CN110016466B - 特异性检测蓝舌病病毒的单抗及其杂交瘤细胞株和应用 - Google Patents
特异性检测蓝舌病病毒的单抗及其杂交瘤细胞株和应用 Download PDFInfo
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Abstract
本发明涉及一种杂交瘤细胞株BTV‑2A4及其分泌的单克隆抗体,及该单克隆抗体所识别的BTV‑NS2蛋白B细胞表位多肽,本发明提供的单克隆抗体与且仅与所述BTV‑NS2蛋白B细胞表位多肽特异性结合,经试验验证,与蓝舌病病毒的同属病毒,如中山病毒CHUV、鹿出血热病毒EHDV‑1及EHDV‑6、广西环状病毒GXOV等并不发生反应,因此在检测鉴定蓝舌病病毒感染时可靠程度较高,对疫病防治有较好的指导意义。本发明同时提供一种上述单克隆抗体制备的特异性检测蓝舌病病毒NS2蛋白的C‑ELISA试剂盒,由于使用了本发明的单克隆抗体,高度特异性地识别含有特定B细胞表位的抗原蛋白,因此检测结果可靠准确,操作简单,有利于疫病防治。
Description
技术领域
本发明涉及蓝舌病的基础研究及防治领域,具体涉及一种杂交瘤细胞株BTV-2A4及其分泌的单克隆抗体,及由上述单克隆抗体所识别的BTV-NS2蛋白B细胞表位多肽;本发明还涉及用于诊断1~24型蓝舌病病毒NS2抗体检测的C-ELISA试剂盒,及所述杂交瘤细胞株、单抗及B细胞表位多肽在蓝舌病病毒检测及预防上的应用。
背景技术
蓝舌病是由蓝舌病病毒(Bluetongue virus,BTV)引起的一种虫媒传播的动物疫病。该病毒主要感染牛、羊、鹿等反刍动物。发病特征为:发热、口腔充血及溃疡、舌充血发绀、蹄叉炎症,可造成跛行、新生动物畸形、流产、死亡。蓝舌病被世界动物卫生组织(OfficeInternational Des Epizooties,OIE)划定为动物A类传染病。
自19世纪在非洲发现首例蓝舌病以来,世界多地(南非、中东、美国、加拿大、南美洲、澳大利亚、欧洲、印度等)均有发生并偶有大规模爆发,给畜牧业造成严重经济损失。1979年在云南省师宗县发现国内首例蓝舌病,之后在湖北、安徽、四川、山西等地零星出现牛羊蓝舌病。
目前已知的BTV血清型共有27种,国内出现的血清型包括1、2、4、7、15、16、21等,其中以1型和16型最为常见。随着国际贸易(包括边境动物跨境走私)规模的扩大,国外BTV入境风险也随之增加。因此,对蓝舌病的诊断、预防、治疗的研究刻不容缓。
BTV是呼肠孤病毒科环状病毒属的一种dsRNA病毒。其基因组由10条不同的dsRNA组成,分别编码7种衣壳蛋白(VP1–VP7)及5种非结构蛋白(NS1–NS4以及NS3a);外围由两层衣壳包裹,通常情况下不含有包膜。BTV的非结构蛋白在不同血清型中具有高度保守性,其中NS2能形成二聚体及多聚体,并能与病毒RNA结合,与病毒的复制及包装密切相关。此外,目前的BTV疫苗以灭活病毒(不含NS2蛋白)为主,被免疫动物的体内不会产生NS2抗体。因此,动物血清是否含有NS2抗体,可作为鉴别被免疫动物是否被野生BTV感染的参考指标,从而对BTV灭活疫苗的效价评估有辅助作用。因此,研发BTV NS2的单克隆抗体,在BTV分子生物学基础研究、阻断NS2功能、鉴定BTV疫苗效价(开发基于NS2mAb的检测血清NS2抗体的ELISA试剂盒)等方面研究及应用具有重要价值。
BTV NS2蛋白是BTV的一种非结构蛋白,是BTV感染宿主细胞时合成的,NS2蛋白主要存在与被感染细胞的细胞核附近,在病毒的复制过程中起重要作用。在BTV流行地区,及时的防控疫病是关键,准确鉴别动物是否被野生BTV感染是指导疫病防治的关键。现有的针对研究BTV NS2抗体的检测技术,存在特异性不够强的缺陷,如中国专利201310478429.7公开了一种蓝舌病病毒NS2蛋白单克隆抗体BTV-4D4及其识别的B细胞表位和应用,为检测蓝舌病疫区动物是否被野生毒株感染提供了一种检测途径,但是在实践中发现,该方法特异性不够强,尤其是与鹿流行性出血热病毒感染容易误判,耽误了疫区防治蓝舌病的时机,而误判的根源在于该专利公开的单克隆抗体BTV-4D4所识别的B细胞表位与EHDV的NS2蛋白相应区段同源性较高。因此,在病毒检测领域,精确找出与且仅与BTV NS2蛋白产生特异性结合的单克隆抗体,才能开发出相应的检测工具,为疫病防治提供依据。
