基于分子识别的新型抗病毒仿生纳米材料的基础研究

发布时间：2018-01-08 02:27

  本文关键词：基于分子识别的新型抗病毒仿生纳米材料的基础研究　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

  更多相关文章： 分子印迹 聚多巴胺 抗病毒效应 f2噬菌体 静电纺丝

【摘要】：病毒是威胁人类健康和生存的重要微生物。随着人类对自然资源不断地开发利用,活动范围的扩大,暴露于未知致命病毒可能性不断增加,往往会带来非常严重的后果。病毒性疾病的诊治与预防是医学领域重要工作,也是社会及个人医疗支出的主要组成部分。病毒性疾病不仅影响人类的健康,还可能引起经济衰退甚至影响社会的稳定。开发有效的乃至于廉价的抗病毒治疗方法具有非常重大的现实意义。随着纳米技术的迅猛发展,纳米材料因其独特的量子效应,小尺寸效应以及大的比表面积而显现出特有的性质,广泛应用于基因治疗,肿瘤治疗和药物靶向运输与释放等诸多生物医学应用领域。可特异性识别并结合病毒的纳米材料在病毒的分离与纯化,病毒检测以及临床治疗等领域均有非常广阔的应用前景。分子印迹聚合物(MIPs)是一种能够模拟抗原-抗体作用的高分子聚合物,具有与模板分子的空间结构相匹配的印迹孔穴,因此对模板分子具有特异性识别能力,被称为人工抗体。以病毒为模板的MIPs制备是当前分子印迹研究的热点以及难点之一。其中生物相容性良好的病毒印迹聚合物因其对靶病毒的特异性识别能力以及高亲和力,在病毒分离与纯化,临床诊断和抗病毒治疗等领域有着极高的潜在应用价值。本研究旨在选用具有良好生物相容性的材料,以f2噬菌体为模式病毒,使用不同的合成技术,制备可中和靶病毒的人工抗体(抗病毒仿生纳米材料),并分析其吸附特性:(1)使用聚乙烯醇为功能单体,利用静电纺丝法制备可特异性吸附f2噬菌体的分子印迹抗病毒仿生纳米纤维膜,分析其病毒吸附性能。(2)使用多巴胺为亲水性功能单体,合成可特异性结合f2噬菌体的人工抗体,并分析其病毒吸附特性;(3)分析所获得的两种抗病毒仿生纳米材料,研究其抗f2噬菌体侵染宿主细胞大肠杆菌的效果和可能机制,并分析其生物相容性和细胞毒性。本研究具体内容主要包括下述三个部分:第一章静电纺丝法制备抗病毒仿生纳米纤维膜的研究目的:利用静电纺丝法结合分子印迹技术合成分子印迹膜,评价其对大肠杆菌f2噬菌体的吸附特性。方法:以大肠杆菌f2噬菌体为模板分子,使用聚乙烯醇(PVA)为聚合物进行电纺合成分子印迹纳米纤维膜。使用戊二醛作为原位交联剂。扫描电镜用于观察所制备的分子印迹膜的形态。静态吸附实验用于评价分子印迹膜的吸附性能。使用f2噬菌体的类似物评价印迹膜的选择性吸附能力。加入一定浓度的f2噬菌体评价分子印迹膜在环境水体中的吸附能力。结果:制备分子印迹膜的较优条件是:0.7gPVA, 8mL纯水,2mLSM缓冲溶液,0.7%的TritonX-100。45mM戊二醛溶液用于此印迹膜的原位交联,并且应在洗脱模板之后进行。静态吸附实验的结果显示,f2噬菌体浓度为1000pfu/mL时,MIMs对f2噬菌体的吸附为52pfu/mL,高于对照组12pfu/mL。相较于f2噬菌体的类似物,如T4, M13, P1噬菌体,MIMs对f2噬菌体的吸附能力远大于类似物,说明MIMs对模板分子有良好的选择性吸附能力。MIMs对噬菌体f2的增殖无明显影响。MIMs在不同环境水体中依然保持着对目标物的良好的吸附能力。结论:静电纺丝法制备的分子印迹膜对f2噬菌体具有良好的选择性,在不同的介质,包括天然水中,均可对f2噬菌体进行准确地识别与分离。第二章聚多巴胺法制备抗病毒仿生印迹聚合物的研究目的:利用聚多巴胺法制备大肠杆菌f2噬菌体分子印迹聚合物,评价所制备的分子印迹聚合物的吸附性能。方法:使用聚多巴胺沉积法对硅胶颗粒进行表面修饰。以大肠杆菌f2噬菌体为模板分子,多巴胺为亲水性功能单体,在Tris缓冲盐溶液中合成MIPs。运用扫描电镜,透射电镜,红外光谱法对MIPs进行表征。静态吸附实验测定MIPs的吸附能力和吸附动力学,优化MIPs的制备条件,评价不同pH环境中的吸附性能。结果:制备MIPs的较优条件是:选择过硫酸铵作为多巴胺聚合反应的氧化剂,反应时间为24小时。电镜结果显示MIPs的厚度在40nm左右。MIPs与NIPs在电镜表征,红外光谱测定中无明显差异,表明MIPs对于f2噬菌体的结合源于印迹孔穴的形成。f2噬菌体浓度为8*102pfu/mL时,MIPs对f2的吸附达到60 pfu/mg,远高于NIPs的10 pfu/mg。MIPs在0.5小时之内达到对f2噬菌体的吸附平衡,说明此MIPs对f2噬菌体有非常迅速的响应能力,有利于对目标物的快速识别。在pH为5到8的范围内,MIPs表现出稳定的性能。结论:以整个病毒颗粒为模板分子所制备的MIPs可非常快速地对目标病毒进行识别,从而有利于对目标物的快速吸附。潜在的病毒去除能力使得MIPs拥有广阔的应用前景。第三章抗病毒仿生纳米材料的抗病毒感染性能研究目的:对MIPs抗病毒感染性能进行评价,分析MIPs的抗病毒机制,并进一步评价其抗干扰能力、重复利用性、生物相容性和细胞毒性,为抗病毒材料的体内应用提供基础数据。方法:噬菌斑生成实验用于评价MIPs和MIMs的抗病毒效能。f2噬菌体的增殖实验中分别加入MIMs和MIPs评价其抗病毒能力。静态吸附实验中加入干扰物质评价MIPs的抗干扰能力。使用去离子水破坏吸附在MIPs上的f2噬菌体,进而重生MIPs的结合位点评价MIPs的吸附性能。使用G噬菌体的类似物作为目标物,评价MIPs的选择性吸附能力。通过测量红细胞裂解出的血红蛋白评价MIPs的生物相容性。MTT实验用于测定MIPs的细胞毒性。结果:噬菌斑生成实验中MIPs对f2噬菌体的抑制率高达90%,而NIPs只有13%。MIMs的抑制率有60%,NIMs的抑制率为10%。被MIPs和MIMs吸附的f2噬菌体丧失了感染宿主细胞的能力,噬菌斑的生成率低于对照组。在增殖实验中,MIPs抑制了f2噬菌体的增殖,并最终延迟了 f2噬菌体到达平台期的时间,而MIMs对f2噬菌体的增殖无影响。MIPs对目标病毒的快速特异性结合使得病毒丧失了侵染宿主细胞的能力,表现出抗病毒增殖作用。MIPs在高盐溶液与高粘稠度溶液中依然对f2噬菌体有很强的吸附能力。使用去离子水作为MIPs的重生试剂,结果表明经过6次循环使用,MIPs没有出现明显的吸附性能下降。相较于f2噬菌体的类似物,如T4, M13, P1噬菌体,MIPs对f2噬菌体的吸附能力远大于类似物,说明MIPs对模板分子有良好的选择性吸附能力。红细胞裂解实验中,MIPs不会破坏红细胞,表现出良好的生物相容性。MTT实验表明MIPs不会引起明显的细胞毒性。结论:以全病毒颗粒为模板分子的制备策略赋予MIPs对目标病毒的高亲和性以及强大的抗干扰能力。MIPs对目标病毒的特异性结合使得病毒丧失了侵染宿主细胞的能力,成为相关材料抗病毒的主要机制。以多巴胺为功能单体的亲水性MIPs表现出很好的生物相容性,预示着病毒印迹在病毒分离与纯化,检测与诊断甚至临床抗病毒治疗领域具有极大的应用潜力。
