由于登革熱病例在全球范圍內(nèi)呈逐年上升的態(tài)勢(shì)，而且目前尚未開發(fā)出相關(guān)疫苗，因此，早期、快速且經(jīng)濟(jì)的檢測(cè)方法成為了攻克這一疾病的唯一途徑。而在現(xiàn)階段，對(duì)登革熱的檢測(cè)仍是一個(gè)懸而未決的問題。NS1是二型登革熱病毒基因組中第一個(gè)被轉(zhuǎn)錄的非結(jié)構(gòu)糖蛋白�，F(xiàn)已發(fā)現(xiàn)，在患者出現(xiàn)臨床癥狀后的1到9天之內(nèi)即可檢測(cè)出NS1的可溶性血清抗原。無論是初次感染還是再次感染患者，其NS1血清抗原都在第3到5天達(dá)到最高值。這些特性使NS1成為了一種極有價(jià)值的分子標(biāo)記，用于快速診斷試劑盒的開發(fā)。我們采用大腸桿菌表達(dá)體系成功表達(dá)并獲得了具有保守結(jié)構(gòu)特征的可溶性rNS1蛋白。進(jìn)而，又通過熒光光譜及圓二色譜對(duì)這些rNS1蛋白的保守結(jié)構(gòu)區(qū)域進(jìn)行了鑒定和驗(yàn)證�？傊@些結(jié)果顯示了重組rNS1蛋白保留了天然病毒蛋白的主要構(gòu)造特征及抗原決定簇，因此對(duì)于診斷試劑盒的開發(fā)具有重要意義。抗體已被廣泛用于各種診斷及免疫療法中。采用傳統(tǒng)的雜交瘤技術(shù)生產(chǎn)針對(duì)rDENV2-NS1的血清型特異的單克隆抗體，基于所分泌單抗與rNS1蛋白的強(qiáng)陽性反應(yīng)，共篩選出了18株穩(wěn)定生產(chǎn)單抗的雜交瘤細(xì)胞株。由于單抗3B3與其它含組氨酸標(biāo)簽的蛋白無交互作用，并在間接... 

【文章來源】：華南理工大學(xué)廣東省 211工程院校 985工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：148 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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
攻讀博士學(xué)位期間取得的研究成果
Acknowledgements
附件


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期刊論文
[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]我國(guó)登革 4型病毒 B5株基因組全序列的測(cè)定及分析（英文）[J]. 王鵬程,秦鄂德,于曼,耿麗卿,趙衛(wèi),胡志君,苑錫同,楊佩英.  中國(guó)生物化學(xué)與分子生物學(xué)報(bào). 2001(02)



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