小反芻獸疫(Peste des petits rumminant,PPR)是一種嚴(yán)重影響小型反芻動(dòng)物經(jīng)濟(jì)的病毒性疾病,其主要感染動(dòng)物的呼吸道和消化道。該病于1942年在西非象牙海岸(科特迪瓦)被首次報(bào)道,由于其傳播風(fēng)險(xiǎn)高,目前已有43個(gè)國(guó)家報(bào)道該病的發(fā)生,呈全球規(guī)模傳播,因此小反芻獸疫病毒Peste des petits ruminant virus,PPRV)抗原的特異性檢測(cè)在該病病控制和根除中起著重要作用。同時(shí)雖然山羊和綿羊是PPR的主要宿主,但不斷有研究證明大型反芻動(dòng)物體內(nèi)依然能檢測(cè)到PPRV抗體的流行,給PPR的有效控制和根除帶來了潛在挑戰(zhàn)。本研究首先以重組山羊SLAM(信號(hào)淋巴細(xì)胞活化分子,PPRV細(xì)胞受體)為捕獲分子,建立了一種間接ELISA方法用于PPRV抗原檢測(cè)。本研究首先成功表達(dá)山羊SLAM受體蛋白胞外區(qū),并對(duì)包被緩沖液、封閉緩沖液、SLAM蛋白包被濃度等參數(shù)進(jìn)行了優(yōu)化和標(biāo)準(zhǔn)化,結(jié)果表明該方法可特異性檢測(cè)PPRV,而與口蹄疫病毒、羊口瘡病毒、綿羊痘和山羊痘病毒均無交叉反應(yīng),且靈敏度為1.56×101TCID50/反應(yīng)(50μL)。隨后對(duì)136份樣品進(jìn)行檢測(cè),結(jié)果表明所...

【文章頁(yè)數(shù)】：86 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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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