猪圆环病毒2型ELISA抗体检测方法的建立及PCV2单克隆抗体的制备

Development and Application of Indirect ELISA Antibody for Porcine Circovirue Type 2 and Production of Monoclonal Antibodies Against PCV2

作者: 专业:细胞生物学 导师:王爱萍 年度:2010 学位:硕士  院校: 郑州大学

Keywords

PCV2, ORF2 gene fragment, Prokaryotic expression, indirect ELISA, monoclonal antibodies

        猪断奶后多系统衰竭综合征(Postweaning multisystemic wasting syndrome, PMWS)是由猪2型圆环病毒(Porcine circovirus type2, PCV2)引起的一种猪的重要传染性疾病。PMWS最早于1991年在加拿大西部被发现,之后便在全世界范围内流行。该病主要侵害断奶仔猪,临床表现为仔猪发热、进行性消瘦、苍白、黄疸、呼吸衰竭及消化系统功能紊乱等症状。猪圆环病毒(Porcine circovirus, PCV)属于圆环病毒科圆环病毒属,属DNA病毒。分为1型(PCV1)和2型(PCV2)。PCV1是细胞污染物,无致病性;PCV2有致病性,PCV2的感染可以造成免疫抑制,引起猪相关疾病的继发和并发感染,如增生性坏死性肺炎(PNP)、猪皮炎与肾炎综合征(PDNS)、以及猪呼吸道综合征(PRDC)、先天性震颤、肠炎、繁殖障碍等。目前,PCV2成为世界范围内对养猪业危害最严重的病原之一。PCV2基因组包含11个开放阅读框,其中ORF1和ORF2是主要的阅读框。ORF1编码Rep蛋白,与病毒复制相关,此蛋白在这两型病毒之间有85%同源性,是引起PCV1和PCV2抗原交叉反应的主要原因。ORF2编码的Cap蛋白是病毒的主要结构蛋白,是型特异性抗原,具有良好的免疫原性,在两型PCV之间不发生抗原交叉反应。本研究建立了PCV2抗体间接ELISA检测方法。这种方法的特点是敏感、快速、特异,并能够有效鉴别2型PCV的感染,对PCV2快速诊断、流行监测和防控具有重大的意义。1.克隆了PCV2 ORF2基因片段,在大肠杆菌原核表达系统中表达ORF2基因片段。结果表明,该基因片段在BL21中成功表达,融合蛋白的相对分子质量约为26kDa,并能为PCV2阳性血清所识别。2.以表达的Cap蛋白作为包被抗原,建立PCV2 Cap蛋白间接ELISA检测方法;利用建立的间接ELISA方法对猪场的80份血清进行检测,并与商品化的试剂盒进行了对照,检出阳性率为60%,两种方法符合率为93%。3.利用PCV2河南株病毒抗原免疫Balb/c小鼠,通过建立的间接ELISA方法检测筛选,最终获得2株稳定分泌抗PCV2单克隆抗体的细胞株:2B11-C1、3F2-A10。特异性实验表明:2B11-C1、3F2-A10不与猪瘟病毒、猪伪狂犬病毒、猪乙脑病毒、猪繁殖与呼吸综合征病毒以及正常PK-15细胞反应,特异性良好。两株PCV2单抗的获得,为进一步建立特异、敏感、准确快速的PCV2检测方法打下了基础。
    Postweaning multisystemic wasting syndrome (PMWS) in pigs was first identified in western Canada in 1991. Thereafter, it has been reported worldwide. The clinical signs of PMWS are fever, progressive weight loss, pallor, jaundice, respiratory and digestive disorders. The main etiological agent of PMWS was porcine circovirus type 2 (PCV2).Porcine circovirus (PCV) is a member of the family Circoviridae. According to the pathogenic and genetic constitution, PCV contains PCV1 and PCV2.PCV1 is a type of contaminant in cells, and it has no pathogenicity. PCV2 has pathogenicity, and it can depress the pig’s immune system. PCV2 has increasingly been associated with various disease syndromes in pigs. So far, the infection of PCV2 has become one of the pathogens which can seriously handicap progress of pig industry worldwide.PCV2 contains eleven open reading frames. ORF1 and ORF2 are the two major open reading frames. ORF1 gene encodes replication-associated proteins (Rep) which is a protein involved in viral replication. Because of Rep protein, PCV1 and PCV2 have cross-reaction of antigenicity. The homology of them is 85%. ORF2 gene encodes the capsid protein (Cap) which is the major constitutive protein. The Cap protein was considered to be type-specific antigen, which has no cross-reaction of antigenicity in two type viruses.The research used the prokaryotic expression protein of ORF2 to establish the indirect ELISA assay, which is a sensitive, rapid, and specific method. The indirect ELISA assay can identify PCV2 efficiently. In a word, the establishment of the method has important significance for controlling the disease and early detection of the disease in pig industry.1. The prokaryotic expression of ORF2 gene fragments in E.coli. The results show as follows. The truncated fragment of PCV2 ORF2 gene was expressed correctly in E.coli BL21. The molecular weight of the expressed recombinant capsid protein was about 26.0kDa. The recombinant capsid protein could react with polyclonal antibody against PCV2.2. The recombinant Cap protein was used as the envelope antigen, and established the indirect ELISA method. We use the method to detect 80 serum samples, and contrast with the commercial kit. The total masculine ratio was 60%. The compliance rate between two methods was 93%.3. We immunized Balb/c mice with PCV2 virus antigen which was condensed and purified, and screened with the indirect ELISA method to obtain monoclonal antibodies specific for PCV. The McAbs were named 2B11-C1 and 3F2-A10. The specific experiments showed that 2B11-C1 and 3F2-A10 had no cross-reactivity with CSFV, PRV, JEV, PRRSV and PK-15 cells. This research provides a basis for setting up a PCV2-testing which has high specificity and sensibility.
        

