抗HCV多肽的筛

发布时间：2018-03-06 08:21

本文选题：丙型肝炎病毒　切入点：抗病毒多肽　出处：《武汉大学》2014年博士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(HCV)是一种正链RNA病毒,在分类学上属于黄病毒科。HCV是慢性肝炎,肝硬化和肝癌等慢性肝病的主要诱因,目前在全球范围内大约有2亿人被感染。多年以来,临床上主要依靠干扰素配合病毒唑的鸡尾酒疗法来治疗HCV的感染,近年来发展了蛋白酶抑制剂配合上述二联疗法的新型抗病毒模式,并取得了一定的治疗效果。但是由于药物副作用、耐药性的产生以及患者的持续病毒学应答存在差异等因素,现行治疗方法并不能满足临床的需要。同时,由于缺乏有效的疫苗来对HCV的感染进行预防,寻找并开发新型的抗HCV药物显得尤为重要。本实验室构建了西藏特里豚蝎和三脊豚蝎毒腺组织cDNA文库,并从中发现了13条具有潜在抗微生物功能的多肽分子。通过化学合成的方法获得了这13条多肽,从中发现了具有抗HCV感染功能的多肽Ctry2459。在体外条件下,Ctry2459多肽能够采用一种“剥膜”的方式将HCV的包膜和核壳结构分离,从而有效抑制HCV的早期感染,但不影响病毒的核壳及其RNA。这种抑制作用具有快速高效的特点,Ctry2459多肽抑制HCV感染的半数有效浓度(EC50)为1.84μg/ml,对Huh7.5.1细胞系的半数毒性浓度(CC50)为79.8μg/ml,药物选择指数(SI)为43.4,半数溶血浓度(HCs0)为137.9μg/ml,表现出了较好的药理学性能。尽管天然蝎毒肽Ctry2459具有一定的抗HCV感染作用,但它缺乏对HCV的感染后抑制功能。这种功能缺陷是由于Ctry2459多肽的细胞摄取量低并受到细胞内含体的限制,通过调节细胞摄取和内含体通路能够有效改善它的生物利用度。在此基础上,以天然多肽Ctry2459为分子模板,设计了两条富含组氨酸的衍生肽Ctry2459-H2和Ctry2459-H3。进一步研究发现,衍生肽Ctry2459-H2和Ctry2459-H3保持了与天然多肽Ctry2459类似的α螺旋结构以及抑制HCV初期感染的功能,它们抑制HCV感染的作用也具有快速高效的特点,EC50分别为1.08μg/ml和0.85μg/ml。通过共聚焦显微镜观察发现Ctry2459衍生肽能够有效地突破细胞摄取和内含体的障碍,从而发挥对HCV感染后的抑制作用。在HCV感染48小时后使用20μg/ml Ctry2459-H2和Ctry2459-H3多肽进行处理,可以分别抑制细胞内40%和70%的HCV含量。此外,研究也发现Ctry2459-H2和Ctry2459-H3多肽具有更低的细胞毒性和溶血性,CC50均大于500μg/ml, HC50分别为203.3μg/ml和416.4μg/ml。因此,与天然多肽Ctry2459相比,衍生肽Ctry2459-H2和Ctry2459-H3展示出了更高的成药性。另外,本研究依据HCVp7离子通道的结构和功能特征,从其跨膜结构域截取了4条可能具有干扰正常通道组装的多肽序列。通过化学合成的方法获得了多肽,并从中发现了对HCV具有感染后抑制作用的多肽p7avp3。体外条件下,p7avp3多肽能够有效抑制HCV的感染后增殖,其EC5o为2.19±0.16μM,CC50为83.64±6.95μM,药物选择指数为38.19。尽管p7avp3多肽具有一定的抗HCV功能,但是它的药物选择指数较低,因此需要对其药理学性能进行优化。在p7avp3多肽的基础上,根据氨基酸的极性以及电荷等特征,设计了10条新的衍生肽,并从中发现了比p7avp3抗病毒功能更强的多肽p7avp5。体外条件下,p7avp5多肽能够高效抑制HCV的感染后增殖,其ECso为1.09±0.13μM,CC50为115.99±16.68μM,药物选择指数为106.41,展示出了更好的药理学性质。此外,p7avp3和p7avp5多肽均表现出了较低的溶血性以及较高的血清稳定性。进一步开展了p7avp5多肽抗HCV的机制研究。通过不同给药方式的抗病毒实验发现,p7avp5多肽可以在多种条件下抵抗HCV的感染,包括作用于细胞和病毒来抑制病毒早期感染,抑制病毒的入侵和感染后增殖。其中,当p7avp5多肽直接作用于HCV时,抗病毒功能最高效,且不依赖于对病毒核酸的破坏。用FITC标记多肽并在共聚焦显微镜下观察发现,p7avp5多肽能够快速吸附到细胞膜上并导致HCV感染的下降,随着时间的推移,p7avp5多肽逐渐在细胞膜上富集并进入细胞质,导致细胞内病毒粒子的感染性下降。因此,p7avp5多肽可能采取了三种作用模式来发挥抗HCV功能,包括封闭细胞和钝化病毒抑制早期感染,以及进入细胞钝化病毒来抑制二次感染。此外,电生理实验结果显示,p7avp5多肽不影响p7离子通道的正常组装及其生理功能,也不阻断其电流,并且不抑制HCV从宿主细胞的释放过程。综上所述,本研究从蝎活性多肽资源库中发现了新的天然抗HCV多肽分子Ctry2459,通过对其合理的优化设计获得了具有高生物利用度以及抗HCV功能的衍生肽。同时,以HCVp7离子通道为基础,设计并鉴定了新的抗HCV多肽分子p7avp5,解析了该多肽的抗HCV功能与机制。本研究发现了具有潜在应用价值的抗HCV多肽分子,丰富了现有的抗病毒多肽开发策略,并为天然动物毒素多肽的开发利用以及新型抗病毒多肽的筛选和设计提供了理论和应用基础。
