山羊痘病毒屬包括綿羊痘病毒（SPPV）,山羊痘病毒（GTPV）和結(jié)節(jié)性皮膚病毒(LSDV。牛是LSDV常見感染宿主,并且在大多數(shù)非洲國家和中東都存在。然而,“SPPV”與“綿羊痘病毒”和“GTPV”與“山羊痘病毒”通常感染綿羊和山羊,主要發(fā)生在非洲,中東和亞洲。而且由“SPPV”與“綿羊痘病毒”感染引起的發(fā)病率和死亡率高達(dá)100%。有效的診斷,是防治該病的重要前提。但目前尚無針對“SPPV”與“綿羊痘病毒”的商業(yè)化和專門的診斷技術(shù)。包膜蛋白P32是“SPPV”與“綿羊痘病毒”的主要結(jié)構(gòu)蛋白,其在“SPPV”與“綿羊痘病毒”的致病過程中發(fā)揮重要作用,并且參與誘導(dǎo)宿主體液和細(xì)胞免疫的產(chǎn)生。值得一提的是,來源于山羊痘病毒屬的P32蛋白具有種屬特異性B細(xì)胞表位,因此,P32蛋白具有作為開發(fā)“SPPV”與“綿羊痘病毒”檢測特異性診斷的有效靶標(biāo)的巨大潛力。本研究將“SPPV”與“綿羊痘病毒”的p32基因分別克隆到pGEX-6p-1和pcDNA3.1載體進(jìn)行原核及真核表達(dá),將純化獲得的P32重組蛋白免疫接種BALB/c小鼠制備抗P32的多克隆抗體;同時建立了基于多肽表位的檢測抗P32抗體的ELISA... 

【文章來源】：揚(yáng)州大學(xué)江蘇省

【文章頁數(shù)】：61 頁

【學(xué)位級別】：碩士

【文章目錄】：
Abstract
摘要
LIST OF ABBREVIATIONS VIII
Review Part
    Sheep and goat pox
        1. Definition
        2. Etiology
        3. Epidemiology
        4. Host range
        5. Clinical signs
        6. Routes of transmission
            6.1 Direct transmission
            6.2 Indirect transmission
        7. Virus infection and replication
        8. Pathogenesis
        9. Diagnosis
            9.1 Polymerase chain reaction (PCR)
            9.2 Enzyme linked immunosorbent assay (ELISA)
            9.3 Western blotting
        10.Virus isolation
        11. Treatment
        12. Control and prevention
        13. Objectives for this study
RESEARCH PART
    Part Ⅰ: Cloning and expression of p32 gene of sheep poxvirus
        1.1 Materials and Methods
            1.1.1 Main materials
            1.1.2 Mice
            1.1.3 Primers and PCR system
                1.1.3.1 Primers
                1.1.3.2 PCR
            1.1.4 Recovery of the PCR fragments
            1.1.5 Digestion and ligation
            1.1.6 Transformation into the DH5a
            1.1.7 Identification of the recombinant plasmids
            1.1.8 Expression of p32 protein induced by IPTG
            1.1.9 SDS-PAGE analysis for the expression of GST-P32
            1.1.10 Western blotting analysis for the of expression of GST-P32
            1.1.11 Purification and identification of recombinant proteins
            1.1.12 Preparation of polyclonal antibodies against P32
            1.1.13 IFA analysis for mouse polyclonal antibody against P32
            1.1.14 Western blot analysis for mouse polyclonal antibody against P32
        1.2 Results
            1.2.1 Construction and identification of the recombinant plasmidpGEX-6p-1-P32 and pcDNA3.1-P32
            1.2.2 Identification of the expression and purification of GST-P32
            1.2.3 Identification of polyclonal antibodies against P32
        1.3 Discussion
    Part Ⅱ: Development monoclonal antibodies against P32 protein of sheeppoxvirus
        2.1 Material and Methods
            2.1.1 Main materials
            2.1.2 A peptide-based ELISA for detection of antibody against P32
            2.1.3 Immunized mice
            2.1.4 Cell fusion
            2.1.5 Screening of positive hybridoma cells by peptide-based ELISA
            2.1.6 Identification of the subclasses of monoclonal antibody
            2.1.7 Monoclonal antibodies against P32 were confirmed by IFA
            2.1.8 Monoclonal antibody against P32 were confirmed by Western blot
            2.1.9 Identification of antigenic epitopes recognized by mAbs against P32
        2.2 Results
            2.2.1 Development of a peptide-based ELISA
            2.2.2 Screening monoclonal antibodies against P32 by the peptide-basedELISA
            2.2.3 mAbs 1D5 and 3A6 efficiently recognized the P32 protein expressed inDF-1 cell
            2.2.4 Identification of antigenic epitopes against P32 mAb
        2.3 Discussion
SUMMARY
REFERENCES
ACKNOWLEDGEMENTS


【參考文獻(xiàn)】：
期刊論文
[1]綿羊睪丸原代細(xì)胞培養(yǎng)及接種羊痘弱毒后的病變觀察[J]. 周建勝,馬慧玲,郭慶山.  中國獸醫(yī)科技. 2004(09)



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