猪圆环病毒-Ⅱ型抗体检测免疫层析试纸的研制及单克隆抗体的筛选与鉴定
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摘要
猪圆环病毒-Ⅱ型（Porcine Circovirus2, PCV2）是引起断奶仔猪多系统衰竭综合征（Postweaning multisystemic wasting syndrome, PMWS）主要及原发病原。由于此病在世界范围内流行，同时能对猪的生长造成严重危害，极大地降低了饲料利用率，对养猪工业造成了重大影响。PCV2基因组大小为1767bp或1768bp，包含11个开放阅读框，其中ORF1、ORF2、ORF3为主要的阅读框。ORF1编码的Rep蛋白主要与病毒的复制相关，ORF3编码蛋白与病毒的致病性相关。ORF2编码的Cap蛋白为病毒的主要结构蛋白，蛋白上存在多个优势抗原表位，具有良好的免疫原性，试验证实Cap蛋白能区分PCV1和PCV2临床血清。
目前，PCV2诊断方法主要为PCR、免疫组化、原位杂交、免疫过氧化物酶单层试验（IPMA）、间接免疫荧光（IFA），这些方法主要用于抗原检测。PCV2抗体检测主要手段为ELISA，目前已有基于Cap蛋白的商品化ELISA试剂盒，但是ELISA检测方法需要专业人员和专门仪器，只适用于实验室检测。当前已经有商品化的猪圆环病毒疫苗用于猪群免疫，且免疫范围较大，因此急需一种简便、快速、敏感的检测方法用于免疫后抗体水平评价。
胶体金免疫层析技术是在胶体金标记技术和免疫层析技术基础上发展而来的，该技术具有简便、快捷的特点，同时不需要任何仪器和专业人员，因此被广泛应用于疫病及药物残留检测。本研究成功制备了PCV2抗体检测试纸，为PCV2抗体临床快速检测提供了有力工具。同时筛选并鉴定了数株PCV2单克隆抗体。
1.猪圆环病毒-Ⅱ型Cap蛋白的原核表达及功能鉴定
为获得具有免疫原性的PCV2Cap蛋白，本试验以含有PCV2ORF2基因的重组质粒PMD18T-ORF2为模板，利用PCR技术扩增得到PCV2ORF2基因片段，将此基因片段与原核表达载体pET28a连接，构建重组表达载体，命名为pET28a-ORF2，经PCR和测序鉴定后，用IPTG诱导ORF2基因的表达，收集诱导的菌液进行SDS-PAGE电泳分析。结果显示在分子量约为26KD处有一条明显的蛋白条带。表达产物上清用Ni柱纯化并透析，Western-Blot鉴定该蛋白能被PCV2阳性血清识别，ELISA检测结果显示蛋白具有较高的活性。研究表明PCV2Cap蛋白在大肠杆菌中得到高效表达，纯化获得的蛋白具有较高生物活性，为开发诊断试剂和亚单位疫苗奠定了基础。
2.猪圆环病毒-Ⅱ型抗体检测免疫层析试纸的研制及性能评价
将胶体金标记猪圆环病毒-Ⅱ型（PCV2）Cap蛋白喷涂于玻璃纤维膜上作为检测探针，金黄色葡萄球菌A蛋白(SPA)和PCV2多克隆抗体IgG固定在硝酸纤维膜上分别作为检测线和质控线，制备组装成免疫层析试纸。运用PCV2标准阳性、阴性血清及猪瘟病毒、蓝耳病病毒、细小病毒、伪狂犬病毒、PCV1阳性血清，对试纸进行特异性、敏感性评价。免疫层析试纸与进口商品化ELISA试剂盒对田间血清样品同时进行检测，评价试纸与ELISA试剂盒的符合率。试验结果显示，该试纸的特异性、敏感性良好，与进口ELISA试剂盒符合率为94.00%(470/500)，灵敏度与ELISA试剂盒相当。表明该试纸具有快速、特异、敏感等优点，操作简便，不需要专业人员与专门仪器，适合田间推广运用。
3.猪圆环病毒-Ⅱ型单克隆抗体的制备及鉴定
用纯化的PCV2免疫Balb/c小鼠，使小鼠对PCV2产生免疫反应并产生抗体，取血清效价较高的小鼠脾细胞与NS0瘤细胞融合。使用基于Cap蛋白的抗体检测试纸对大量杂交瘤细胞培养上清进行检测和鉴定，同时采用病毒阻断方法评价抗体与病毒的反应情况。最终筛选出5株分泌高亲和力单克隆抗体的杂交瘤细胞：3C9、6A5、8F9、9F4、15E4，其中6A5、8F9、9F4三株能与病毒反应。结果表明，该单抗的腹水能很好地识别PCV2病毒及其Cap蛋白，效价分别为：1:4×105、1:3×105、1:4×105。具有高度的特异性，与其他病毒和蛋白无交叉反应性。该单抗的成功制备，为PCV2抗原快速检测试纸条的制备奠定了基础。
Porcine circovirus type2(PCV2) is the main and primary causative agent ofPostweaning Multisystemic Wasting Syndrome (PMWS), which has causedsignificant economic losses in the swine industry. The genome of PCV2is1767bp or1768bp in length and consists of11ORFs. Three major ORFs have beencharacterized for PCV2: ORF1, ORF2, and ORF3. ORF1encodes a replication-associated protein (Rep), while ORF2encodes a major structural protein (Cap), themain antigenic determinant of the virus, and ORF3is involved in PCV2pathogenesis.Cap protein contains several immune-dominant epitopes and is confirmed to be type-specific.