猪圆环病毒2型ELISA抗体检测方法的建立及PCV2单克隆抗体的制备

摘要4-6
Abstract6-7
目录8-12
引言12-13
第一部分 文献综述13-27
    1 病原学13-14
        1.1 猪圆环病毒的发现与命名13
        1.2 PCV的分类地位及分型13-14
        1.3 PCV的形态及理化特征14
        1.4 PCV的增殖与培养特性14
    2 PCV的分子生物学特性14-18
        2.1 PCV的基因组特征14-15
        2.2 PCV编码蛋白及功能15-16
        2.3 PCV的复制及转录16-18
    3 流行病学18-19
    4 致病机理19-20
    5 PCV2检测方法研究进展20-24
        5.1 电镜法21
        5.2 免疫组织化学检测技术21
        5.3 免疫荧光技术21-22
        5.4 酶联免疫吸附试验诊断方法22
        5.5 免疫过氧化物酶单层细胞试验22
        5.6 免疫胶体金技术22
        5.7 聚合酶链反应22-23
        5.8 原位杂交方法23
        5.9 基因芯片检测技术23-24
    6 PCV2感染的防治24
    7 猪圆环病毒单克隆抗体研究进展24-25
        7.1 单克隆抗体技术24-25
        7.2 PCV2单克隆抗体的研究25
    8 本研究的意义25-27
第二部分 猪圆环病毒2型ORF2基因片段的原核表达27-40
    1 材料与试剂27-29
        1.1 病毒、载体及菌株27
        1.2 主要试剂和酶27-28
        1.3 主要试剂配制28-29
    2 方法29-34
        2.1 引物设计与合成29
        2.2 PCR模板的制备29-30
        2.3 PCR扩增30
        2.4 DNA片段回收30
        2.5 重组质粒pMD18-T-ORF2的构建30-32
        2.6 重组原核表达质粒的构建32-33
        2.7 重组融合蛋白PET-28a-ORF2的诱导表达及检测33-34
    3 结果与分析34-38
        3.1 目的基因片段的扩增34-35
        3.2 pMD18-T-ORF2的酶切鉴定35-36
        3.3 重组质粒pET-28a-ORF2的PCR和酶切鉴定36-37
        3.4 重组蛋白的SDS-PAGE分析37
        3.5 重组蛋白的Western-Blot分析37-38
    4 讨论38-40
第三部分 PCV2 Cap蛋白间接ELISA检测方法的建立40-55
    1 材料与试剂40-43
        1.1 主要实验仪器和设备40
        1.2 主要试剂配制40-43
    2 方法43-46
        2.1 包涵体的制备和纯化43-44
        2.2 包涵体的复性44
        2.3 间接ELISA检测方法的建立44-46
    3 结果与分析46-53
        3.1 重组蛋白抗原最佳包被浓度与血清稀释度的确定46-47
        3.2 重组抗原包被条件的确定47-48
        3.3 封闭液的选择48
        3.4 一抗血清最佳反应时间的确定48-49
        3.5 酶标二抗最佳工作浓度的确定49
        3.6 酶标二抗最佳工作时间的确定49-50
        3.7 显色时间的确定50
        3.8 间接ELISA阴阳性临界值的确定50-51
        3.9 特异性试验51-52
        3.10 重复性试验52
        3.11 临床血清样品检测52-53
    4 讨论53-55
第四部分 PCV2单克隆抗体的制备55-65
    1 材料与试剂55-56
        1.1 病毒、实验动物与细胞55
        1.2 主要试剂配制55-56
    2 方法56-61
        2.1 免疫原的制备56-58
        2.2 免疫58
        2.3 抗原最佳包被浓度和最佳血清稀释度的确定58
        2.4 杂交瘤细胞株的建立58-60
        2.5 单克隆抗体的生产60-61
        2.6 杂交瘤细胞株的鉴定61
    3 结果61-63
        3.1 PCV2的PCR检测61-62
        3.2 PCV2抗原的含量62
        3.3 免疫结果62
        3.4 ELISA抗原最佳包被浓度和血清最佳稀释度的确定62
        3.5 细胞融合筛选结果62
        3.6 杂交瘤细胞培养上清效价的测定62-63
    4 讨论63-65
        4.1 病毒培养63
        4.2 抗原制备63
        4.3 动物免疫63-64
        4.4 细胞融合64
        4.5 关于细胞培养中的问题64-65
第五部分 全文总结65-66
参考文献66-71
硕士期间发表的学术论文与研究成果71-72
致谢72
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