绵羊痘病毒p32基因的克隆，表达及其单克隆抗体的制备

发布时间：2021-05-15 20:28

　　山羊痘病毒属包括绵羊痘病毒（SPPV）,山羊痘病毒（GTPV）和结节性皮肤病毒(LSDV。牛是LSDV常见感染宿主,并且在大多数非洲国家和中东都存在。然而,“SPPV”与“绵羊痘病毒”和“GTPV”与“山羊痘病毒”通常感染绵羊和山羊,主要发生在非洲,中东和亚洲。而且由“SPPV”与“绵羊痘病毒”感染引起的发病率和死亡率高达100%。有效的诊断,是防治该病的重要前提。但目前尚无针对“SPPV”与“绵羊痘病毒”的商业化和专门的诊断技术。包膜蛋白P32是“SPPV”与“绵羊痘病毒”的主要结构蛋白,其在“SPPV”与“绵羊痘病毒”的致病过程中发挥重要作用,并且参与诱导宿主体液和细胞免疫的产生。值得一提的是,来源于山羊痘病毒属的P32蛋白具有种属特异性B细胞表位,因此,P32蛋白具有作为开发“SPPV”与“绵羊痘病毒”检测特异性诊断的有效靶标的巨大潜力。本研究将“SPPV”与“绵羊痘病毒”的p32基因分别克隆到pGEX-6p-1和pcDNA3.1载体进行原核及真核表达,将纯化获得的P32重组蛋白免疫接种BALB/c小鼠制备抗P32的多克隆抗体;同时建立了基于多肽表位的检测抗P32抗体的ELISA... 

【文章来源】：扬州大学江苏省

【文章页数】：61 页

【学位级别】：硕士

【文章目录】：
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


【参考文献】：
期刊论文
[1]绵羊睾丸原代细胞培养及接种羊痘弱毒后的病变观察[J]. 周建胜,马慧玲,郭庆山.  中国兽医科技. 2004(09)



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