To date, several commercial PCV2vaccines were under use in the market for theprevention and control of PCV2. Therefore, a method for specific serologic detectionin routine field practice to monitor PCV2antibody titers induced by vaccines isessential. Immunoperoxidase monolayer assay (IPMA), indirect immunofluorescentassay (IFA), and enzyme linked immunosorbent assay (ELISA) are the mostcommonly diagnostic methods for detecting PCV2antibodies. However, thesemethods require specialized equipment and technical expertise, and are suitable forlaboratory use only. Furthermore, IPMA and IFA require the cultivation of PCV2onPK-15cell, which is time-consuming and labor-intensive.
The membrane-based immunochromatographic lateral flow strip test represents awell-established and appropriate technique for a variety of point-of-care andfield-use applications, which is used in the detection of pathogen and durgs. Thistechnology has several advantages over traditional immunoassays, such as simplicityof procedure, rapid operation and immediate results, low cost, no requirements fortechnical expertise or specialized equipment. In this study, a simple and rapidimmunochromatographic strip was developed for the detection of PCV2antibodies inswine. And several mAbs were screened and identificated.
1. Prokaryotic expression and characterization of Cap protein
The ORF2gene, truncated nuclear localization signal (NLS) sequence, wasamplified by PCR from the recombinant plasmid pMD18T-ORF2. The purified PCRproduct was digested with BamHI and HindIII, and cloned into the pET28a vector.Then the recombinant plasmid was transformed to Escherichia coli (E. coli) BL21competent cells. The positive clone was grown at37℃in Luria-Bertani (LB)medium supplemented with100μg/mL kanamycin to the optical density of0.6at600nm, and then isopropyl-β-D-thiogalactopyranoside was added to a final concentrationof1mM. After8h of induction at30℃, cells were harvested by centrigugation(8000rpm for30min) and resuspended in20mL100mM Tris-HCl (pH8.0). Thecells were disrupted by ultra-sonication on ice and centrifuged at12000rpm for25min. The supernatant were collected and purified using nickel-nitrilotriacetic acid(Ni-NTA) resin following the manufacturer’s protocol. SDS-PAGE showed a newprotein band with a molecular weight of26KD. Western blot indicated that it couldreact specifically with PCV2positive sera. In ELISA, the protein was demonstrated tobe of high bioactivity. Thus, Cap protein successfully expressed in E. coli could beused as a diagnostic reagent and provides a basis for the study of subunit vaccine.
2. Development and evaluation of an immunochromatographic strip for therapid detection of antibodies against Porcine circoviurs2
A rapid (less than5min) immunochromatographic strip using gold-antigen probewas successfully developed and applied in the detection of porcine circovirus2(PCV2) antibodies in swine. Recombinant Cap protein truncated nuclear localizationsignal of PCV2was expressed and labeled with colloidal gold. This conjugate wasdispensed on conjugate pad as the detector. The staphylococcal protein A (SPA) andpurified PCV2antibodies (porcine origin) were blotted on the nitrocellulosemembrane for the test and control lines, respectively. Sensitivity and specificity of thestrip was evaluated using PCV2antisera as well as other antisera from pigs infectedwith different swine viruses.500clinical swine serum samples were detected both by strip and the commercial ELISA kit. The agreement of immunochromatographic stripand ELISA kit was94.00%. It showed that this strip possesses high sensitivity andspecificity and may be useful for clinical laboratories and rapid diagnosis in the field.