[Abstract]:Hepatitis C virus (HCV) is a positive strand RNA virus belonging to the Flaviviridae family, in the taxonomy of.HCV is main cause of chronic hepatitis, cirrhosis and liver cancer and other chronic liver disease worldwide, currently about 200 million people have been infected. Over the years, mainly rely on clinical infection cocktail therapy with interferon ribavirin treatment HCV, a new antiviral model developed in recent years with the two protease inhibitor combination therapy, and achieved a certain therapeutic effect. But due to the side effects of the drug resistance, and the patients with sustained viral response to existence differences and other factors, the current treatment methods can not meet the clinical needs. At the same time, due to the lack of effective the HCV vaccine to prevent infection, to find and develop new anti HCV drugs is particularly important.
The cDNA library was constructed for the Tibet dolphins and three ridge dolphin Terry scorpion scorpion BmK organization, and found 13 potential anti microbial peptides from. By the method of chemical synthesis for the 13 peptides, found with anti HCV infection function of peptide Ctry2459. in vitro, Ctry2459 peptides can adopts a "stripping" will be coated HCV and core-shell structure separation, which can effectively inhibit early HCV infection, but does not affect the nuclear shell virus RNA. and this inhibition is fast and efficient, Ctry2459 inhibit Iabour HCV infection (EC50) for 1.84 g/ml. The median toxic concentrations of Huh7.5.1 cell lines (CC50) for 79.8 g/ml, the drug selection index (SI) was 43.4, half hemolytic concentration (HCs0) of 137.9 g/ml, showing the pharmacology of good performance.
