病毒细胞受体SLAM在小反刍兽疫诊断中的应用研究

发布时间：2023-04-23 01:45

　　小反刍兽疫(Peste des petits rumminant,PPR)是一种严重影响小型反刍动物经济的病毒性疾病,其主要感染动物的呼吸道和消化道。该病于1942年在西非象牙海岸(科特迪瓦)被首次报道,由于其传播风险高,目前已有43个国家报道该病的发生,呈全球规模传播,因此小反刍兽疫病毒Peste des petits ruminant virus,PPRV)抗原的特异性检测在该病病控制和根除中起着重要作用。同时虽然山羊和绵羊是PPR的主要宿主,但不断有研究证明大型反刍动物体内依然能检测到PPRV抗体的流行,给PPR的有效控制和根除带来了潜在挑战。本研究首先以重组山羊SLAM(信号淋巴细胞活化分子,PPRV细胞受体)为捕获分子,建立了一种间接ELISA方法用于PPRV抗原检测。本研究首先成功表达山羊SLAM受体蛋白胞外区,并对包被缓冲液、封闭缓冲液、SLAM蛋白包被浓度等参数进行了优化和标准化,结果表明该方法可特异性检测PPRV,而与口蹄疫病毒、羊口疮病毒、绵羊痘和山羊痘病毒均无交叉反应,且灵敏度为1.56×101TCID50/反应(50μL)。随后对136份样品进行检测,结果表明所...

【文章页数】：86 页

【学位级别】：博士

【文章目录】：
摘要
abstract
List of Abbreviations
Chapter 1 Introduction
    1.1 Peste des Petits Ruminant(PPR)
        1.1.1 Background Information
        1.1.2 Etiology
        1.1.3 Sign and Symptoms
        1.1.4 Pathological Lesions
        1.1.5 History and Geographical distribution
    1.2 The Viral genome
        1.2.1 Structural and Accessory Proteins
        1.2.2 Attachment and Entry
        1.2.3 Transcription and Replication
    1.3 Structural Proteins
        1.3.1 Nucleocapsid(N)protein
        1.3.2 Phospo(P)protein
        1.3.3 Matrix(M)protein
        1.3.4 Fusion(F)protein
        1.3.5 Haemagglutinin-Neuraminidase(HN)Protein
        1.3.6 Large Protein
    1.4 PPRV Accessory Proteins
        1.4.1 C protein
        1.4.2 V protein
    1.5 PPRV Host Cellular Receptors
        1.5.1 Signaling Lymphocyte Activation Molecule(SLAM/CD150)
        1.5.2 Nectin-4/PVRL4
        1.5.3CD46
        1.5.4 Other Putative Receptors
    1.6 The Potential Role of Virus-Receptor Interaction in Host Range Restriction
    1.7 The Impact of Virus-Receptor Interactions on the Immune Response against PPRV
        1.7.1 Innate Immune Response
        1.7.2 Adaptive Immune Response
        1.7.3 Inhibitory Impact of Viral Protein-SLAM Interactions on the Immune Response
    1.8 Diagnosis
        1.8.1 Advances in Diagnosis
            1.8.1.1 Conventional Methods/PPRV Antigen Detection
            1.8.1.2 Immunochromatographic Test
            1.8.1.3 Molecular Diagnostic Techniques
            1.8.1.4 Control
    1.9 Rationale and Objectives of Study
Chapter 2 Development of SLAM/CD150 based ELISA for the detection of PPRV
    2.1 Introduction
        2.1.1 Enzyme Linked Immunosorbent Assay(ELISA)
        2.1.2 SLAM/CD 150
    2.2 Materials and Methods
        2.2.1 Cells and Viruses
        2.2.2 Virus Titration
        2.2.3 Virus Purification
    2.3 Cloning and Vector Construction
        2.3.1 Designing of Primers
        2.3.2 Reverse Transcription PCR for the Synthesis of c DNA
        2.3.3 Transformation of Competent Cells
        2.3.4 SDS-PAGE and Western Blot Analysis
    2.4 Preparation of PPRV Antisera
    2.5 Optimization of coating buffer,blocking buffer and rg SLAM
    2.6 Indirect ELISA
    2.7 Samples and Real-time RT-q PCR assay
    2.8 Results
        2.8.1 Cloning and Vector Construction
        2.8.2 Purification of Recombinant SLAM Protein
        2.8.3 Optimization of Coating buffer,Blocking Buffer and rg SLAM
        2.8.4 Determination of Cut-off
        2.8.5 Analytical Sensitivity of i-ELISA
        2.8.6 Analytical Specificity of i-ELISA
        2.8.7 Results of RT-q PCR assay
        2.8.8 Performance of PPRV SLAM-i ELISA on clinical samples
    2.9 Discussion
Chapter 3 Serological investigations of Peste des Petits Ruminants in cattle of Nepal
    3.1 Introduction
    3.2 Methods
    3.3 Results
    3.4 Discussion
Chapter 4 Conclusion
References
Appendix A
Appendix B
Appendix C
Appendix D
Appendix E
Appendix F
Acknowledgement
Author Resume



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