鉴别一种可被蛋白衣壳包裹的短肽

发布时间：2021-11-23 15:14

　　分子间或分子内通过非共价键结合在一起形成的复合物为超分子。某些天然超分子通过自组装形成一种通常包括八面体、十二面体或者二十面体等不同的形状的壳结构,而这些壳结构为超分子提供了一些广泛的应用。其中之一就是用这些衣壳作为承载其他小分子的容器,包载物的衣壳在外界条件改变下将包裹的小分子释放出来发挥自身的功能,因此这些承载了外来分子的衣壳可以用在材料、酶学催化或者药物传递等多领域。酶不稳定且易降解的特点众所周知,如果用某种特定的壳将酶包裹起来就可以防止酶在发挥作用过程中外界条件对酶的影响。在药物传递领域,某些可以杀死细胞的毒性无机小分子或者生物大分子在杀死癌细胞的同时也会伤害正常细胞,如果用衣壳将其包裹,在传递的过程中就可以避免对正常细胞的伤害,到达特定靶位时释放包裹的分子,将癌细胞杀死从而提高生物性能。病毒是自然界中最常见的一种天然分子容器,使用病毒衣壳来传递药物也是近年来很常见也是很成熟的一种手段。除病毒外,许多非病毒的天然蛋白衣壳也可看作是承载小分子的容器。例如,在枯草芽孢杆菌中存在着一种由六十个亚基组成的具有十二面体衣壳结构的二氧四氢喋啶合酶（BsLS）,在衣壳内部包裹着一个三聚体的核... 

【文章来源】：天津大学天津市 211工程院校 985工程院校 教育部直属院校

【文章页数】：109 页

【学位级别】：硕士

【文章目录】：
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



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