本文關(guān)鍵詞：基于分子識別的新型抗病毒仿生納米材料的基礎(chǔ)研究　出處：《華中科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 分子印跡 聚多巴胺 抗病毒效應(yīng) f2噬菌體 靜電紡絲

【摘要】：病毒是威脅人類健康和生存的重要微生物。隨著人類對自然資源不斷地開發(fā)利用,活動范圍的擴大,暴露于未知致命病毒可能性不斷增加,往往會帶來非常嚴(yán)重的后果。病毒性疾病的診治與預(yù)防是醫(yī)學(xué)領(lǐng)域重要工作,也是社會及個人醫(yī)療支出的主要組成部分。病毒性疾病不僅影響人類的健康,還可能引起經(jīng)濟衰退甚至影響社會的穩(wěn)定。開發(fā)有效的乃至于廉價的抗病毒治療方法具有非常重大的現(xiàn)實意義。隨著納米技術(shù)的迅猛發(fā)展,納米材料因其獨特的量子效應(yīng),小尺寸效應(yīng)以及大的比表面積而顯現(xiàn)出特有的性質(zhì),廣泛應(yīng)用于基因治療,腫瘤治療和藥物靶向運輸與釋放等諸多生物醫(yī)學(xué)應(yīng)用領(lǐng)域。可特異性識別并結(jié)合病毒的納米材料在病毒的分離與純化,病毒檢測以及臨床治療等領(lǐng)域均有非常廣闊的應(yīng)用前景。分子印跡聚合物(MIPs)是一種能夠模擬抗原-抗體作用的高分子聚合物,具有與模板分子的空間結(jié)構(gòu)相匹配的印跡孔穴,因此對模板分子具有特異性識別能力,被稱為人工抗體。以病毒為模板的MIPs制備是當(dāng)前分子印跡研究的熱點以及難點之一。其中生物相容性良好的病毒印跡聚合物因其對靶病毒的特異性識別能力以及高親和力,在病毒分離與純化,臨床診斷和抗病毒治療等領(lǐng)域有著極高的潛在應(yīng)用價值。本研究旨在選用具有良好生物相容性的材料,以f2噬菌體為模式病毒,使用不同的合成技術(shù),制備可中和靶病毒的人工抗體(抗病毒仿生納米材料),并分析其吸附特性:(1)使用聚乙烯醇為功能單體,利用靜電紡絲法制備可特異性吸附f2噬菌體的分子印跡抗病毒仿生納米纖維膜,分析其病毒吸附性能。(2)使用多巴胺為親水性功能單體,合成可特異性結(jié)合f2噬菌體的人工抗體,并分析其病毒吸附特性;(3)分析所獲得的兩種抗病毒仿生納米材料,研究其抗f2噬菌體侵染宿主細(xì)胞大腸桿菌的效果和可能機制,并分析其生物相容性和細(xì)胞毒性。本研究具體內(nèi)容主要包括下述三個部分:第一章靜電紡絲法制備抗病毒仿生納米纖維膜的研究目的:利用靜電紡絲法結(jié)合分子印跡技術(shù)合成分子印跡膜,評價其對大腸桿菌f2噬菌體的吸附特性。方法:以大腸桿菌f2噬菌體為模板分子,使用聚乙烯醇(PVA)為聚合物進行電紡合成分子印跡納米纖維膜。使用戊二醛作為原位交聯(lián)劑。掃描電鏡用于觀察所制備的分子印跡膜的形態(tài)。靜態(tài)吸附實驗用于評價分子印跡膜的吸附性能。使用f2噬菌體的類似物評價印跡膜的選擇性吸附能力。加入一定濃度的f2噬菌體評價分子印跡膜在環(huán)境水體中的吸附能力。結(jié)果:制備分子印跡膜的較優(yōu)條件是:0.7gPVA, 8mL純水,2mLSM緩沖溶液,0.7%的TritonX-100。45mM戊二醛溶液用于此印跡膜的原位交聯(lián),并且應(yīng)在洗脫模板之后進行。靜態(tài)吸附實驗的結(jié)果顯示,f2噬菌體濃度為1000pfu/mL時,MIMs對f2噬菌體的吸附為52pfu/mL,高于對照組12pfu/mL。相較于f2噬菌體的類似物,如T4, M13, P1噬菌體,MIMs對f2噬菌體的吸附能力遠(yuǎn)大于類似物,說明MIMs對模板分子有良好的選擇性吸附能力。MIMs對噬菌體f2的增殖無明顯影響。MIMs在不同環(huán)境水體中依然保持著對目標(biāo)物的良好的吸附能力。結(jié)論:靜電紡絲法制備的分子印跡膜對f2噬菌體具有良好的選擇性,在不同的介質(zhì),包括天然水中,均可對f2噬菌體進行準(zhǔn)確地識別與分離。第二章聚多巴胺法制備抗病毒仿生印跡聚合物的研究目的:利用聚多巴胺法制備大腸桿菌f2噬菌體分子印跡聚合物,評價所制備的分子印跡聚合物的吸附性能。方法:使用聚多巴胺沉積法對硅膠顆粒進行表面修飾。