【作者】 常天明；

【导师】 仇华吉；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 硕士

【摘要】 猪瘟病毒(classical swine fever virus,CSFV)属于黄病毒科(Flaviviridae)瘟病毒属(Pestivirus),为单股正链RNA病毒。CSFV含有4种结构蛋白:C、Erns、E1和E2。其中,Erns和E2作为囊膜蛋白存在于成熟病毒粒子表面,能够诱导机体产生中和抗体;而E1与Erns通过二硫键形成异源二聚体,包埋于病毒囊膜内,不能诱导猪产生中和抗体。猪瘟病毒进入宿主细胞是通过病毒囊膜蛋白与细胞受体蛋白之间相互作用介导的内吞作用,Erns作用的受体蛋白为硫酸乙酰肝素,而E2作用的受体蛋白目前尚不清楚。抗独特型抗体(anti-idiotype antibody,anti-Id)是指机体针对Ab1抗原抗体结合区的独特位产生的抗体,其中Ab2β具有“内影像”特性,能够模拟病毒抗原,产生针对初始抗原的特异性抗体或细胞免疫应答。抗独特型抗体相当于病毒粒子的探针,通过其与宿主细胞之间的相互作用可以鉴定出病毒感染过程中所需的受体蛋白。我们以针对猪瘟病毒E2蛋白的单克隆抗体6E10为免疫原,经皮下免疫新西兰白兔后制备了抗独特型多克隆抗体。竞争抑制ELISA证明其能够模拟E2抗原,免疫组化试验及Western blot发现anti-Id能够识别猪只外周血淋巴细胞以及不同组织中一个分子量大小约54 kDa的蛋白,命名为p54,质谱分析其与IgG同源性最高。为了进一步揭示p54蛋白的特征及其生物学功能,我们参考大量文献后发现p54蛋白分子量大小与CD46蛋白十分接近,因此我们根据NCBI提供的猪CD46基因设计一对引物,从猪只血液中扩增得到目的基因,经测序分析后分别将其克隆至原核表达载体pET32a和真核表达载体pcDNA3.1,并利用原核表达的蛋白制备了兔抗CD46高免血清,阻断试验证明,病毒感染的抑制作用与抗CD46多克隆抗体表现出剂量依赖性。但我们发现兔anti-Id不与CD46蛋白反应,同样利用抗CD46多抗也无法在PBMC中检测到p54,由此我们认为p54与CD46蛋白是两种截然不同的蛋白质。在此基础之上,我们进行了受体重建工作:将已经构建的pcDNA-CD46转染猪瘟病毒非敏感细胞BHK-21,该细胞经RT-PCR检测是不存在猪CD46基因的。转染后加G418筛选,传至第三代后的BHK-CD46细胞进行病毒感染试验与激光共聚焦试验。病毒感染试验显示,猪瘟病毒能够吸附或进入BHK-CD46细胞但不增殖。激光共聚焦结果显示,猪瘟病毒能够吸附在表达CD46蛋白的BHK-CD46细胞膜表面。免疫共沉淀试验证明猪瘟病毒E2蛋白能够沉淀PK15细胞表面的CD46分子。以上结果表明,CD46蛋白与猪瘟病毒E2蛋白之间存在相互作用,可能是猪瘟病毒吸附过程中所必需的受体蛋白之一。更多还原

【Abstract】 Classical swine fever virus (CSFV) is classified within the genus Pestivirus in the family Flaviviridae. It is an enveloped, non-segmented, positive-stranded RNA virus encoding four structural proteins: C, Erns (formerly known as E0), E1, and E2. Erns and E2 reside on the outer surface of CSFV virion and transmembrane glycoprotein E1 is present as an E1-E2 heterodimer by disulfide linkage. Glycoprotein E2 is the most immunogenic protein of CSFV and glycoprotein Erns is the second target for neutralizing antibodies. Glycoproteins, which present in the viral envelope, are involved in the attachment to susceptible cells and mediate the CSFV entry. Interaction of Erns with heparin sulfate (HS) immobilizes CSFV at the cell surface but E2 also associates with cellular receptor(s) by an as yet unknown mechanism.Anti-idiotype antibodies (anti-Ids) elicited by an antibody molecule are directed against antigenic determinants in or close to the antibody’s combing site. Besides using anti-Ids as immunogens for a protective response against a particular infectious agent, Ab2βinternal image antibodies have been used as probes for receptors by mimicking viral epitopes that bind to cell receptors.Polyclonal anti-Ids were produced in New Zealand White rabbits by immunizing monoclonal antibody (MAb) 6E10 via subcutaneous injection. Characterization of anti-Ids, which can mimic an epitope on the CSFV glycoprotein E2, was performed by competitive ELISA. In immunohistochemistry assay and Western blot analysis, a 54 kDa cell surface protein (p54) was detected by the anti-Ids from PBMC. Microchemical analysis showed that the most similar molecule of p54 was IgG-like. To identify the structure and function of p54, we searched a mass of literatures and found that the molecular masses between p54 and CD46 are similar. To verify our hypothesis, the CD46 gene of pig was cloned and expressed in vectors pET32a and pcDNA3.1. Polyclonal rabbit anti-CD46 sera were produced and they could block CSFV infection in porcine kidney cells. We also found that p54 and CD46 were different proteins because anti-Ids could not recognize CD46 and p54 could not be recognized by anti-CD46 serum. In further study, BHK-21 cells were transfected with the expression plasmid pcDNA-CD46. Stable CD46-expressing cell lines (BHK-CD46) were selected by adding G418 to the culture medium after transfection. CSFV infection on BHK-CD46 cells expressing CD46 was detected by antigen-capture ELISA and laser confocal light microscopy. Co-IP showed that CSFV E2 could precipitate CD46 from PK15 cells. Our data showed that the expression of CD46 did not confer full susceptibility to non-permissive cells but binding. This study indicates that CD46 is a cellular receptors that is involved in attachment to susceptible cells of CSFV.更多还原