CN114874995A - 猪瘟病毒2型Erns蛋白的单克隆抗体杂交瘤细胞株及应用 - Google Patents
猪瘟病毒2型Erns蛋白的单克隆抗体杂交瘤细胞株及应用 Download PDFInfo
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Abstract
本发明属于兽用生物制品领域，本发明公开了一株猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1，其保藏编号为CCTCC NO：C 202247。该单抗可与CSFV2型Erns蛋白发生特异反应，不与牛病毒性腹泻病毒1型(BVDV1型)Erns蛋白反应。本发明适用于CSFV2型毒株感染猪与RecC‑M1或E2蛋白免疫猪血清抗体的鉴别检测，也可用于区分CSFV2型毒株感染猪与BVDV1型病毒感染猪血清抗体。
Description
技术领域
本发明涉及猪瘟病毒2型Erns蛋白特异性单克隆抗体细胞株的制备；基于该单抗建立阻断ELISA体系，用于区分猪瘟病毒2型野毒株感染猪的血清抗体和标记疫苗或E2蛋白亚单位疫苗免疫猪的血清抗体，属于兽用生物制品领域。
背景技术
猪瘟(classical swine fever，CSF)是感染家猪及野猪的重要病毒病，具有高传染性和高致病性的特点，是世界动物卫生组织(OIE)规定必需报告的疫病之一，该病由黄病毒科(Flaviviridae)瘟病毒属(Pestivirus)的猪瘟病毒(classical swine fever virus，CSFV)引起，在我国属一类动物传染病。
CSFV包含三个基因型(1型、2型和3型)，基因1型(CSFV1型)较为经典，而上世纪八十年代以来分离的毒株大多属于基因2型(CSFV2型)。CSFV弱毒C株(以下简称C株)是由CSFV1型强毒株在兔体连续传代致弱后得到，猪在接种C株后不会出现体温反应，仅出现轻微的病毒血症。C株被公认为安全有效的CSF疫苗，但不具备为相关检测试剂盒提供鉴别CSFV野毒感染血清抗体和疫苗C株免疫血清抗体的能力。国内生猪主产区的CSFV流行的毒株以CSFV2型为主，如QZ14株、HZ08株、HuN23株、HLJ1株等，CSFV2型等不同型毒株的流行可能影响基于基因1型的C株疫苗免疫保护效果。因此，具有鉴别能力、对基因2型等流行毒株的新型疫苗研制对更有效地控制CSF流行至关重要。
CSFV同属其他重要病原包括牛病毒性腹泻病毒1型及2型(Bovine ViralDiarrhea Virus，BVDV；其1型和2型分别简称BVDV1和BVDV2)，它们与CSFV在血清学上有一定的交叉反应性。我国不少省份有猪感染BVDV的报道，主要可能为BVDV1型毒株，BVDV2型鲜有报道。CSFV和BVDV囊膜蛋白Erns均由227个氨基酸残基组成，具有高保守性，其上有中和表位，能产生的中和抗体，可用于开发基因工程亚单位疫苗。CSFV和BVDV的Erns之间同源性较高，但在特定区域有一定差异，可能是两者互相鉴别的靶点；在新型标记疫苗研制中，具有鉴别疫苗免疫动物和野毒感染动物的潜力。由欧盟多个科研机构联合研发的重组疫苗CP7_E2alf及其配套检测体系是研究较为深入的猪瘟标记疫苗，已获欧洲药品管理局许可(商品名
CSF Marker)。CP7_E2alf的骨架为BVDV1型CP7株，通过反向遗传学方法将其E2基因替换为CSFV Alfort/187毒株的相应区域构建而成，具有较好的免疫原性。此重组疫苗的弊端在于，虽然目前已有与之相配套的以BVDV1型Erns为靶点的血清学鉴别检测体系，但由于其与CSFV血清存在一定的交叉反应性，限制了其适用性。同时，由于该疫苗株基因组中包含了CSFV强毒株Alfort/187的基因组分，使得重组疫苗与野毒株之间有可能通过同源或非同源重组方式突变为强毒株，有潜在的生物安全隐患。
酶联免疫吸附试验(Enzyme-linked Immunosorbent Assays,ELISA)主要用于抗体检测，广泛应用于临床，快速便捷且灵敏度高，不需要特殊的仪器设备。目前CSFV抗体检测的商品化试剂盒多采用间接ELISA或阻断ELISA方法，其中阻断ELISA主要基于纯度较高的特异性单克隆抗体，与特定的包被抗原结合，可显著提高检测的特异性，减少间接ELISA方法可能出现的非特异性反应。
本实验室将CSFV弱毒C株基因组中的Erns置换为BVDV1型的Erns，E2基因对应区域置换为CSFV2型毒株QZ14的E2高变区，利用反向遗传系统，获得了基于弱毒C株的重组标记疫苗候选株RecC-M1(授权专利号ZL201910551633.4)。
发明内容
本发明要解决的技术问题是提供一株针对猪瘟病毒(classical swine fevervirus,CSFV)2型(CSFV2型)Erns蛋白的单克隆抗体杂交瘤细胞株9B1及其应用。
为解决上述技术问题，本发明提供一株猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1，其保藏编号为CCTCC NO：C 202247。
说明：猪瘟病毒2型(CSFV2型)Erns蛋白的杂交瘤细胞株9B1，是一株分泌抗CSFV2型Erns蛋白单克隆抗体的杂交瘤细胞株9B1(简称单抗9B1)。
作为本发明的猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1的改进：该细胞株分泌的单抗属于κ型轻链的IgG1亚型。
本发明还同时提供了上述猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1构建方法：以CSFV2型Erns蛋白免疫小鼠，取其脾细胞与杂交瘤细胞SP2/0融合和筛选获得。
作为本发明构建方法的改进：