Although natural scorpion venom peptide Ctry2459 has certain anti HCV infection, but its lack of inhibition function of HCV after infection. This defect is due to the cellular uptake of Ctry2459 polypeptide was low and cell inclusions constraints, improve its bioavailability through regulation of cell uptake and inclusion based pathways. On the natural peptide Ctry2459 is a template for the design of the two histidine rich peptides derived from Ctry2459-H2 and Ctry2459-H3. further study found that Ctry2459-H2 and Ctry2459-H3 derived peptides maintain alpha helix and natural polypeptide Ctry2459 similar function and inhibition of HCV infection early, they inhibit HCV infection also has the characteristics of fast and efficient. EC50 were 1.08 g/ml and 0.85 g/ml. by confocal microscopy observation showed that Ctry2459 derived peptide can effectively break cell uptake and containing The obstacles to exert inhibitory effect on HCV after infection were treated using 20 g/ml Ctry2459-H2 and Ctry2459-H3 polypeptide in HCV after 48 hours of infection, can the content of HCV in cells 40% and 70% were inhibited. In addition, the study also found that Ctry2459-H2 and Ctry2459-H3 polypeptides with lower cytotoxicity and hemolysis, CC50 more than 500 g/ml, HC50 were 203.3 g/ml and 416.4 g/ml., therefore, compared with the natural peptide Ctry2459 derived peptide, Ctry2459-H2 and Ctry2459-H3 showed a higher resistance.
In addition, on the basis of the structure and function of the HCVp7 ion channel characteristics, from its transmembrane domain from 4 may interfere with normal channel assembly of peptide sequence. By the method of chemical synthesis of peptides obtained, and from that of HCV after infection has inhibitory effect of polypeptide p7avp3. in vitro, p7avp3 peptide can effectively inhibit HCV proliferation after infection, the EC5o was 2.19 + 0.16 M, CC50 83.64 + 6.95 M, drug selection index was 38.19. while the p7avp3 polypeptide with anti HCV function, but its drug selection index is low, so the need for its pharmacological properties were optimized. Based on p7avp3 polypeptide according to the amino acid, polarity and charge characteristics, we design 10 new derived peptides, and find the more powerful than p7avp3 antiviral polypeptide p7avp5. in vitro, p7avp5 peptides can inhibit HCV high efficiency After infection, the ECso was 1.09 + 0.13 M, CC50 was 115.99 + 16.68 M, and the drug selection index was 106.41, showing better pharmacological properties. In addition, p7avp3 and p7avp5 peptides showed low hemolysis and high serum stability.
Further research on the mechanism of p7avp5 anti HCV polypeptide. The antiviral effect of different ways of administration found that peptide p7avp5 in a variety of conditions against HCV infection, including the effect on the cell and virus to inhibit early infection, inhibit the virus invasion and infection after proliferation. Among them, when the p7avp5 peptide directly on the HCV and the most efficient antiviral function, and does not depend on the viral nucleic acid damage. Observed by confocal microscopy and FITC labeled peptide, p7avp5 peptide can rapidly adsorbed to the cell membrane and lead to a decline in HCV infection, with the passage of time, gradually p7avp5 polypeptides in the cell membrane into the cytoplasm and enrichment of lead to infection. Viral particles in cells decreased. Therefore, p7avp5 polypeptide may adopt three kinds of mode of action to play the function of anti HCV, including closed cell and virus infection during early suppression of passivation, And enter the cell passivation virus to inhibit the two infection. In addition, the electrophysiological experiment results showed that p7avp5 polypeptide did not affect the normal assembly and physiological function of P7 ion channel, nor blocked its current, and did not inhibit the release process of HCV from host cells.
In summary, this study found a new natural anti HCV peptide Ctry2459 from scorpion peptides in the repository, by the reasonable optimization design is obtained with high bioavailability of derived peptides and anti HCV function. At the same time, the HCVp7 ion channel basis, design and identification of anti HCV peptide p7avp5 the analysis of the function and mechanism of anti HCV polypeptide. This study found that the anti HCV peptide has potential application value, enrich the development strategy of the existing antiviral polypeptide, and provides a theoretical and practical basis for the development and utilization of natural animal toxin polypeptides and new antiviral peptide selection and design.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.63;R3416

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