对虾抗脂多糖因子ALF的功能分析和开发利用

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	对虾抗脂多糖因子ALF的功能分析和开发利用
	杨辉
学位类型	博士
导师	李富花
	2016-05-21
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	对虾抗菌肽抗脂多糖因子alf 白斑综合征病毒重组表达
摘要	对虾是重要的水产养殖品种之一，由于各类病害的爆发，对对虾养殖产业造成了巨大的经济损失。对虾抵御病原入侵的主要方式是启动先天免疫应答系统，通过产生免疫效应分子，来清除各种病原。抗菌肽作为对虾免疫应答反应产生的一类重要的效应分子，在清除感染的外来病原的过程中起着重要作用。抗脂多糖因子（Anti-lipopolysaccharride factor, ALF）作为对虾的一类重要的抗菌肽，在先天性免疫系统中起着重要作用。前期的研究表明，在中国对虾体内存在7种不同的ALF基因，其抗菌和抗WSSV活性存在着明显差别，但是对于不同ALF的结构与功能的关系，以及抑菌和抗病毒的作用机理了解较少，亟待深入研究。另外，由于传统抗生素导致的细菌耐药性的问题日益引起大家的关注，寻找新型抗菌活性物质来替代传统抗生素的需求更加迫切。抗菌肽由于其抗菌机理与传统抗生素存在明显差别，不会引起细菌耐药性的问题，被认为是抗生素的理想替代物。本论文为解决未来可能的应用问题，通过对ALF的结构进行改造，以提高ALF的抗菌活性；同时也通过利用合成生物学，原核和真核重组表达等方法，获得了具有较高生物学活性的抗菌肽重组蛋白。本研究为抗菌肽产品的研发，实现其在医药或水产养殖中的应用奠定了重要基础。主要研究进展如下： 1. 通过分析中国对虾7种不同ALF的脂多糖结合结构域(LPS binding domain, LBD)短肽的理化特征和功能活性的关系，推测LBD短肽中碱性氨基酸的数量以及亲水和疏水氨基酸的空间排布共同决定着LBD短肽的抑菌活性。为证实此推断，按照以上原则设计合成了一系列短肽，并对其最小抑菌浓度（MIC）和最小杀菌浓度（MBC）进行了检测，结果表明，有些结构改造后的短肽杀菌能力明显提高，在2-4 μM的浓度下即可杀灭大肠杆菌，溶藻弧菌和哈维氏弧菌等，且对大部分革兰氏阴性菌和革兰氏阳性菌都具有一定的抑制效果。对这些改造后的LBD短肽的生物学活性和杀菌机理进一步分析，结果表明，这些短肽具有很好的热稳定性，即使经过了热处理仍能在短时间内杀灭细菌，其活性未发生明显改变。通过对LBD短肽处理过的细菌进行观察，发现LBD短肽是通过破坏细菌的细胞外膜，导致胞质外溢而杀灭细菌的。圆二相色谱分析的结果表明，LBD短肽呈典型的β-sheet结构。以上研究结果为高活性抗菌肽的设计和改造提供了重要依据。 2. 针对中国对虾7种ALFs中的一种类型—FcALF5，其对大肠杆菌无抑制活性，而对WSSV有抑制活性的特点，利用原核重组表达系统成功获得了具有明显抗WSSV活性的重组蛋白，并进一步探讨了其抗WSSV的作用机理。以重组的FcALF5为诱饵蛋白，利用Far Western Blot获得了与其存在相互作用的WSSV蛋白，经过LC-MS/MS分析鉴定，发现该蛋白与WSSV的囊膜蛋白VP24具有最高的相似性。进一步利用原核重组表达获得了VP24重组蛋白，利用点杂交和Far Western Blot验证了FcALF5与WSSV的VP24存在相互作用，同时利用FcALF5的LBD短肽也证实了其与VP24的相互作用。由于VP24是WSSV入侵细胞的一种重要的囊膜蛋白，因此推断FcALF5发挥抗WSSV作用主要是通过LBD结构域和VP24的相互作用来实现的。 3. 对FcALF2的LBD进行结构改造，抗菌活性明显提高的一种类型（LBDv），替换对虾FcALF2基因原有的结构功能域，并结合酵母系统密码子的偏好性，利用生物合成的方法合成该基因，并对该基因在宿主菌毕赤酵母GS115进行重组表达，成功获得了目的蛋白。对重组表达获得的蛋白进行抗菌活性分析，结果表明，该蛋白具有明显的抗菌和抗WSSV活性。此系统的建立为利用体外真核表达系统获得重组蛋白并在生产上应用奠定了重要基础，也为抗菌肽药物的开发和利用提供了重要参考。
其他摘要	Shrimp is one of the major species in aquaculture. Shrimp diseases have brought a great economic loss to the production of shrimp aquaculture. Shrimp relies on the innate immune system to defend against the invaded pathogens by producing kinds of immune effectors. Anti-lipopolysaccharide factors (ALFs), as one type of antimicrobial peptides (AMPs), are a kind of immune effectors produced by the innate immune system of shrimp. Previously, we have isolated seven ALF isoforms from Chinese shrimp Fenneropenaeus chinensis with different antimicrobial and antiviral (WSSV) activities, but the relationship between ALF structure and activities, and their antimicrobial or antiviral mechanism have not been well studied till present. Moreover, with the appearance of the drug-resistant bacteria caused by antibiotics abuse, AMPs are regarded as potential candidates for the development of novel antimicrobial drugs in recent years. In order to explore the feasibility for the future application of ALF in aquaculture or other areas, we have modified the structure of ALFs in order to btain peptides with high activity. At the same time, synthetic biology approach and prokaryotic or eukaryotic expression system were used to obtain the recombinant AMPs protein with expected activity. The present study provides new strategies for