[Abstract]:The virus is an important microbial threat to human health and survival. With the continuous development of the use of natural resources, expand the scope of activities, exposed to the possibility of unknown deadly virus continues to increase, often have very serious consequences. Treatment and prevention of viral diseases is an important work in the medical field, is also a major component of social and personal health expenditure. Viral diseases not only affect human health, but also may cause economic recession and even affect the stability of society. The development of an effective and inexpensive method of antiviral therapy has very important practical significance. With the rapid development of nanotechnology, nano materials because of its unique quantum effect, small size effect and large surface the area shows the unique properties, are widely used in gene therapy, tumor therapy and targeted drug release and transport and other biological medicine should be With the field. Specifically recognizing and binding to separation and purification of nano materials in virus virus, virus detection and clinical application prospects are very broad. As in the treatment of molecularly imprinted polymer (MIPs) is a polymer which can mimic antigen antibody interaction, imprinted cavities with spatial structure and template matching therefore, with specific recognition ability to template molecules, known as artificial antibodies to the virus. As a template for the preparation of MIPs is the current research hotspot of molecular imprinting and one of the difficulties. The biocompatibility of the virus imprinted polymer because of the target virus specific recognition ability and high affinity purification in virus isolation and the clinical diagnosis and antiviral treatment, and other fields has a high potential application value. The purpose of this study is to use good biocompatible materials, F2 phage As a model virus, using different synthesis techniques, the preparation of artificial antibody neutralizing target virus (anti biomimetic nano materials), and analyze its adsorption characteristics: (1) using polyvinyl alcohol as functional monomer, molecular imprinting bionic nano fiber membrane preparing antiviral specific adsorption of phage F2 by electrostatic spinning method, analysis the virus adsorption performance. (2) using dopamine as hydrophilic monomer, synthesis of artificial antibody specifically bound phage F2, and analyze the virus adsorption characteristics; (3) two kinds of antiviral bionic analysis of nano materials, study its anti F2 phage infection of Escherichia coli host cells and the possible mechanism of the effect analysis, and its biological compatibility and cytotoxicity. The main content of this study includes three parts: the first chapter electrospinning of biomimetic nano fiber membrane antiviral research objective: using electrostatic spinning Wire method combined with molecular imprinting technique and synthesis of molecularly imprinted membrane, to evaluate the adsorption characteristics of Escherichia coli phage F2. Methods: using Escherichia coli phage F2 as template molecule, using polyvinyl alcohol (PVA) was electrospun nanofibrous membrane for synthesis of molecularly imprinted polymers. Using two amyl aldehyde as crosslinking agent. The in situ observation of molecular imprinting the