【作者】 商绍彬；

【导师】 周继勇；

【作者基本信息】 浙江大学 ， 微生物学， 2007， 博士

【摘要】 猪圆环病毒(Porcine circovirus, PCV)属圆环病毒科,为单链、环状DNA病毒,包括猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2)。PCV1无致病性,PCV2有致病性。PCV2基因组全长1768nt或1767nt,PCV1基因组全长1759nt,均具有潜在的11个阅读框。目前已知,ORF1编码与病毒复制相关的Rep蛋白,ORF2编码与宿主免疫相关的Cap蛋白,ORF3编码与复制无关、与致病性有关的蛋白。PCV2是引发猪圆环病毒病(PCVD),导致猪群免疫抑制,给养猪业造成了巨大的经济损失。尽管目前众多学者以PCV2 Cap蛋白为靶抗原构建了各种各样的PCV2的疫苗,但对其如何诱导免疫反应以及PCV1、2 ORF2诱导的免疫反应之间的差异在哪里不甚清楚。为此,本课题对PCV1和PCV2 Cap蛋白的抗原特性进行了较为深入的分析,并建立了用于PCV2感染血清学检测的ELISA方法。采用去除PCV Cap蛋白核定位信号(Nuclear localization signal)的策略,以PCV DNA为模板,设计特异性引物,分别扩增了截短的PCV2衣壳蛋白(PCV2-dCap)和PCV1衣壳蛋白(PCV1-dCap)基因,进而分别克隆至原核表达载体pGEX-4T-1和pET28a(+)中,成功构建重组载体pGEX-PCV2-dCap和pET-PCV1-dCap。经表达条件优化,PCV2重组GST-dCap融合蛋白以可溶形式表达,最佳表达条件为0.1mM IPTG,37℃诱导4h；PCV1重组His-dCap蛋白以包涵体形式表达,在37℃、经1mM IPTG终浓度诱导4h可获得充分表达。采用GST亲和层析纯化,获得了48kD的PCV2 GST-dCap融合蛋白和切去GST的dCap蛋白(22kD)；采用Ni-NTA亲和柱纯化,获得了28kDa的变性PCV1 His-dCap蛋白。PCV2 GST-dCap蛋白和PCV1His-dCap蛋白的表达量分别可达6.14mg/L菌液和36mg/L菌液。Western blot结果表明PCV2GST-dCap融合蛋白和dCap蛋白都能与猪PCV2阳性血清发生特异性反应；而PCV1 His-dCap蛋白与抗His标签单克隆抗体发生特异性反应,与选用的猪PCV2阳性血清不发生反应。以PCV2重组dCap蛋白、PCV2病毒粒子和PCV1 His-dCap蛋白为抗原分别免疫小鼠,应用杂交瘤细胞融合和有限稀释克隆技术制备针对PCVCap蛋白的特异性单抗。分别采用间接ELISA、IFA和Western blot筛选及鉴定杂交瘤细胞上清。结果,以PCV2重组Cap蛋白为免疫原,获得了5株(1B3、1B9、8A12、8B12、8C12)针对PCV2天然Cap蛋白的单克隆抗体(mAb)分泌细胞,4株(3F6、5A3、5E11、6E1)针对PCV1、2天然Cap蛋白的单克隆抗体分泌细胞,1株(4C3)仅针对PCV1、2重组表达Cap蛋白的单克隆抗体分泌细胞；以纯化的PCV2病毒粒子为免疫原,获得了4株(1E3、2B1、6H9、7F5)针对PCV1、2天然Cap蛋白、但不与PCV1重组表达Cap蛋白反应的单克隆抗体分泌细胞；以PCV1重组表达的Cap蛋白为免疫原,获得10株(1A11、4A2、1D11、2D4、2F5、4F5、3F11、2G9、3G7、3H1)针对PCV1天然Cap蛋白的单克隆抗体分泌细胞,1株(4H7)针对PCV1、2天然Cap蛋白的单克隆抗体分泌细胞。除mAb 5A3、3F11、3G7、2G9、1A11为IgG2b和mAb 7F5、2F5为IgG2a外,其余mAb均为IgG1亚类。所有单抗的轻链均为κ链。中和试验表明,制备的mAb中,来源于PCV2重组Cap蛋白的mAb 3F6和来源于纯化PCV2病毒的mAb 6H9具有阻止PCV2病毒感染细胞的能力,来源于纯化PCV2病毒的mAb 1E3、7F5则能同时中和PCV1和PCV2粒子,说明mAb 1E3、7F5是识别PCV1、2共同抗原表位的功能性单克隆抗体,而mAb 3F6、6H9是仅识别PCV2特有抗原表位的功能性单抗。采用合成肽技术,利用已获得的针对PCV衣壳蛋白的单克隆抗体以及PCV2多克隆抗体,对PCV衣壳蛋白的线性B细胞抗原表位进行了鉴定。试验结果表明,所试验的25株抗Cap蛋白的mAb中,仅5个线性B细胞表位为14株单克隆抗体识别,分别为：mAb 8A12、8B12和8C12识别PCV2 Cap蛋白C末端231LNP233表位；mAb 1B3和1B9识别PCV2 Cap蛋白的195HVGLGTAF202表位；mAb 4C3识别PCV1、2 Cap蛋白的175QPNNKRNQLWLRLQTAGN192表位；mAb 3G7识别PCV1、2 Cap蛋白的156YHSRYFT162表位；mAbs 4F5、4H7、3F6、1E3同时识别PCV1、2 Cap蛋白156YHSRYFT162和175QPNNKRNQLWLRLQTAGN192两个表位；mAbs 3F11、2G9和2D4识别PCV1 Cap蛋白的92LPFQYYRIRKAK103表位。氨基酸突变分析表明,231LNP233表位中233位的脯氨酸是该表位的和PCV2型特异性的关键氨基酸。利用PCV2猪多抗分析了PCV2 Cap的线性B细胞抗原表位谱,其B细胞抗原表位主要位于氨基酸残基25-42,55-72,95-112,115-142,156-192,216-233之间；对PCV2感染或PCV2 Cap真核质粒免疫后不同时期采集的猪血清中各抗原表位抗体的动力曲线分析表明,不同个体对各抗原表位的免疫反应性不同,其中,表位115DRGVGSSAVILDDNFVTK132、156YHSRYFT162、175QPNNKRNQLWLRLQTAGN192、231LNP233为免疫优势表位,但均只在感染后28-35天以后才可检测到相应抗体。对表位231LNP233检测PCV2 Cap抗体的潜力评估表明,231LNP233是PCV2Cap潜在的新的血清学标志。以重组表达的GST-dCap融合蛋白为抗原,建立了检测PCV2抗体的间接ELISA方法。试验的最佳反应条件为：抗原包被浓度为1.24μg/ml,包被液为0.05M pH8.5的Tris-HCl缓冲液；待检血清1：400稀释,酶标抗体为1：2000；5%脱脂奶为封闭液；含0.05%吐温-20的磷酸缓冲液作样品稀释液;待检血清和酶标二抗反应时间为30min,底物反应时间10min。以S/P值等于0.16作为PCV抗体阴、阳性血清判定的临界值。特异试验表明：建立的方法不与猪瘟病毒、猪繁殖与呼吸综合症病毒、伪狂犬病毒等阳性血清发生交叉反应。