分子間或分子內(nèi)通過(guò)非共價(jià)鍵結(jié)合在一起形成的復(fù)合物為超分子。某些天然超分子通過(guò)自組裝形成一種通常包括八面體、十二面體或者二十面體等不同的形狀的殼結(jié)構(gòu),而這些殼結(jié)構(gòu)為超分子提供了一些廣泛的應(yīng)用。其中之一就是用這些衣殼作為承載其他小分子的容器,包載物的衣殼在外界條件改變下將包裹的小分子釋放出來(lái)發(fā)揮自身的功能,因此這些承載了外來(lái)分子的衣殼可以用在材料、酶學(xué)催化或者藥物傳遞等多領(lǐng)域。酶不穩(wěn)定且易降解的特點(diǎn)眾所周知,如果用某種特定的殼將酶包裹起來(lái)就可以防止酶在發(fā)揮作用過(guò)程中外界條件對(duì)酶的影響。在藥物傳遞領(lǐng)域,某些可以殺死細(xì)胞的毒性無(wú)機(jī)小分子或者生物大分子在殺死癌細(xì)胞的同時(shí)也會(huì)傷害正常細(xì)胞,如果用衣殼將其包裹,在傳遞的過(guò)程中就可以避免對(duì)正常細(xì)胞的傷害,到達(dá)特定靶位時(shí)釋放包裹的分子,將癌細(xì)胞殺死從而提高生物性能。病毒是自然界中最常見(jiàn)的一種天然分子容器,使用病毒衣殼來(lái)傳遞藥物也是近年來(lái)很常見(jiàn)也是很成熟的一種手段。除病毒外,許多非病毒的天然蛋白衣殼也可看作是承載小分子的容器。例如,在枯草芽孢桿菌中存在著一種由六十個(gè)亞基組成的具有十二面體衣殼結(jié)構(gòu)的二氧四氫喋啶合酶（BsLS）,在衣殼內(nèi)部包裹著一個(gè)三聚體的核... 

【文章來(lái)源】：天津大學(xué)天津市 211工程院校 985工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：109 頁(yè)

【學(xué)位級(jí)別】：碩士

【文章目錄】：
摘要
Abstract
Chapter 1 Introduction
    1.1 Natural encapsulation using protein capsids
    1.2 Engineered Capsids as containers
    1.3 Lumazine synthase
    1.4 Aim of this thesis
Chapter 2 Materials and Methods
    2.1 Ecoli cells and plasmids
    2.2 Reagents
    2.3 Primers
    2.4 Instruments
    2.5 Plasmid construction
        2.5.1 Materials for plasmid of GFP variants
        2.5.2 Methods
    2.6 Protein production and purification
        2.6.1 Materials
        2.6.2 Methods
    2.7 SDS-PAGE
        2.7.1 Materials for SDS-PAGE
        2.7.2 Method for SDS-PAGE
        2.7.3 Yield detection by SDS-PAGE
        2.7.4 Measurement of protein concentration
    2.8 Analytical size-exclusion chromatography
    2.9 Fluorescence spectroscopy
    2.10 UV detection for loading yield
    2.11 MS detection of co-purifying proteins
    2.12 The characterization of BsLS with His6 tag
        2.12.1 Site directed mutagenesis
        2.12.2 BsLS-H6 protein production and purification
        2.12.3 SDS-PAGE analysis for BsLS-H6 capsid loading
    2.13 ELISA Detection of GFP11
    2.14 Western blotting analysis for Abrin A-HA-BsRS11
    2.15 Guest release from the capsid
    2.16 TEM
    2.17 Attempts at capsid loading in vitro
    2.18 AaLS-switch-pH capsid become pentamer
    2.19 AaLS-switch-pH pentamer go back to capsid
    2.20 Phage display
Chapter 3 Results
    3.1 Design of fusion proteins
    3.2 Analysis of capsid-guest complexes
        3.2.1 SEC and A280 analysis
        3.2.2 Fluorescence spectroscopy
        3.2.3 SDS-PAGE analysis
        3.2.4 UV detection for loading yield
        3.2.5 Detection of GFP by ELISA
        3.2.6 The specificity and generality of the encapsulation system
        3.2.7 Guest release from the capsid
        3.2.8 TEM
        3.2.9 Attempted in vitro loading
    3.3 Selection of encapsulation peptide tags for AaLS
        3.3.1 Confirming of AaLS-switch-pH
        3.3.2 Phage display selection round
Chapter 4 Discussion
    4.1 The loading yield of other engineering capsids
    4.2 The encapsulation yield of GFPs
    4.3 The specificity and generality of the encapsulation system
    4.4 The analysis of 24 LS and RS
    4.5 The mild release condition of BsLS capsid
    4.6 conclusion and future work
References
Publication in scientific research
Acknowledgements



本文編號(hào)：3514144