designing AMPs drugs, and makes the important foundation for the healthful aquaculture of shrimp production. The main progresses are as follows: 1. The physicochemical characteristic and functional activity of seven LPS-binding domains (LBDs) were analyzed. The content of basic amino acids and distribution of hydrophilic or hydrophobic amino acid were regarded as the main factors influencing the antimicrobial activity of LBDs. Based on these principles, we have designed and synthesized some peptides by solid-phase synthesis. Through the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) test, the antimicrobial activity of the modified LBDs have been greatly enhanced with broad spectrum of activities against Gram-positive and Gram-negative bacteria. To learn the prospect of modified LBD application, the characteristics of these peptide were also analyzed. These peptides were stable after heat treatment and could kill bacteria in a short time. The LBD peptides can bind to bacteria, and cause bacterial agglutination, and then kill bacteria by damaging their membrane integrity. Structure analysis showed that LBD peptides hold the β-sheet structure. Collectively, these findings highlighted the potential antimicrobial mechanism of LBD peptides, and provided important information for the commercial use of LBD in future. 2. From the seven ALF isoforms, we found one isoform of ALF (named FcALF5) showed apparent antiviral (WSSV) activity but no activity to E.coli. We have produced a recombinant protein rFcALF5 with biological activity through a prokaryotic expression system. To learn the antiviral mechanism how rFcALF5 influences WSSV infection, the interaction between WSSV and rFcALF5 was analyzed by Far Western Blot. The data showed that rFcALF5 had direct interaction with the envelope protein VP24 of WSSV. To verify this interaction, the VP24-GST recombinant protein were produced. Far Western Blot and Dot Blot analysis further confirmed the interaction between rFcALF5 or LBD-FcALF5 with VP24 protein of WSSV. VP24 is one of the major envelope proteins of WSSV and plays important roles during WSSV infection. Therefore we infer that the antiviral activity of FcALF5 might be achieved through the binding of its LBD to VP24 of WSSV. 3. For the potential commercial use of ALFs, the sequence of the LBD domain of FcALF2 were replaced by the sequence of LBDv. Based on the differences in codon usage preference in yeast, the modified FcALF2 (named mFcALF2) gene was synthesized. The mFcALF2 was expressed through a yeast Pichia pastoris GS115 eukaryotic expression system. The recombinant target protein was purified and confirmed by Western-blot and mass spectrometry analysis. It exhibited apparent antibacterial and antiviral activities. The present study have made important foundation, and provided important information for the commercial use of the antimicrobial peptides in the future.
学科领域	地球科学 ; 海洋科学 ; 海洋生物学
语种	英语
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112549
专题	实验海洋生物学重点实验室
推荐引用方式 GB/T 7714	杨辉. 对虾抗脂多糖因子ALF的功能分析和开发利用[D]. 北京. 中国科学院大学,2016.

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