抗二型登革热病毒非结构蛋白1的单域重链抗体和单克隆抗体的制备及应用于免疫检测的比较研究

发布时间：2021-03-24 10:58

　　由于登革热病例在全球范围内呈逐年上升的态势，而且目前尚未开发出相关疫苗，因此，早期、快速且经济的检测方法成为了攻克这一疾病的唯一途径。而在现阶段，对登革热的检测仍是一个悬而未决的问题。NS1是二型登革热病毒基因组中第一个被转录的非结构糖蛋白。现已发现，在患者出现临床症状后的1到9天之内即可检测出NS1的可溶性血清抗原。无论是初次感染还是再次感染患者，其NS1血清抗原都在第3到5天达到最高值。这些特性使NS1成为了一种极有价值的分子标记，用于快速诊断试剂盒的开发。我们采用大肠杆菌表达体系成功表达并获得了具有保守结构特征的可溶性rNS1蛋白。进而，又通过荧光光谱及圆二色谱对这些rNS1蛋白的保守结构区域进行了鉴定和验证。总之，这些结果显示了重组rNS1蛋白保留了天然病毒蛋白的主要构造特征及抗原决定簇，因此对于诊断试剂盒的开发具有重要意义。抗体已被广泛用于各种诊断及免疫疗法中。采用传统的杂交瘤技术生产针对rDENV2-NS1的血清型特异的单克隆抗体，基于所分泌单抗与rNS1蛋白的强阳性反应，共筛选出了18株稳定生产单抗的杂交瘤细胞株。由于单抗3B3与其它含组氨酸标签的蛋白无交互作用，并在间接... 

【文章来源】：华南理工大学广东省 211工程院校 985工程院校 教育部直属院校

【文章页数】：148 页

【学位级别】：博士

【文章目录】：
摘要
Abstract
Table of Contents
Abbreviations
Chapter 1 Introduction
    1.1 Historical Background
    1.2 Symptoms
    1.3 Genome
    1.4 Protein Expression System
    1.5 Dengue Diagnosis
        1.5.1 Current Protocols for the Diagnosis of Dengue Infections
            1.5.1.1 Serological Detection
            1.5.1.2 Virus Detection
            1.5.1.3 Antigen Detection
            1.5.1.4 Genome Detection
            1.5.1.5 Main Problems for Dengue Detection
        1.5.2 Requirements for Better Diagnosis
    1.6 Immunoglobulins, A Universal Tool
        1.6.1 Antibody Structure
        1.6.2 IMGT Unique Numbering System
        1.6.3 Surface Plasmon Resonance （SPR）
        1.6.4 Monoclonal Antibody （MAb）
        1.6.5 Nanobodies （VHH Antibodies）
            1.6.5.1 Increased Solubility
            1.6.5.2 Tissue Penetration
            1.6.5.3 Targeting Cryptic Epitopes / Enzyme Inhibitor
            1.6.5.4 Low Immunogenicity
            1.6.5.5 VHH Antibodies Preparation
    1.7 Phage Display Technology
    1.8 Epitope Mapping
    1.9 Development of Rapid Diagnostic Kit
    1.10 Main Objectives for Present Study
Chapter 2 Development of Dengue type 2 Recombinant NS1 Antigen
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 NS1 Gene Preparation
            2.2.1.1 TA Cloning
            2.2.1.2 Colony Culturing and Colony PCR
        2.2.2 Double Digestion of Plasmid and Vector
        2.2.3 Ligation and Transformation
        2.2.4 Colony Culturing and Colony PCR
        2.2.5 Protein Expression and Purification
        2.2.6 Western Blot Analysis
        2.2.7 Indirect ELISA
        2.2.8 Fluorescence Spectroscopy and Circular Dichroism
    2.3 Results
        2.3.1 Amplification, Cloning, Plasmid Construction and Identification of DENV2 NS1
        2.3.2 Expression and Purification of the Recombinant DENV2 NS1 Protein
        2.3.3 rNS1 refolding
        2.3.4 Spectroscopic Analysis of rNS140
    2.4 Discussion
Chapter 3 Development of monoclonal and VHH antibodies against rNS1: A comparison study
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Preparation of Monoclonal Antibody
            3.2.1.1 Indirect ELISA to Analyze the Sera of Mice
            3.2.1.2 Cell Fusion
            3.2.1.3 Screening of clones
            3.2.1.4 Limited Dilution
            3.2.1.5 Amplification and Preservation of Clones
            3.2.1.6 Ascites and Monoclonal Antibody Production
            3.2.1.7 Activity of Ascites and anti-rNS1 Monoclonal Antibodies
            3.2.1.8 SDS-PAGE and Western Blot Analysis
        3.2.2 Construction of Non-Immune Llama Library
        3.2.3 Llama Heavy Chain Antibody Screening
            3.2.3.1 M13K07 Phage Proliferation
            3.2.3.2 Phage Titration
            3.2.3.3 VHH Antibody Screening
            3.2.3.4 Phage Binding Analysis
            3.2.3.5 Sequence Analysis of Plasmids （pCANTAB5E-VHH）
            3.2.3.6 Purification and Double Digestion of Plasmids （pCANTAB5E-VHH） and Vectors
            3.2.3.7 Ligation and Transformation
            3.2.3.8 VHH Antibody Expression and Purification
        3.2.4 Surface Plasmon Resonance of VHH and Monoclonal antibodies
    3.3 Results
        3.3.1 Development of Monoclonal Antibody
        3.3.2 Development of VHH antibody
        3.3.3 Binding Affinity Analysis of VHH and Monoclonal Antibodies
    3.4 Discussion
Chapter 4 Epitope Mapping of VHH and Monoclonal Antibodies
    4.1 Introduction
    4.2 Materials and Methods
        4.2.1 Phage Titering
        4.2.2 Surface Panning Procedure （Direct target Coating）
        4.2.3 Plaque Amplification for Sequencing
        4.2.4 Rapid Purification of Sequencing Templates for Sequencing of Phage DNA
        4.2.5 Sequencing Guidelines
    4.3 Results
    4.4 Discussion
Chapter 5 Development and comparison study of VHH and Monoclonal Antibodies Immobilized Rapid Diagnostic Kits
    5.1 Introduction
    5.2 Materials and Methods
        5.2.1 Synthesis of Colloidal Gold for Rapid Diagnostic Test
        5.2.2 Test Strip Preparation
        5.2.3 Principle of Immunochromatographic Lateral-Flow Test Strip
    5.3 Results
    5.4 Discussion
Conclusions
References
Appendix
攻读博士学位期间取得的研究成果
Acknowledgements
附件


【参考文献】：
期刊论文
[1]Strategies for production of active eukaryotic proteins in bacterial expression system[J]. Orawan Khow,Sunutcha Suntrarachun.  Asian Pacific Journal of Tropical Biomedicine. 2012(02)
[2]我国登革 4型病毒 B5株基因组全序列的测定及分析（英文）[J]. 王鹏程,秦鄂德,于曼,耿丽卿,赵卫,胡志君,苑锡同,杨佩英.  中国生物化学与分子生物学报. 2001(02)



本文编号：3097600