以CSFV2型毒株cDNA为模板，用一对特异引物PCR扩增获得Erns蛋白编码区序列；引物对如SEQ ID NO.1、SEQ ID NO.2所示；
Erns蛋白编码区cDNA序列如SEQ ID NO.3所示；
将Erns蛋白编码区序列克隆于载体pFastBacHTB，获得重组转移质粒pFastBac-Erns；pFastBac-Erns转座至E.coli DH10Bac感受态细胞，获得重组杆粒Bacmid-Erns；该重组杆粒转染Sf9细胞，获得表达CSFV2型Erns蛋白的重组杆状病毒，其表达产物简称CErns(“C”表示CSFV)。
本发明提供的猪瘟病毒2型Erns截短蛋白CtErns表达载体的构建方法及其表达为：
以原核表达质粒pET-30a(+)为载体，构建CSFV2型Erns蛋白截短片段编码区与GST(谷胱甘肽S-转移酶)编码区串联的重组质粒，经IPTG(异丙基硫代半乳糖苷)诱导表达，用镍柱纯化，获得CSFV2型Erns蛋白截短片段与GST的融合蛋白CtErns(“C”表示CSFV；“t”表示截短，以区别于Erns为全长的CErns)。
本发明构建所得的融合蛋白CtErns：
融合蛋白CtErns中的Erns蛋白截短片段的第104至170位氨基酸(aa104～170)，如SEQ ID NO.15所示。
本发明还同时提供了上述猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1的用途：
单抗9B1(保藏编号为CCTCC NO：C 202247的猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1所分泌)与融合蛋白CtErns发生特异反应，但不与牛病毒性腹泻病毒1型(BVDV1型)Erns蛋白反应。
作为本发明用途的改进：
单抗9B1识别CSFV2型Erns蛋白的第119-122位氨基酸区域为119Asn-Thr-Asp-Val122或119Asn-Ala-Asp-Val122；BVDV1型Erns蛋白该区域序列为119Asp-Ser-Asp-Leu122，所以单抗9B1不与其反应。
本发明还同时提供了一种检测CSFV2型感染猪血清抗体的阻断ELISA试剂盒，包括以下：
单抗9B1的辣根过氧化物酶(HRP)标记抗体HRP-9B1，包含CSFV2型Erns截短片段aa104～170的CtErns蛋白及其预包被的酶标板(0.5μg/孔)。
实际使用时：待检猪血清按1:4稀释，与等体积HRP-9B1(1:4000稀释)混合。
作为本发明的阻断ELISA试剂盒的改进：
该阻断ELISA试剂盒检测CSFV2型毒株感染猪的血清抗体为阳性，而CSFV标记疫苗候选株RecC-M1免疫猪的血清抗体为阴性(RecC-M1是以CSFV弱毒C株为骨架，将其Erns蛋白编码区置换为BVDV1型Erns蛋白编码区所获得的嵌合弱毒，授权专利号ZL201910551633.4)；CSFV E2蛋白亚单位疫苗免疫的猪血清，该阻断ELISA方法检测为阴性，而CSFV2型感染猪的血清抗体则为阳性。
说明：该阻断ELISA试剂盒用于区分CSFV2型野毒感染血清和疫苗免疫血清。
因此，本发明筛选得到了一株稳定分泌抗CSFV2型Erns蛋白的杂交瘤细胞株9B1，基于该单克隆抗体建立了与猪瘟标记疫苗候选株RecC-M1或CSFV E2亚单位疫苗配套的阻断ELISA检测体系，可用于CSFV2型毒株感染猪与RecC-M1或E2蛋白免疫猪血清抗体的鉴别检测，也可用于区分CSFV2型毒株感染猪与BVDV1型病毒感染猪血清抗体。
本发明中：
构建表达CSFV2型Erns蛋白重组杆状病毒，感染Sf9细胞获得CSFV2型Erns蛋白，以该蛋白免疫BALB/c小鼠，通过细胞融合和亚克隆筛选，获得了一株分泌抗CSFV2型Erns蛋白的单克隆抗体杂交瘤细胞株9B1，该细胞株分泌的单克隆抗体(以下简称“单抗9B1”)可与CSFV2型Erns蛋白发生特异性反应，不与牛病毒性腹泻病毒1型(BVDV1型)Erns蛋白反应。该细胞株已于2022年2月22日送交湖北省武汉市武昌区武汉大学中国典型培养物保藏中心，CCTCC NO:C202247。
鉴定了单抗9B1的识别表位：通过分析Erns蛋白序列，将单抗识别位点定位在第119-122位氨基酸，CSFV2型代表性毒株的序列为119NTDV122或119NADV122，而BVDV1型毒株的序列为119DSDL122。1)构建重组原核表达质粒，表达并纯化获得了CSFV2型Erns aa104～170(第104～170位氨基酸)与谷胱甘肽S-转移酶(GST)的融合蛋白(以下简称CtErns)。2)对CSFV2型119NTDV122这4个位点逐个进行丙氨酸置换，探明单抗9B1识别的抗原表位位于CSFV2型Erns蛋白第119～122氨基酸区域，其中第121位天冬氨酸是关键位点，其次是第122位缬氨酸和119位天冬酰胺。
使用辣根过氧化物酶(HRP)标记单抗9B1(以下简称“HRP-9B1”)，以CtErns作为包被抗原，建立了阻断ELISA体系，以该体系检测CSFV2型毒株感染猪血清、BVDV1型人工感染猪血清和两种免疫血清(猪瘟标记疫苗候选株RecC-M1和E2亚单位疫苗)。结果表明，HRP-9B1对CSFV2型毒株感染血清的阻断率较高(65％以上)，对RecC-M1和E2亚单位疫苗免疫血清以及BVDV1型感染血清的阻断率较低(30％以下)。该阻断ELISA体系可以与RecC-M1标记疫苗或E2蛋白亚单位疫苗配合，适用于CSFV2型毒株感染猪与RecC-M1或E2蛋白免疫猪血清抗体的鉴别检测，也可用于区分CSFV2型毒株感染猪与BVDV1型病毒感染猪血清抗体。