同批抗原制备的不同检测板变异系数(CV)值在1-13%之间,不同批抗原制备的检测板CV值在5-22%之间。本方法与间接免疫荧光试验相比,阳性符合率为95.28%、阴性符合率为94.09%、总符合率为95.09%。与PCV2全病毒ELISA相比,本试剂盒的阳性符合率为93.3%,阴性符合率为84.2%,总符合率为91.14%。与基于PCV2型特异的多肽ELISA相比,本试剂盒的阳性符合率可达93.8%,阴性符合率为88.9%,总符合率为92.4%。与西班牙INGENASA公司的PCV2抗体试剂盒比较,本方法的诊断敏感性为91.1%,特异性为90.2%,二者符合率达90.5%；应用建立的PCV2抗体检测方法检测人工感染猪PCV2抗体消长规律发现,感染猪PCV2抗体在感染后14-21天阳转,于35-49天达到高峰,与IFA检测结果相符。表明dCAP-ELISA检测方法能够体现PCV2感染后真实的抗体动态变化。上述结果表明,所建立的PCV2抗体ELISA检测技术具有高度的特异性和敏感性。更多还原

【Abstract】 Porcine circovirus (PCV), a member of the family Circoviridae, is a small, non-enveloped virus, with a circular, single-stranded DNA genome. PCV includes nonpathogenic Porcine circovirus type 1 (PCV1) and pathogenic Porcine circovirus type 2 (PCV2). The circular PCV2 viral genome contains 1767-1768 nucleotides while the PCV1 genome contains 1759 nucleotides. However, the genomic organization of both PCV1 and PCV2 is similar, containing 11 potential open reading frame (ORF). Recently, the ORF1-encoding replicase (Rep) involved in viral replication, the ORF2-encoding capsid (Cap) involved in host immune response and the product encoded by ORF3 associated with pathogenicity of PCV2 were verified. PCV2 was believed to be the primary causative agent of porcine circovirus-associated diseases (PCVD), especially postweaning multisystemic wasting syndrome (PMWS) which caused economically great loss in swine industry. Many researchers have developed ORF2-based vaccines for prevention of PCV2-associated diseases. However, the mechanism by which those vaccines against PCV2 induced immune response was unclear and remained to be understood for developing more effective vaccines to PCV2. In this study, the antigenic characterization of the capsid protein of PCV1 and PCV2 was identified, and an enzyme-linked immunosorbant assay for serodiagnosis of PCV2 infection was developed using the recombinant capsid protein of PCV2 as antigen.The nuclear localization signal-defected capsid protein genes of PCV2 and PCV1 were amplified with specific primers and cloned into prokaryotic vector pGEX-4T-l and pET28a(+), respectively, to construct recombinant plasmid pGEX-PCV2-dCap and pET-PCVl-dCap. The recombinant PCV2 dCap protein fused with glutathione S-transferase (GST-dCap) was expressed as a soluble form, and the expression conditions were optimized to be that recombinant bacteria containing pGEX-PCV2-dCap were induced by a final concentration of 0.1 mM of IPTG for 4 h at 37℃. While the 6×His-tagged PCV1 dCap (His-dCap) was expressed as inclusion bodies after induction by a final concentration of 1 mM of IPTG for 4 h at 37℃. The recombinant PCV2 GST-dCap protein with a mass of 48kD and PCVI His-dCap protein with a mass of 28kD were purified respectively by GST affinity chromatography and Ni-NTA affinity chromatography. And the 22-kD PCV2 recombinant dCap protein was obtained through cleavage from GST-dCap with