the activity of SAH hydrolase, which indicate that the skin activity peptide has broad- spectrum activity;(3) The maximum nontoxic concentration is 150mg/ml. there are comparison group, infection group, killing group, and therapy group and prevent group in this experiment. Compared with infection group, the titer of therapy group and prevent group, the killing group differ significant(P<0.01), the HA of killing guoup reduced from (10±0.82) to (3 ±0. 82), the HA of prevent group reduce by (5. 5 ± 0. 50) , HA of therapy group reduce to (5.3 ± 0.58) , compared with therapy group and prevent group, the killing group shows distinct effect on the avian influenza virus H9N2.Conclusion: (1)We establish antiviral filtration model in vitro— SAH hydrolase model, and examine its activity from the synthesizes direction with ultraviolet spectrophotometer, it is our innovation. This method has high accuracy, short periods and can be easy operated, it is adapt to be spread out;(2) the skin peptide of Northeast forest frogs regress the activity of SAH hydrolase, which indicate that the skin activity peptide has broad- spectrum activity;(3)the skin AMPs of northeast forest frogs have inactive properties to H9N2 avian influenza virus, indicate that AMPs of northeast forest frogs can inactive evoloped virus.更多还原