综上，本发明用CSFV2型Erns蛋白免疫BALB/c小鼠，通过细胞融合和亚克隆筛选，获得了一株分泌CSFV2型Erns蛋白特异性单克隆抗体杂交瘤细胞株9B1。该单抗可与CSFV2型Erns蛋白发生特异反应，不与牛病毒性腹泻病毒1型(BVDV1型)Erns蛋白反应；单抗9B1识别位点在CSFV2型Erns蛋白第119-122位氨基酸(119NTDV122或119NADV122)，其中第121位天冬氨酸是关键位点，其次是第122位缬氨酸和119位天冬酰胺。使用辣根过氧化物酶标记单抗9B1，以CSFV2型Erns蛋白截短片段(第104-170位氨基酸)作为包被抗原，建立了阻断ELISA体系，可以与RecC-M1标记疫苗或E2蛋白亚单位疫苗配合，适用于CSFV2型毒株感染猪与RecC-M1或E2蛋白免疫猪血清抗体的鉴别检测，也可用于区分CSFV2型毒株感染猪与BVDV1型病毒感染猪血清抗体。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为Sf9细胞感染重组杆状病毒的形态观察及免疫荧光(IFA)鉴定图；其中，左列为带有CSFV2型Erns基因的重组杆状病毒穿梭质粒转染Sf9细胞，右列为空载体转染Sf9细胞对照；上两张图为转染72h后的Sf9细胞形态观察，下两张图为转染72h后Sf9细胞的荧光检测结果。
图2为CSFV2型Erns真核表达蛋白的免疫印迹鉴定图；泳道1～泳道5均为含300mM咪唑的50mM PBS目的蛋白洗脱液。
图3为单克隆抗体9B1亚类鉴定结果。
图4为单克隆抗体9B1与真核表达蛋白BVDV1型Erns(BErns)及CSFV2型Erns(CErns)的反应性鉴定图。
图5为单克隆抗体9B1纯化前后的PAGE电泳鉴定。
图6为NCBI数据库中不同来源CSFV2型和BVDV1型Erns蛋白氨基酸序列对比。
图7为CSFV2型Erns肽段aa104-170与GST融合蛋白(CtErns)及其1-4#突变蛋白的PAGE电泳鉴定。
图8为单克隆抗体9B1对CSFV2型CtErns蛋白及其1-4#突变蛋白反应性比较(即单抗9B1的Erns蛋白识别位点鉴定)；其中，A为免疫印迹结果，B为ELISA结果；“**”表示与CtErns相比，P<0.01。
图9为抗原最适包被浓度和HRP标记9B1单抗最适工作浓度的优化。
图10为阻断ELISA针对不同来源血清的特异性检测结果；QZ14-1～QZ14-3、HZ08-1和HZ08-2为野毒株感染猪血清，RecC-M1～RecC-M5为标记疫苗株免疫猪血清，CSFV-E2-1～CSF-E2-3为E2蛋白亚单位疫苗免疫猪血清，BVDV1-1和BVDV1-2为BVDV1型人工感染猪血清；
图10中，A为间接ELISA检测血清与CSFV2型E2或BVDV1型E2蛋白的反应性，B为间接ELISA检测血清与CSFV2型Erns或BVDV1型Erns蛋白的反应性；C为阻断ELISA检测血清与CSFV2型CtErns蛋白的反应性。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1、CSFV2型Erns重组蛋白的制备
1.携带CSFV2型Erns蛋白编码区的重组杆状病毒质粒构建
将CSFV2型毒株QZ14的Erns蛋白编码区序列克隆于pFastBacHTB，得到重组转移载体，转座于E.coli DH10Bac受体菌获得重组杆粒：
(1).载体酶切：选用BamHI和HindIII作为酶切位点，对pFastBacHTB空载体进行双酶切，酶切体系按照表1配置，37℃孵育2h。酶切产物按OMEGA的PCR产物回收试剂盒进行纯化，回收产物用NanoDrop1000进行浓度测定。
表1、pFastBacHTB载体酶切体系
(2).CSFV2型Erns蛋白编码区序列的PCR扩增和一步克隆：先用特异引物从CSFV2型QZ14毒株反转录和PCR扩增获得其Erns蛋白编码区序列的cDNA(SEQ ID NO.3，以下简称Erns片段)，所用引物序列(SEQ ID NO.1和SEQ ID NO.2)如下：
F：GGGCGCCATGGGATCCGAGAATATAACTCAATGGAACCTGAGTG
R：ACTTCTCGACAAGCTTGGCATAGGCACCGAACCAG
(3).扩增产物纯化与重组杆状病毒转移质粒构建：用1％琼脂糖凝胶电泳鉴定步骤(2)所得的目的产物，呈现约700bp的单一目的条带；用OMEGA产物纯化试剂盒Cycle-pureKit对PCR产物进行纯化和回收，具体操作步骤见说明书。包含Erns片段的回收产物用NanoDrop1000进行浓度测定。将步骤(1)所得的双酶切线性化的pFastBacHTB与回收的Erns片段进行一步克隆，体系如表2，反应条件为37℃水浴30min后冰上静置5min，获得的一步克隆产物即为重组杆状病毒转移质粒(简称pFastBac-Erns)。
表2.一步克隆的连接体系
(4).重组转移质粒pFastBac-Erns转化与鉴定：将感受态细胞E.coli DH5α从-80℃取出，置于冰浴上融化后，加入20μL一步克隆产物(pFastBac-Erns)，轻轻混匀，置于冰浴上30min。反应完毕后42℃水浴热击60s，立即将热击后的EP管置于冰上2min。加入900μL无抗性LB液体培养基，置于37℃摇床180rpm震荡培养60min；5000rpm离心5min，弃掉900μL上清液，用剩余培养基重悬菌体沉淀，均匀涂布于含50μg/mL氨苄青霉素的LB平板上，37℃静置培养过夜。挑取数个单菌落分别于含50μg/mL氨苄青霉素的LB液体培养基中，37℃180rpm振荡培养，待菌液浑浊后用pFastBacHTB通用引物进行菌液PCR鉴定(说明：菌液PCR产物与6×Gel loading dye混合后进行核酸电泳，得到大小约为2800bp[实际为2825bp]的单一条带，此菌液鉴定为阳性菌液)，鉴定阳性菌液送测序，序列正确后将菌液保存。