以大腸桿菌f2噬菌體為模板分子,多巴胺為親水性功能單體,在Tris緩沖鹽溶液中合成MIPs。運用掃描電鏡,透射電鏡,紅外光譜法對MIPs進行表征。靜態(tài)吸附實驗測定MIPs的吸附能力和吸附動力學(xué),優(yōu)化MIPs的制備條件,評價不同pH環(huán)境中的吸附性能。結(jié)果:制備MIPs的較優(yōu)條件是:選擇過硫酸銨作為多巴胺聚合反應(yīng)的氧化劑,反應(yīng)時間為24小時。電鏡結(jié)果顯示MIPs的厚度在40nm左右。MIPs與NIPs在電鏡表征,紅外光譜測定中無明顯差異,表明MIPs對于f2噬菌體的結(jié)合源于印跡孔穴的形成。f2噬菌體濃度為8*102pfu/mL時,MIPs對f2的吸附達(dá)到60 pfu/mg,遠(yuǎn)高于NIPs的10 pfu/mg。MIPs在0.5小時之內(nèi)達(dá)到對f2噬菌體的吸附平衡,說明此MIPs對f2噬菌體有非常迅速的響應(yīng)能力,有利于對目標(biāo)物的快速識別。在pH為5到8的范圍內(nèi),MIPs表現(xiàn)出穩(wěn)定的性能。結(jié)論:以整個病毒顆粒為模板分子所制備的MIPs可非常快速地對目標(biāo)病毒進行識別,從而有利于對目標(biāo)物的快速吸附。潛在的病毒去除能力使得MIPs擁有廣闊的應(yīng)用前景。第三章抗病毒仿生納米材料的抗病毒感染性能研究目的:對MIPs抗病毒感染性能進行評價,分析MIPs的抗病毒機制,并進一步評價其抗干擾能力、重復(fù)利用性、生物相容性和細(xì)胞毒性,為抗病毒材料的體內(nèi)應(yīng)用提供基礎(chǔ)數(shù)據(jù)。方法:噬菌斑生成實驗用于評價MIPs和MIMs的抗病毒效能。f2噬菌體的增殖實驗中分別加入MIMs和MIPs評價其抗病毒能力。靜態(tài)吸附實驗中加入干擾物質(zhì)評價MIPs的抗干擾能力。使用去離子水破壞吸附在MIPs上的f2噬菌體,進而重生MIPs的結(jié)合位點評價MIPs的吸附性能。使用G噬菌體的類似物作為目標(biāo)物,評價MIPs的選擇性吸附能力。通過測量紅細(xì)胞裂解出的血紅蛋白評價MIPs的生物相容性。MTT實驗用于測定MIPs的細(xì)胞毒性。結(jié)果:噬菌斑生成實驗中MIPs對f2噬菌體的抑制率高達(dá)90%,而NIPs只有13%。MIMs的抑制率有60%,NIMs的抑制率為10%。被MIPs和MIMs吸附的f2噬菌體喪失了感染宿主細(xì)胞的能力,噬菌斑的生成率低于對照組。在增殖實驗中,MIPs抑制了f2噬菌體的增殖,并最終延遲了 f2噬菌體到達(dá)平臺期的時間,而MIMs對f2噬菌體的增殖無影響。MIPs對目標(biāo)病毒的快速特異性結(jié)合使得病毒喪失了侵染宿主細(xì)胞的能力,表現(xiàn)出抗病毒增殖作用。MIPs在高鹽溶液與高粘稠度溶液中依然對f2噬菌體有很強的吸附能力。使用去離子水作為MIPs的重生試劑,結(jié)果表明經(jīng)過6次循環(huán)使用,MIPs沒有出現(xiàn)明顯的吸附性能下降。相較于f2噬菌體的類似物,如T4, M13, P1噬菌體,MIPs對f2噬菌體的吸附能力遠(yuǎn)大于類似物,說明MIPs對模板分子有良好的選擇性吸附能力。紅細(xì)胞裂解實驗中,MIPs不會破壞紅細(xì)胞,表現(xiàn)出良好的生物相容性。MTT實驗表明MIPs不會引起明顯的細(xì)胞毒性。結(jié)論:以全病毒顆粒為模板分子的制備策略賦予MIPs對目標(biāo)病毒的高親和性以及強大的抗干擾能力。MIPs對目標(biāo)病毒的特異性結(jié)合使得病毒喪失了侵染宿主細(xì)胞的能力,成為相關(guān)材料抗病毒的主要機制。以多巴胺為功能單體的親水性MIPs表現(xiàn)出很好的生物相容性,預(yù)示著病毒印跡在病毒分離與純化,檢測與診斷甚至臨床抗病毒治療領(lǐng)域具有極大的應(yīng)用潛力。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R318.08;TB383.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 翟丁;周勇;張余;高從X&;;聚乙烯醇縮糠醛納濾膜的制備[J];水處理技術(shù);2011年02期