3. Preparation and characterization of monoclonal antibody against PCV2
Antibodies against PCV2were elicited by immunizing Balb/c mice with purifiedPCV2. MAbs were prepared routinely by cell fusion. The above-developed strip wasused to screen hybirdoma lines. Five positive hybridoma lines of3C9,6A5,8F9,9F4,15E4were screened out. Yet only6A5,8F9,9F4could react with PCV2. The striptiter for these hybridoma lines were1:4×105,1:3×105,1:4×105, respectively. ThesemAbs possess high-specificity, show on cross reaction with other proteins or virusesand lay the foundation for the development of strip test for the rapid detection ofPCV2.
目前，PCV2诊断方法主要为PCR、免疫组化、原位杂交、免疫过氧化物酶单层试验（IPMA）、间接免疫荧光（IFA），这些方法主要用于抗原检测。PCV2抗体检测主要手段为ELISA，目前已有基于Cap蛋白的商品化ELISA试剂盒，但是ELISA检测方法需要专业人员和专门仪器，只适用于实验室检测。当前已经有商品化的猪圆环病毒疫苗用于猪群免疫，且免疫范围较大，因此急需一种简便、快速、敏感的检测方法用于免疫后抗体水平评价。
胶体金免疫层析技术是在胶体金标记技术和免疫层析技术基础上发展而来的，该技术具有简便、快捷的特点，同时不需要任何仪器和专业人员，因此被广泛应用于疫病及药物残留检测。本研究成功制备了PCV2抗体检测试纸，为PCV2抗体临床快速检测提供了有力工具。同时筛选并鉴定了数株PCV2单克隆抗体。
1.猪圆环病毒-Ⅱ型Cap蛋白的原核表达及功能鉴定
为获得具有免疫原性的PCV2Cap蛋白，本试验以含有PCV2ORF2基因的重组质粒PMD18T-ORF2为模板，利用PCR技术扩增得到PCV2ORF2基因片段，将此基因片段与原核表达载体pET28a连接，构建重组表达载体，命名为pET28a-ORF2，经PCR和测序鉴定后，用IPTG诱导ORF2基因的表达，收集诱导的菌液进行SDS-PAGE电泳分析。结果显示在分子量约为26KD处有一条明显的蛋白条带。表达产物上清用Ni柱纯化并透析，Western-Blot鉴定该蛋白能被PCV2阳性血清识别，ELISA检测结果显示蛋白具有较高的活性。研究表明PCV2Cap蛋白在大肠杆菌中得到高效表达，纯化获得的蛋白具有较高生物活性，为开发诊断试剂和亚单位疫苗奠定了基础。
2.猪圆环病毒-Ⅱ型抗体检测免疫层析试纸的研制及性能评价
将胶体金标记猪圆环病毒-Ⅱ型（PCV2）Cap蛋白喷涂于玻璃纤维膜上作为检测探针，金黄色葡萄球菌A蛋白(SPA)和PCV2多克隆抗体IgG固定在硝酸纤维膜上分别作为检测线和质控线，制备组装成免疫层析试纸。运用PCV2标准阳性、阴性血清及猪瘟病毒、蓝耳病病毒、细小病毒、伪狂犬病毒、PCV1阳性血清，对试纸进行特异性、敏感性评价。免疫层析试纸与进口商品化ELISA试剂盒对田间血清样品同时进行检测，评价试纸与ELISA试剂盒的符合率。试验结果显示，该试纸的特异性、敏感性良好，与进口ELISA试剂盒符合率为94.00%(470/500)，灵敏度与ELISA试剂盒相当。表明该试纸具有快速、特异、敏感等优点，操作简便，不需要专业人员与专门仪器，适合田间推广运用。
3.猪圆环病毒-Ⅱ型单克隆抗体的制备及鉴定
用纯化的PCV2免疫Balb/c小鼠，使小鼠对PCV2产生免疫反应并产生抗体，取血清效价较高的小鼠脾细胞与NS0瘤细胞融合。使用基于Cap蛋白的抗体检测试纸对大量杂交瘤细胞培养上清进行检测和鉴定，同时采用病毒阻断方法评价抗体与病毒的反应情况。最终筛选出5株分泌高亲和力单克隆抗体的杂交瘤细胞：3C9、6A5、8F9、9F4、15E4，其中6A5、8F9、9F4三株能与病毒反应。结果表明，该单抗的腹水能很好地识别PCV2病毒及其Cap蛋白，效价分别为：1:4×105、1:3×105、1:4×105。具有高度的特异性，与其他病毒和蛋白无交叉反应性。该单抗的成功制备，为PCV2抗原快速检测试纸条的制备奠定了基础。
Porcine circovirus type2(PCV2) is the main and primary causative agent ofPostweaning Multisystemic Wasting Syndrome (PMWS), which has causedsignificant economic losses in the swine industry. The genome of PCV2is1767bp or1768bp in length and consists of11ORFs. Three major ORFs have beencharacterized for PCV2: ORF1, ORF2, and ORF3. ORF1encodes a replication-associated protein (Rep), while ORF2encodes a major structural protein (Cap), themain antigenic determinant of the virus, and ORF3is involved in PCV2pathogenesis.Cap protein contains several immune-dominant epitopes and is confirmed to be type-specific.