(5).携带CSFV2型Erns片段的重组杆状病毒穿梭质粒制备：将测序正确的pFastBac-Erns转座至E.coli DH10Bac感受态细胞，置于37℃摇床180rpm震荡培养5h；取培养物50μL均匀地涂布于预先制备好的蓝白斑选择性培养基制备上。配置方法：LB琼脂中加终浓度为50μg/mL卡那霉素、7μg/mL庆大霉素和10μg/mL四环素(以下简称“三抗”)，琼脂凝固后在37℃培养箱倒置放置适当时间，将100μL(20mg/mL)X-gal和5μL(1mol/L)IPTG混合后均匀涂在板上，将琼脂板置于37℃下培养48h。挑取数个白色单菌落分别于含三抗的LB液体培养基中，37℃180rpm振荡培养，待菌液浑浊后用通用引物M13(-40)进行PCR鉴定；鉴定正确的菌液再次用制备好的三抗LB平板进行菌落划线纯化，获得携带CSFV2型Erns片段重组杆状病毒穿梭质粒(重组杆粒，简称Bacmid-Erns)。
2.表达CSFV2型Erns蛋白的重组杆状病毒制备
(1).重组杆粒Bacmid-Erns转染：挑取上述鉴定正确的阳性菌落至含三抗的LB液体培养基中，37℃180rpm培养过夜，抽提Bacmid-Erns备用。将Sf9细胞于28℃条件下贴壁培养在多个25-cm2细胞培养瓶中，培养基为含1％Antibiotic-Antimycotic(100x)的Sf-900TMIII SFM培养基，当单层细胞密度达到80％～90％(总数达到约5×106个)时进行转染。取Bacmid-Erns2μg加入200μL不含血清的Sf-900TM III SFM培养基中，吹打混匀，获得A液；另取6μL
II Reagent加入另一份200μL不含血清的Sf-900TM III SFM培养基中，振荡混匀，，获得B液；将A液与B液混合，室温静置30min。将此转染混合液逐滴加入弃去培养基的上述细胞培养物中，后补充1mL不含血清的Sf-900TM III SFM培养基，28℃培养5h，每半小时轻轻晃动液体使其与细胞接触均匀；吸出转染混合物，加入5mL完全培养基SF900Ⅲ。
(2).重组杆状病毒获取：上述转染后的Sf9细胞28℃培养，直至细胞呈囊泡状(通常在转染后72～96h，细胞生长停滞、颗粒状外观、有从培养皿解离的倾向)，直接收集含有病毒的培养基，转移至无菌的15mL离心管中，4000rpm离心收集上清，此为P1代重组杆状病毒，分装至-80℃保存；吸取500μL P1代杆状病毒感染一个1瓶新的T75 Sf9细胞，72h收获P2代重组杆状病毒；以此类推，收获P3代重组杆状病毒。
(3).表达CSFV2型Erns蛋白的重组杆状病毒IFA鉴定：将重组杆状病毒P2代病毒接种于Sf9细胞贴壁培养24孔板的孔中，培养48h，同时设立空白对照；用磷酸缓冲盐溶液(PBS)洗1次，吸干；每孔加入80μL 80％的丙酮(-20℃预冷)，-20℃固定20min，吸干；用PBS洗3次，吸干；每孔加入500μL按1:1000稀释(稀释液为含0.5％脱脂奶粉的PBS)鼠抗His单抗，37℃作用1h。用PBS洗2次，吸干。每孔加入500μL按1:1500稀释的Alexa Fluor 555标记的羊抗鼠IgG，37℃避光作用1h。用PBS洗3次，吸干，倒置荧光显微镜下观察荧光并拍照。如图1所示，重组杆状病毒感染Sf9细胞后，其细胞形态与正常Sf9细胞形态相比有明显差异，如细胞生长减缓、散在细胞数增加等，部分细胞膜不完整，表明重组杆状病毒感染。感染重组杆状病毒的Sf9细胞被抗His抗体特异识别(细胞呈现明显的荧光)，而未感染的对照细胞无荧光，表明带His标签的目的蛋白获得了表达。
3.CSFV2型Erns蛋白表达与纯化
将3个铺满75-cm2细胞培养皿的Sf9细胞转入含有200mL Sf-900TM III SFM完全培养基的1L容量的玻璃三角烧瓶中，27℃培养箱中培养至细胞密度为1.2×106/mL；根据重组杆状病毒滴度，以感染比(MOI)0.5的比例接种到悬浮培养的Sf9细胞中，继续于28℃下135rpm振荡培养96h收获细胞，10000rpm 5min得到上清和细胞沉淀，目的蛋白即在上清中。吸取1.5mL镍柱凝胶颗粒加入纯化柱空柱，用5个柱床体积的50mM PBS平衡镍柱；重复以上平衡镍柱步骤1次；将平衡去除乙醇残留后的镍柱凝胶颗粒加入蛋白样品，在试管翻转混合器上4℃结合过夜；将镍柱结合后的蛋白样品加入纯化柱空柱，打开控速阀开关使液体流出，收集流穿液；用30ml含10mM咪唑的50mM PBS洗涤杂蛋白，流速先慢后快，收集洗涤液；用5mL含300mM咪唑的50mM PBS分次洗脱目的蛋白，收集洗脱液，BCA法测定蛋白浓度；用His抗体进行免疫印迹鉴定：结果如图2所示，获得CSFV2型Erns真核表达蛋白(以下简称为CErns，“C”代表CSFV)，大小约为36kDa。
实施例2、CSFV2型Erns蛋白单克隆抗体杂交瘤细胞株的制备
1.动物免疫
取6周龄的BALB/C雌性小鼠3只，将实施例1获得的真核表达纯化CSFV2型Erns蛋白与等量弗氏完全佐剂混合乳化后，按200μg/只的剂量在小鼠背部多点注射；首次免疫后14天和28天分别用与等体积弗氏不完全佐剂混合乳化的抗原皮下多点注射，免疫剂量为每次100μg；酶标板用CErns(5μg/mL，即0.5μg/孔)作为抗原包被，首次免疫后第35天小鼠眶下静脉采血，分离获得血清进行倍比稀释作为一抗，以1:10000稀释的HRP-羊抗鼠IgG作为二抗，用间接ELISA方法测定小鼠血清效价，同时设置阴性对照。取效价高于1:8000的小鼠腹腔注射CSFV2型Erns蛋白100μg，3天后，取脾细胞进行融合。
2.脾细胞制备