morphology of the film prepared for scanning electron microscopy. The static adsorption experiment for evaluating adsorption performance of molecularly imprinted membrane. The adsorption selectivity of analogues was evaluated using the F2 phage imprinted membrane. The adsorption capacity of bacteriophage F2 evaluation of molecularly imprinted membrane with certain concentration in environmental water. Results: the optimal conditions for the preparation of molecularly imprinted membrane is 0.7gPVA, 8mL water, 2mLSM buffer, TritonX-100.45mM 0.7% glutaraldehyde for in situ crosslinking this imprinted membrane, and should be carried out in the template. After the static elution The results of adsorption experiments showed that F2 phage concentration is 1000pfu/mL, the adsorption of MIMs on F2 phage was 52pfu/mL, higher than that of the control group compared to the 12pfu/mL. analogues of bacteriophage F2 such as T4, M13, P1 phage, phage F2 adsorption capacity of MIMs is far greater than the counterpart, that MIMs has good selective adsorption ability of.MIMs the proliferation of phage F2 to template molecule has no obvious effect on the target.MIMs is still a good adsorption capacity in different water environment. Conclusion: the molecularly imprinted membrane prepared by electrospinning method has good selectivity for F2 phage, in different media, including natural water, can carry out the accurate identification and separation of objective to study the phage F2. The second chapter polydopamine bionic preparation of antiviral imprinted polymer using prepared coliphage F2 molecularly imprinted polymer polydopamine method, evaluation The adsorption properties of molecularly imprinted polymer preparation. Methods: using polydopamine deposition method for surface modification of the silica particles. The Escherichia coli bacteriophage F2 as template molecules, dopamine is a hydrophilic functional monomer in Tris buffer solution in the synthesis of MIPs. by scanning electron microscopy, transmission electron microscopy, the MIPs was characterized by infrared spectroscopy. The static adsorption capacity and adsorption kinetics of MIPs determination experiment, preparation conditions optimization of MIPs system, the adsorption performance evaluation of different pH environment. Results: the optimal conditions for the preparation of MIPs is: oxidant ammonium persulfate as dopamine polymerization reaction, the reaction time was 24 hours. The results of SEM showed that the thickness of the MIPs.MIPs at about 40nm and NIPs in electron microscopy, infrared spectroscopy showed that MIPs had no significant differences, for the combination of source F2 phage in phage.F2 imprinted cavities formed at the concentration of 8* 102pfu/mL, MIP The adsorption of s on F2 was 60 pfu/mg, much higher than the NIPs 10 pfu/mg.MIPs to reach adsorption equilibrium of F2 phage within 0.5 hours, the MIPs has a very rapid response capability of bacteriophage F2, is conducive to the rapid identification of the target. In the pH range of 5 to 8, MIPs showed a stable conclusion: in the whole performance. The virus particles as template molecules prepared by MIPs can very quickly