To date, several commercial PCV2vaccines were under use in the market for theprevention and control of PCV2. Therefore, a method for specific serologic detectionin routine field practice to monitor PCV2antibody titers induced by vaccines isessential. Immunoperoxidase monolayer assay (IPMA), indirect immunofluorescentassay (IFA), and enzyme linked immunosorbent assay (ELISA) are the mostcommonly diagnostic methods for detecting PCV2antibodies. However, thesemethods require specialized equipment and technical expertise, and are suitable forlaboratory use only. Furthermore, IPMA and IFA require the cultivation of PCV2onPK-15cell, which is time-consuming and labor-intensive.
The membrane-based immunochromatographic lateral flow strip test represents awell-established and appropriate technique for a variety of point-of-care andfield-use applications, which is used in the detection of pathogen and durgs. Thistechnology has several advantages over traditional immunoassays, such as simplicityof procedure, rapid operation and immediate results, low cost, no requirements fortechnical expertise or specialized equipment. In this study, a simple and rapidimmunochromatographic strip was developed for the detection of PCV2antibodies inswine. And several mAbs were screened and identificated.
1. Prokaryotic expression and characterization of Cap protein
The ORF2gene, truncated nuclear localization signal (NLS) sequence, wasamplified by PCR from the recombinant plasmid pMD18T-ORF2. The purified PCRproduct was digested with BamHI and HindIII, and cloned into the pET28a vector.Then the recombinant plasmid was transformed to Escherichia coli (E. coli) BL21competent cells. The positive clone was grown at37℃in Luria-Bertani (LB)medium supplemented with100μg/mL kanamycin to the optical density of0.6at600nm, and then isopropyl-β-D-thiogalactopyranoside was added to a final concentrationof1mM. After8h of induction at30℃, cells were harvested by centrigugation(8000rpm for30min) and resuspended in20mL100mM Tris-HCl (pH8.0). Thecells were disrupted by ultra-sonication on ice and centrifuged at12000rpm for25min. The supernatant were collected and purified using nickel-nitrilotriacetic acid(Ni-NTA) resin following the manufacturer’s protocol. SDS-PAGE showed a newprotein band with a molecular weight of26KD. Western blot indicated that it couldreact specifically with PCV2positive sera. In ELISA, the protein was demonstrated tobe of high bioactivity. Thus, Cap protein successfully expressed in E. coli could beused as a diagnostic reagent and provides a basis for the study of subunit vaccine.
2. Development and evaluation of an immunochromatographic strip for therapid detection of antibodies against Porcine circoviurs2
A rapid (less than5min) immunochromatographic strip using gold-antigen probewas successfully developed and applied in the detection of porcine circovirus2(PCV2) antibodies in swine. Recombinant Cap protein truncated nuclear localizationsignal of PCV2was expressed and labeled with colloidal gold. This conjugate wasdispensed on conjugate pad as the detector. The staphylococcal protein A (SPA) andpurified PCV2antibodies (porcine origin) were blotted on the nitrocellulosemembrane for the test and control lines, respectively. Sensitivity and specificity of thestrip was evaluated using PCV2antisera as well as other antisera from pigs infectedwith different swine viruses.500clinical swine serum samples were detected both by strip and the commercial ELISA kit. The agreement of immunochromatographic stripand ELISA kit was94.00%. It showed that this strip possesses high sensitivity andspecificity and may be useful for clinical laboratories and rapid diagnosis in the field.
3. Preparation and characterization of monoclonal antibody against PCV2
Antibodies against PCV2were elicited by immunizing Balb/c mice with purifiedPCV2. MAbs were prepared routinely by cell fusion. The above-developed strip wasused to screen hybirdoma lines. Five positive hybridoma lines of3C9,6A5,8F9,9F4,15E4were screened out. Yet only6A5,8F9,9F4could react with PCV2. The striptiter for these hybridoma lines were1:4×105,1:3×105,1:4×105, respectively. ThesemAbs possess high-specificity, show on cross reaction with other proteins or virusesand lay the foundation for the development of strip test for the rapid detection ofPCV2.
引文
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