将免疫完全的小鼠吸入麻醉后，摘眼球采血(后续作为ELISA阳性对照)后脱颈致死；将尸体在75％酒精中浸泡2min，取出后仰卧固定于干净泡沫板上，放入生物安全柜；75％酒精清洁小鼠体表，从腹中线靠右处用灭过菌的剪刀镊子依次剪开皮肤、肌肉层和腹膜，暴露脾脏；另取无菌剪刀和镊子分离脾脏，置于含RPMI 1640的无菌培养皿中，分离脾脏表面结缔组织和脂肪，用新的RPMI 1640润洗脾脏3次；取无菌200目铜网于直径100-mm的无菌培养皿中，RPMI 1640浸润网面，将分离好的脾脏置于其上，用无菌平底试管碾磨脾脏，并不断用RPMI 1640冲洗，直至脾脏被碾碎，滤过铜网；用RPMI 1640将铜网冲洗干净；收集培养皿中脾脏细胞悬液于无菌圆底离心管中，室温1200rpm离心10min，沉淀用20mL RPMI1640重悬待用。
3.细胞融合
取2个75-cm2细胞培养瓶对数生长期的小鼠骨髓瘤细胞SP2/0，约为脾细胞数目的1/5～1/10，弃去培养基，用RPMI 1640润洗一遍后各用5mL RPMI 1640将贴壁细胞重悬。收集SP2/0悬液于另一个无菌圆底离心管，28℃1200rpm离心10min，弃去上清，沉淀用20mLRPMI 1640重悬，反复洗2次，弃上清。将收集的脾细胞与SP2/0混合，室温1200rpm离心10min，弃去上清，轻轻敲击圆底离心管底部使细胞混匀至稀泥状。将离心管置于含37℃无菌水的烧杯中水浴保持温度，1min内逐滴加入提前在37℃预温的1mL 50％PEG1450，边滴加边晃动使细胞充分混匀，静置30s，梯度稀释终止融合：即分步加入提前于37℃预温的不同体积RPMI 1640(分别为1mL、2mL、3mL、4mL、5mL和6mL)，每个体积在1min内加完，边加边晃动，每次加液间静置30s；28℃1200rpm离心10min，弃去上清，用100mL HT筛选培养基(含RPMI1640、20％灭活新生牛血清、1％Antibiotic-Antimycotic以及1％HAT)重悬细胞沉淀，均匀铺至10块96孔细胞培养板中。
4.间接ELISA筛选阳性克隆
(1).阳性单克隆细胞株的筛选：当融合成功后的杂交瘤细胞分裂聚集约达到孔底面积的1/3时(8-10天)，取杂交瘤上清进行ELISA筛选。阳性克隆筛选的体系为包被CErns的间接ELISA：抗原包被浓度为1μg/mL；一抗为杂交瘤细胞培养上清，二抗为HRP羊抗鼠IgG(1:10000稀释使用)，阳性对照为小鼠血清200倍稀释，阴性对照为1640筛选培养基，以P/N＞2.1为阳性，筛选含阳性杂交瘤细胞的孔。选取OD450nm读数较大的数十个孔扩增并进行后续单克隆化，具体策略为混匀阳性孔细胞并计数，取约100个细胞至10mL的HT筛选培养基铺一块96孔细胞培养板，观察并记录每孔细胞克隆数。2周后取培养上清进行间接ELISA检测，标记阳性细胞孔，如此进行3轮筛选，获得疑似阳性单克隆杂交瘤细胞株。
(2).阳性单克隆细胞株的鉴定：细胞计数后取约60个细胞铺一块96孔细胞板，2周后取上清进行ELISA检测，若所有含细胞的孔均为阳性，则此细胞株为单克隆杂交瘤。取此细胞株培养上清进行CSFV2型Erns的特异性鉴定，即酶标板分别包被CErns和BErns(后者为本实验室保存的携带His标签的BVDV1型Erns重组杆状病毒表达产物，BVDV1型Erns序列参考GenBank上发表的VEDEVAC株[GenBank：KC695814.1]，BErns中的“B”表示BVDV，以区别于CErns)，杂交瘤细胞培养上清作为一抗分别与这两种蛋白进行间接ELISA，目的是排除与BErns和His标签的交叉反应性。只与CErns反应，不与携带His标签的BErns反应，则证明其为CSFV2型Erns特异性抗体，完成单克隆筛选过程。继续通过2轮筛选，最终获得一株稳定分泌CSFV2型Erns蛋白特异的单克隆抗体杂交瘤细胞株9B1，其分泌的抗体在本发明中简称单抗9B1。
该稳定分泌CSFV2型Erns蛋白特异的单克隆抗体杂交瘤细胞株9B1进行了保藏，保藏信息如下：
保藏名称：猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1，保藏单位：中国典型培养物保藏中心，保藏地址：中国武汉武汉大学，保藏编号：CCTCC NO：C202247，保藏时间2022年2月22日。
5.单抗9B1腹水制备
取8周龄的雌性BALB/c小鼠，腹腔注射1mL无菌液体石蜡；7天后将对数生长期的杂交瘤细胞9B1腹腔注射小鼠，每只5×106；每日观察小鼠精神状况及腹部大小变化，7天后腹部明显膨大，采集腹水；腹水于4℃12000rpm离心10min，弃去上层油脂及下层细胞沉淀，澄黄色腹水分装于无菌试管，-20℃保存。
实施例3、单克隆抗体9B1的鉴定
1.单克隆抗体9B1的亚型测定
根据Proteintech抗体亚型鉴定试剂盒说明书对上述实施例所得到的单克隆抗体9B1进行鉴定。抗体亚型鉴定的原理是利用酶联免疫吸附实验(ELISA)，测定待测抗体与不同亚型特异性抗体(鉴定试剂盒中提供)的结合情况，鉴定出与待测抗体特异性结合的抗体，依据结合抗体的特异性可以得出待测抗体的亚型。鉴定方法：按照试剂盒说明，依据酶标仪检测吸光度来判断被测单克隆抗体的亚类，结果见图3，单抗9B1为κ型轻链的IgG1亚型。
2.免疫印迹鉴定
分别将CErns和BErns的真核表达蛋白与5×蛋白上样缓冲液按照4:1的体积混匀(蛋白上样量为0.5μg)，沸水浴中放置10min后进行SDS-PAGE电泳(电泳程序为150V，75min)；将分离的蛋白转印至硝酸纤维素膜上，经5％脱脂奶粉封闭后，加入杂交瘤细胞9B1的培养上清原液，37℃孵育1h，用含0.05％Tween-20的Tris盐缓冲液(TBST)漂洗3次；加入按1:5000稀释的HRP羊抗鼠IgG，37℃孵育1h，用TBST漂洗3次；按照FDbio-Pico ECL发光液说明书进行显色。结果如图4所示，9B1单抗只与CErns蛋白发生结合，在36kDa处出现特异性条带，而不与BErns蛋白结合，说明9B1只与CSFV2型Erns发生特异性反应。