thrombin. The yield of recombinant PCV2 GST-dCap protein and PC VI His-dCap was about 6.14 mg per liter cell culture and 36 mg per liter cell culture, respectively. Western blot analysis indicated that the recombinant PCV2 GST-dCap protein and dCap were recognized by swine anti-PCV2 serum, while PCV1 His-dCap protein was recognized by anti-His tag monoclonal antibody, but not by the swine anti-PCV2 serum.Thereafter, the recombinant PCV2 GST-dCap, purified PCV2 pariticles and PCV1 His-dCap were used to immunize Balb/c mice for production of monoclonal antibodies (mAb) against PCV Cap. After screening by ELISA and IFA, cloning by limiting dilution and identification by western-blotting assay, five hybridomas, named 1B3,1B9,8A12,8B12 and 8C12 whose supernatant only recognize PCV2 native capsid protein, four hybridomas, named 3F6,5A3,5E11, and 6E1 whose supernatant could recognize the native capsid proteins of PCV2 and PCVI and one hybridomas, named 4C3 whose supernatant could recognize recombinant PCV2 GST-dCap and PCV1 His-dCap, were developed using PCV2 GST-dCap as immunogen; And using the purified PCV2 particles as immunogen, four hybridomas named 1E3,2B1,6H9 and 7F5, whose supernatant could react with the native capsid proteins of PCV2 and PCV1 but not with recombinant PCV1 His-dCap were obtained; While using the PCV1 His-dCap as immunogen, ten hybridomas named 1A11,4A2,1D11,2D4,2F5,4F5,3F11,2G9,3G7 and 3H1, respectively whose supernatant could only recognize PCV1 native capsid protein and one hybridomas named 4H7 whose supernatant could recognize both PCV2 capsid and PCVI capsid were developed. Among these mAbs, mAb 5A3,3F11,3G7,2G9 and 1A11 belong to IgG2b, mAb 7F5 and 2F5 belong to IgG2a, the other mAbs belong to IgGl subtype. All the mAbs haveκ-light chain of immunoglobulin. Neutralization assay indicated that mAb 3F6,1E3,6H9 and 7F5 to PCV2 Cap had the neutralizing ability to PCV2, and mAbs 1E3 and 7F5 also showed neutralizing ability to PCV1, while all the mAbs to PCVI His-dCap had no neutralizing ability to PCVI and PCV2.Utilizing those monoclonal antibodies to PCV Cap protein, swine anti-PCV2 or-Cap sera as well as synthesized overlapping peptides from PCV2 Cap, mapping and localization of B-cell epitopes on capsids of PCV was performed. Five linear B-cell epitopes on PCV Cap were identified and localized respectively at aa 231-233 (231LNP233) recognized by PCV2-specific mAbs 8A12,8B12 and 8C12, aa 195-202 (195HVGLGTAF202) recognized by PCV2-specific mAb 1B3 and 1B9, aa 175-192 (175QPNNKRNQLWLRLQTAGN192) recognized by mAb 4C3 to recombinant PCV Cap protein, aa 156-162 (156YHSRYFT162) recognized by PCVl-specific mAb 3G7, and aa 92-103 (92LPFQYYRIRKAK103) recognized by PCVl-specific mAbs 3F11,2G9 and 2D4. While mAb 3F6,1E3,4H7 and 4F5 againt PCV2 and PCV1 Cap recognized two epitopes of aa 156-162 and aa 175-192. Thus, the epitope,231LNP233, was