2 張云;張強;劉燕;代瑞華;劉翔;;飲用水中病毒的消毒技術(shù)研究進展[J];衛(wèi)生研究;2010年05期

3 汪洋;萬隆;劉小磐;劉宣勇;;靜電紡絲參數(shù)對聚乳酸超細(xì)纖維支架形貌的影響[J];湖南大學(xué)學(xué)報(自然科學(xué)版);2009年07期

4 李珍;王軍;;靜電紡絲可紡性影響因素的研究成果[J];合成纖維;2008年09期

5 李梅,胡洪營;F-RNA噬菌體及其作為水中腸道病毒指示物的研究進展[J];生態(tài)環(huán)境;2005年04期

相關(guān)博士學(xué)位論文 前3條

1 王欣梅;大腸桿菌f2噬菌體分子印跡膜的制備、評價和應(yīng)用[D];華中科技大學(xué);2011年

2 劉軍義;海產(chǎn)諾如病毒（norovirus）檢測和養(yǎng)殖水體生物修復(fù)研究[D];中國科學(xué)院研究生院（武漢病毒研究所）;2007年

3 鄭耀通;閩江流域福州區(qū)段水體環(huán)境病毒污染、存活規(guī)律與滅活處理[D];福建農(nóng)林大學(xué);2002年

相關(guān)碩士學(xué)位論文 前2條

1 陳亮;活性誘導(dǎo)牙周組織再生材料制備與表征[D];浙江大學(xué);2006年

2 貼金鳳;噬菌體作為病毒滅活指標(biāo)毒株的探索研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2006年

，

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