说明：杂交瘤细胞9B1的培养上清原液的制备方法为：将杂交瘤细胞9B1接种至T-25细胞培养瓶中，细胞融合度达到80％时，用无菌枪头吸取细胞培养上清液，转移至15mL离心管中，4℃800rpm离心5min，将上清液分装至1.5-mL离心管中，于-80℃保存。
3.单克隆抗体的纯化
将“实施例2”所获9B1小鼠腹水为上海优宁维生物科技股份有限公司使用ProteinA/G法进行纯化，SDS-PAGE鉴定纯化前后的9B1抗体，用考马斯亮蓝染色液对蛋白胶进行染色，脱色后结果见图5。大约在分子量55kDa和25kDa处分别可见纯化后9B1抗体重链和轻链两个条带，符合预期的大小。
4.单抗9B1的表位鉴定
(1).CSFV2型Erns蛋白截短片段及其特定氨基酸突变蛋白表达载体构建：利用本实验室已有的CSFV2型Erns第1～110位氨基酸(aa1～110)、第50～150位氨基酸(aa55～150)和第100～207位氨基酸(aa100～207)(分别为SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6)三个不同截短片段原核表达产物作为包被抗原进行间接ELISA，发现单抗9B1不与aa1～100片段反应，而与aa55～150和aa100～207这两个片段反应，表明该单抗的识别区在aa101-150之间。利用DNASTAR MegAlign软件，分析了不同基因型CSFV毒株与BVDV1和BVDV2型毒株的差异氨基酸位点，初步确定第119～122位氨基酸为单抗9B1潜在的识别位点(图6)。以pET-30a(+)为载体(N端带6个His标签)，构建CSFV2型Erns蛋白第104～170位氨基酸片段的编码区与GST编码区串联的重组质粒(以下简称pCtErns)，即用特异性引物(pET30a-F/pET30a-R，SEQ ID NO.7和SEQ ID NO.8)扩增pET-30a(+)质粒获得线性化载体，再用特异引物(gst-F/gst-R，SEQ ID NO.9和SEQ ID NO.10)从携带gst基因的pGEX-4T-1商品化质粒作为模板进行PCR扩增，获得GST的编码基因，与线性化的pET-30a(+)经一步克隆获得重组质粒(以下简称pGST)，转化至感受态E.coli DH5α，筛选阳性克隆，扩大培养并抽提质粒用于后续亚克隆。用特异性引物(pGST-F/pGST-R，SEQ ID NO.11和SEQ ID NO.12)以pGST质粒为模板进行PCR扩增获得线性化的pGST，再从携带CSFV2型毒株QZ14 Erns蛋白编码区全长的质粒pFastBac-Erns中用特异性引物(Erns-F/Erns-R，SEQ ID NO.13和SEQ ID NO.14)扩增第104～170位氨基酸编码区序列(SEQ ID NO.15)，与线性化的质粒pGST经一步克隆后获得基于pET-30a(+)的重组质粒pCtErns(“C”表示CSFV；“t”表示截短，即Erns蛋白截短片段aa104～170)，转化至感受态E.coli DH5α，筛选获得的阳性克隆进行测序确认。再通过设计氨基酸单点突变的引物，以重组质粒pCtErns为模板进行PCR扩增引入第119～122位氨基酸的单点突变，线性化质粒在同源重组酶作用下自连成环，获得单点突变质粒，转化至感受态E.coliDH5α，筛选获得的阳性克隆进行测序确认。获得的第119～122位氨基酸单点突变质粒分别为pCtErns-1、pCtErns-2、pCtErns-3和pCtErns-4(表3)，即将CSFV2型Erns的119NTDV122相应位点的氨基酸逐个置换为丙氨酸(A)：N119A、T120A、D121A和V122A。重组质粒构建和点突变引物见表4(序列表中的SEQ ID NO.16～SEQ ID NO.23)。
表3.携带CSFV2型Erns蛋白截短片段编码区的突变质粒构建策略§
§Erns蛋白截短片段为Erns第104～170位氨基酸片段。
*三联密码子中携带下划线的碱基为点突变位点，将pCtErns中与“NTDV”对应的编码子逐一置换为丙氨酸编码子。
表4.携带CSFV2型Erns蛋白截短片段编码区与GST编码区串联的重组质粒和氨基酸单点突变质粒构建所用引物§
§Erns蛋白截短片段为Erns第104～170位氨基酸片段，单点突变位点见表3。
(2).CSFV2型Erns蛋白截短片段及其突变蛋白表达：将pCtErns、pCtErns-1、pCtErns-2、pCtErns-3和pCtErns-4重组突变质粒转化至感受态E.coli Rosetta，扩大培养至OD600nm＝0.6，加入终浓度为1mM的IPTG诱导，离心收集菌体；用20mL 50mM PBS重悬，按照超声4s，间隔4s的程序超声30min；4℃10000rpm离心15min，取上清液(含携带His标签的目的蛋白)与Ni-NTA树脂4℃结合过夜，第二天上柱进行洗涤和纯化，获得CSFV2型Erns蛋白截短片段与GST的融合蛋白(以下简称CtErns，“C”表示CSFV；“t”表示截短，即Erns蛋白截短片段aa104～170)及其与突变质粒对应的1～4号突变蛋白(CtErns-1、CtErns-2、CtErns-3和CtErns-4)，并进行SDS-PAGE电泳验证，经考马斯亮蓝染色及脱色液处理后观察，纯化后的蛋白见图7，可用于后续相关试验。