specific for PCV2 Cap; 175QPNNKRNQLWLRLQTAGN192 and 156YHSRYFT162 was the common epitopes of PCV1 Cap and PCV2 Cap and 92LPFQYYRIRKAK103 was specific for PCV1 Cap. Analysis of substitution with alanine showed that the proline at aa 233 was a vital and type-specific residue for the epitope 231LNP233 and PCV2 capsid protein. Epitope mapping of the capsid of PCV2 was also achieved by reaction of synthesized peptides with swine anti-PCV2 sera. The resulting immunoactive regions of the capsid of PCV2 were located between amino acids (aa) 25-42,55-72,95-112,115-142, 156-192,216-233, respectively. Further analysis of the kinetic curves of antibodies to those epitopes in swine anti-PCV2 or-Cap polyclonal antibody revealed that the immune responses to these antigenic determinants in individual pig were different, and the four epitopes, aa 115-132, 156-162,175-192 and 230-233 were immunodominant in PCV2 capsid, however, the corresponding antibody was detectable after 28 days post-inoculation. And the epitope 231LNP233 was showed to have the potential as serological marker for PCV2.Using the PCV2 GST-dCap as antigen, an alternative indirect enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of PCV2 infection was developed. The optimal antigen concentration and serum sample dilution were set at 1.24μg/ml and 1:400; The optimal dilution of the conjugate was 1:2000; The 0.05 M Tris-HCl buffer at pH 8.5 was the best coating buffer; The 5%skimmed milk was the optimal blocking buffer; The phosphate buffer containing 0.05% tween-20 was the best diluent; The optimal reaction time for serum samples or conjugate and chromatogenic substrate was 30 min and 10 min, respectively. A S/P ratio of 0.16 was set as a negative-positive cutoff. Repeatability tests revealed that the coefficient of variation of positive sera within and between runs were 1-13% and 5-22%. Cross-reactivity assay showed that this assay was PCV-specific. This assay was validated by comparison with an indirect immuno-fluorescence assay (IIF), a PCV2-based ELISA, a PCV2-specific peptide-based ELISA and a commercial PCV2 ELISA kit. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the PCV2 CAP ELISA were 95.3%,93.9% and 95.1%, compared with IIF on 1080 field serum samples, and 93.3%,84.2%and 91.1%, compared with the PCV2-based ELISA on 79 field sera, and 93.8%,88.9%and 92.4%by comparison with the PCV2 peptide-based ELISA on 92 field sera, and 91.1%,90.2%and 90.5%by comparison with the commercial PCV2 ELISA kit on 85 field sera, respectively. The kinetic curve of PCV2 antibody after infection or vaccination was ploted by this ELISA, indicating that this assay could detected the seroconversation to PCV2. These data indicated that this ELISA is highly specific and sensitive for large scale surveys of PCV2 infection at low cost and the evaluation of the efficiency of various vaccines against PCV2.更多还原