identify the target virus, so as to facilitate the rapid adsorption of the target. The potential virus removal ability makes MIPs have broad application prospects. In the third chapter, antiviral biomimetic nano materials antiviral research objective: To evaluate performance the MIPs virus infection performance, analyze the antiviral mechanism of MIPs, and further evaluate its anti-interference ability, reusability, biocompatibility and cytotoxicity, provide a basis for in vivo antiviral materials Basic data. Methods: the proliferation of experimental antiviral effectiveness evaluation MIPs and MIMs.F2 in the phage were added to MIMs and MIPs to evaluate the antiviral ability for plaque formation experiment. The static adsorption material to evaluate the anti-jamming ability of the MIPs to join the interference experiment. Destruction of adsorption of phage F2 MIPs in the use of deionized water, combined with adsorption properties site evaluation of MIPs. Then the rebirth of MIPs analogues using G phage as a target, selective adsorption ability to evaluate MIPs. Through the measurement and evaluation of hemoglobin red blood cell lysis of the biocompatibility of MIPs.MTT experiment by cytotoxic assay of MIPs. Results: MIPs plaque formation experiment in inhibition of bacteriophage F2 the rate of up to 90%, and the NIPs inhibition rate of only 13%.MIMs 60%, the inhibition rate of NIMs lost the ability to infect host cells for F2 phage 10%. by MIPs and MIMs adsorption, plaque Generation rate lower than the control group. The proliferation experiment, MIPs inhibited the proliferation of F2 phage, and ultimately delayed the time of phage F2 to the platform, while MIMs had no effect on the proliferation of F2 phage binding specificity of.MIPs to target the virus quickly make the virus lost the ability to infect host cells, showed antiviral proliferation.MIPs in high salt solution with high viscosity solution of bacteriophage F2 still has a strong adsorption ability. Using deionized water as regeneration reagent of MIPs. The results showed that after 6 cycles, MIPs did not appear obvious adsorption would decline. Compared to the F2 phage analogues, such as T4, M13, P1 phage the adsorption capacity of F2 phage MIPs, far more than similar, indicating that MIPs has good selective adsorption capacity for template molecules. Red blood cell lysis experiments, MIPs does not destroy red blood cells, showing a good student Biocompatibility of.MTT showed that MIPs did not induce significant cytotoxicity. Conclusion: the full preparation strategy gives the virus particles as template molecule MIPs on target virus specific high affinity and strong anti-jamming ability of.MIPs to target the virus the virus infected host cells lose the ability, become the main mechanism materials related to antiviral. Using dopamine as functional monomers, hydrophilic MIPs exhibit good biocompatibility, indicates that the virus in virus isolation and purification of imprinting, has great potential application in detection and diagnosis and clinical antiviral treatment.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;TB383.1

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