(3).针对CSFV2型Erns蛋白单抗9B1识别位点鉴定：利用免疫印迹及ELISA法分析单抗9B1识别的关键氨基酸位点。免疫印迹：将上述收获的不同突变蛋白进行SDS-PAGE和转膜(条件同“实施例3”)，按1:1000稀释的纯化后单抗9B1作一抗(稀释液为含0.5％脱脂奶粉的TBST)，按1:5000稀释的HRP-驴抗猪IgG作为二抗，根据超敏显色试剂盒说明书进行显色。ELISA：利用上述收获的不同突变蛋白作为包被抗原，按1:2000稀释的纯化后单抗9B1作为一抗，按1:10000稀释的HRP-驴抗猪IgG作为二抗，TMB(3,3',5,5'-四甲基联苯胺)显色液进行显色，用酶标仪读取OD450nm值。
免疫印迹结果显示，与亲本蛋白CtErns比较，单抗9B1与CtErns-3(第121位D→A)突变蛋白不发生反应，与CtErns-1(第119位N→A)和CtErns-4(第122位V→A)突变蛋白反应性降低，而第120位突变(T→A，即CtErns-2突变蛋白)则不影响与单抗9B1的反应性(图8A)。ELISA检测表明，单抗9B1与CtErns-3和CtErns-4突变蛋白的反应性显著低于CtErns，与CtErns-1的反应性略有降低，而与突变蛋白CtErns-2的反应性则高于CtErns(图8B)。综合上述两种方法的结果，单抗9B1识别的抗原表位位于CSFV2型Erns蛋白第119～122氨基酸区域，其中第121位天冬氨酸是关键位点，其次是第122位缬氨酸和119位天冬酰胺。结合CSFV2型Erns第120位氨基酸为苏氨酸或丙氨酸，第119、121和122位相同，即单抗9B1识别CSFV2型Erns的表位为119Asn-Thr-Asp-Val122(SEQ ID NO.24)或119Asn-Ala-Asp-Val122(SEQ ID NO.25)；第121位天冬氨酸在CSFV2型和BVDV1型毒株中保守，而BVDV1型Erns蛋白该区域序列为119Asp-Ser-Asp-Leu122，综合图4和图8结果，说明第122位氨基酸可能是单抗9B1差异识别CSFV2型及BVDV1型的关键氨基酸，第119位氨基酸也可能相关。
实施例4、基于CtErns蛋白和单抗9B1的阻断ELISA方法建立及应用
1.单克隆抗体9B1的标记
纯化的单抗9B1由上海优宁维生物科技股份有限公司进行辣根过氧化物酶(HRP)标记，标记后抗体(以下简称HRP-9B1)浓度为1mg/mL。
2.抗原最适包被浓度和标记单抗HRP-9B1最适工作浓度的确定
以“实施例3”的CtErns作为包被抗原，ELISA测定HRP-9B1的反应效价。采用方阵法，优化各反应条件：CtErns蛋白包被浓度为1.25、2.5、5和10μg/mL，HRP-9B1稀释度为1:1000、1:2000、1:4000、1:8000和1:16000，加入HRP-9B1后，37℃反应1h，PBST洗3次，用TMB显色10min，2M硫酸终止显色，OD450nm读数。图9所示，后续选择CtErns蛋白包被浓度5μg/mL，标记抗体HRP-9B1稀释比1:4000用于阻断ELISA方法的建立。
3.猪血清浓度优化
选择CtErns蛋白包被浓度5μg/mL(每孔100μL，即0.5μg/孔，下同)，HRP-9B1稀释比1:4000，分别将CSFV2型毒株QZ14攻毒两头猪所采集的第28天血清(待检血清)及一份CSFV阴性猪血清按1:2、1:4、1:8和1:16稀释，与稀释后的等体积HRP-9B1抗体液混合后孵育，37℃反应1h，PBST洗3次，每孔加入100μL TMB显色10min，2M硫酸终止显色。测定的OD450nm值，按如下公式计算：阻断率(％)＝(1–待检血清OD450nm)/阴性血清OD450nm。结果表明，单抗9B1能够阻断1:2、1:4和1:8稀释的CSFV2型毒株QZ14攻毒的猪血清与CtErns蛋白结合，阻断率均大于50％(表5)，因此以1:2或1:4稀释的猪血清稀释度可以用于阻断ELISA体系。
表5.单抗9B1对CSFV2型毒株QZ14攻毒猪血清不同稀释度的阻断率
按5μg/mL的浓度包被CtErns抗原，分别将两头猪在CSFV2型毒株QZ14攻毒后第7、14、21和28天所收获的血清及CSFV阴性猪血清按1:4稀释，与稀释后的等体积HRP-9B1抗体液混合，37℃孵育1h，PBST洗3次，加入TMB显色10min，2M硫酸终止显色。结果显示，该阻断ELISA体系可以检出CSFV2型毒株QZ14攻毒后第14天及以后的血清抗体(表6)。
表6.单抗9B1对CSFV2型毒株QZ14攻毒后不同时间点血清的阻断率
4.阻断ELISA的特异性检测
取15份猪血清(包含3份CSFV2型毒株QZ14感染、2份CSFV2型毒株HZ08感染、2份BVDV1型人工接种感染，5份RecC-M1标记疫苗株免疫，3份CSFV E2亚单位疫苗免疫)，1份CSFV/BVDV阴性猪血清(Negative)，采用上述建立的阻断ELISA体系进行检测(设立阴性血清与不含血清的单抗对照)。主要操作步骤如下：包被抗原CtErns浓度为5μg/mL，血清按1:4稀释，HRP-9B1按1:4000稀释，稀释的血清和单抗9B1等比例混合后37℃反应1h，PBST洗3次，TMB显色10min，2M硫酸终止显色，用酶标仪读取OD450nm值；结果如图10所示，15份血清与相应的包被抗原(CSFV和BVDV1的E2或Erns)的间接ELISA显示这些血清是对应病毒的阳性血清(图10A和10B)；仅CSFV2型毒株QZ14及HZ08攻毒猪血清的阻断率达65％以上，BVDV1型血清以及携带BVDV1型Erns的RecC-M1免疫猪血清和CSFV E2亚单位疫苗免疫血清的阻断率均小于30％(图10C)。
上述结果表明，本发明建立的基于CtErns蛋白和单抗9B1的阻断ELISA方法可以特异性检出CSFV2型毒株QZ14和HZ08感染的血清抗体，与BVDV1型感染的血清抗体以及RecC-M1和CSFV E2亚单位疫苗免疫的血清抗体不发生明显的竞争性阻断。RecC-M1是本实验室获得的CSFV标记疫苗候选株，是以猪瘟弱毒疫苗C株为骨架，将其基因组中的Erns置换为BVDV1型的Erns，其E2蛋白编码序列高变区置换为CSFV2型的E2高变区，利用反向遗传技术构建而成。该阻断ELISA体系可以与RecC-M1标记疫苗或E2蛋白亚单位疫苗配合，适用于CSFV2型毒株感染猪与RecC-M1或E2蛋白免疫猪血清抗体的鉴别检测，也可用于区分CSFV2型毒株感染猪与BVDV1型病毒感染猪血清抗体。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
序列表
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Claims (10)
1.猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1，其特征在于保藏编号为CCTCC NO：C202247。
2.根据权利要求1所述的猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1，其特征在于：该细胞株分泌的单抗属于κ型轻链的IgG1亚型。
3.如权利要求1所述的猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1构建方法，其特征在于：以CSFV2型Erns蛋白免疫小鼠，取其脾细胞与杂交瘤细胞SP2/0融合和筛选获得。
4.根据权利要求3所述的构建方法，其特征在于：
以CSFV2型毒株cDNA为模板，用一对特异引物PCR扩增获得Erns蛋白编码区序列；引物对如SEQ ID NO.1、SEQ ID NO.2所示；
Erns蛋白编码区cDNA序列如SEQ ID NO.3所示；
将Erns蛋白编码区序列克隆于载体pFastBacHTB，获得重组转移质粒pFastBac-Erns；pFastBac-Erns转座至E.coli DH10Bac感受态细胞，获得重组杆粒Bacmid-Erns；该重组杆粒转染Sf9细胞，获得表达CSFV2型Erns蛋白的重组杆状病毒，其表达产物简称CErns。
5.猪瘟病毒2型Erns截短蛋白CtErns表达载体的构建方法及其表达，其特征在于：
以原核表达质粒pET-30a(+)为载体，构建CSFV2型Erns蛋白截短片段编码区与GST编码区串联的重组质粒，经IPTG诱导表达，用镍柱纯化，获得CSFV2型Erns蛋白截短片段与GST的融合蛋白CtErns。
6.如权利要求5构建所得的融合蛋白CtErns，其特征在于：
融合蛋白CtErns中的Erns蛋白截短片段的第104至170位氨基酸，如SEQ ID NO.15所示。
7.如权利要求1或2所述的猪瘟病毒2型Erns蛋白的杂交瘤细胞株9B1的用途，其特征在于：
单抗9B1与融合蛋白CtErns发生特异反应，但不与牛病毒性腹泻病毒1型Erns蛋白反应。
8.根据权利要求7所述的用途，其特征在于：
单抗9B1识别CSFV2型Erns蛋白的第119-122位氨基酸区域为119Asn-Thr-Asp-Val122或119Asn-Ala-Asp-Val122；BVDV1型Erns蛋白该区域序列为119Asp-Ser-Asp-Leu122，所以单抗9B1不与其反应。
9.一种检测CSFV2型感染猪血清抗体的阻断ELISA试剂盒，其特征在于包括：
单抗9B1的辣根过氧化物酶标记抗体HRP-9B1，包含CSFV2型Erns截短片段aa104～170的CtErns蛋白及其预包被的酶标板。
10.根据权利要求9所述的阻断ELISA试剂盒，其特征在于：
该阻断ELISA试剂盒检测CSFV2型毒株感染猪的血清抗体为阳性，而CSFV标记疫苗候选株RecC-M1免疫猪的血清抗体为阴性；CSFV E2蛋白亚单位疫苗免疫的猪血清，该阻断ELISA方法检测为阴性，而CSFV2型感染猪的血清抗体则为阳性。
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| CN116165379A (zh) * | 2022-12-07 | 2023-05-26 | 浙江洪晟生物科技股份有限公司 | 一种猪瘟病毒鉴别检测试剂盒及其制备方法和应用 |
| CN116554282A (zh) * | 2023-04-17 | 2023-08-08 | 浙江大学 | 用于鉴别猪瘟病毒2型感染的单克隆抗体制备方法及应用 |
| CN117886906A (zh) * | 2024-01-22 | 2024-04-16 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一种猪瘟病毒Erns蛋白的抗原表位、单克隆抗体及其应用 |
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| CN116165379B (zh) * | 2022-12-07 | 2023-09-05 | 浙江洪晟生物科技股份有限公司 | 一种猪瘟病毒鉴别检测试剂盒及其制备方法和应用 |
| CN116554282A (zh) * | 2023-04-17 | 2023-08-08 | 浙江大学 | 用于鉴别猪瘟病毒2型感染的单克隆抗体制备方法及应用 |
| CN117886906A (zh) * | 2024-01-22 | 2024-04-16 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 一种猪瘟病毒Erns蛋白的抗原表位、单克隆抗体及其应用 |
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