发明内容
为解决上述问题,本发明提供了一种分泌抗蓝舌病病毒NS2蛋白单克隆抗体的杂交瘤细胞株,本发明同时提供了一个蓝舌病病毒NS2蛋白的B细胞表位多肽;本发明提供的杂交瘤细胞株BTV-2A4分泌的抗蓝舌病病毒NS2蛋白的单克隆抗体,且仅与本发明提供的蓝舌病病毒NS2蛋白的B细胞表位多肽发生特异性结合,从而为精确检测BTV NS2蛋白提供了依据。
本发明提供的分泌抗蓝舌病病毒NS2蛋白单克隆抗体的杂交瘤细胞株,命名为BTV-2A4,保藏在中国典型培养物保藏中心,菌种保藏编号:CCTCC NO:C2018232。
上述杂交瘤细胞株所分泌抗蓝舌病病毒NS2蛋白的单克隆抗体。
一个与上述单克隆抗体发生特异性结合的蓝舌病病毒NS2蛋白的B细胞表位多肽,所述表位多肽的氨基酸序列在91~138区间内,所述氨基酸序列如SEQ ID NO.4所示。
进一步,上述抗蓝舌病病毒NS2蛋白的单克隆抗体在蓝舌病病毒诊断或检测中的应用。
进一步,上述蓝舌病病毒NS2蛋白B细胞表位多肽在制备诊断或检测蓝舌病病毒感染中的应用。
本发明同时提供一种以上述单克隆抗体制备的特异性检测蓝舌病病毒NS2蛋白的C-ELISA试剂盒,所述C-ELISA试剂盒包括酶标板,还包括上述的抗蓝舌病病毒NS2蛋白的单克隆抗体,及抗鼠的酶标二抗、阳性对照、阴性对照、稀释液、TMB显色液、洗涤液和终止液。
进一步,所述C-ELISA试剂盒的酶标板由抗原蛋白包被,所述抗原蛋白为细菌TrxA蛋白与蓝舌病病毒NS2蛋白片段的融合蛋白,其氨基酸序列如SEQ ID NO.3所示;所述蓝舌病病毒NS2蛋白片段为1型蓝舌病病毒Seg8基因编码的1~228位氨基酸序列,其氨基酸序列如SEQ ID NO.1所示。
进一步,所述酶标二抗为HRP标记的羊抗鼠IgG,所述所述阳性对照为自然感染蓝舌病病毒1型的BTV抗体阳性血清;所述阴性对照为蓝舌病病毒抗体阴性的血清;所述稀释液为含有5%(W/V)脱脂奶的PBS缓冲液;所述洗涤液为PBS缓冲液。
进一步,所述包被酶标板的TrxA-NS2抗原蛋白为经过钴离子树脂纯化的多肽,其包被量为100ng/孔;所述单克隆抗体的稀释倍数为160倍;所述酶标二抗的稀释倍数为2000倍;孵育时间均为1h,TMB显色时间为10min。
进一步,所述C-ELISA试剂盒,待测血清用量为25μl/孔。
本发明是通过以下技术方案来实现的:
本发明采用Trizol法提取BTV-1总RNA,逆转录获得cDNA;通过高保真PCR克隆NS2基因的1~864bp的DNA片段;经双酶切连接插入pET-32a质粒,从而构建出能表达含有BTVNS2蛋白高保守区域(N端1~228AA)的融合蛋白其氨基酸序列如SEQ ID NO.1所示。通过原核表达获得NS2蛋白1~228AA与细菌TrxA的融合蛋白,原核表达载体中编码TrxA-NS2融合蛋白的核苷酸序列如SEQ ID NO.2所示,利用融合蛋白的6×His标签,通过TALON树脂富集及咪唑溶液洗脱,获得高纯度目的蛋白。透析除咪唑后的融合蛋白用于免疫Balb/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合。经96孔板有限稀释传代培养分离单克隆细胞株及ELISA鉴定,最终获得一株稳定分泌抗BTV NS2单克隆抗体的杂交瘤细胞株,命名为BTV-2A4,保藏在中国典型培养物保藏中心(地址:武汉市武昌区八一路299号武汉大学校内,典型培养物保藏中心,邮编:430072),其菌种保藏编号为CCTCC NO.C2018232。所述C-ELISA方法,以纯化所得TrxA-NS2多肽(氨基酸序列见SEQ ID NO.3)作为抗原包被酶标板,并以单克隆抗体BTV-2A4作为竞争抗体。
本发明的有益效果如下:
1、提供了一种可以分泌抗蓝舌病病毒NS2蛋白单克隆抗体的杂交瘤细胞株。
2、提供了一种抗蓝舌病病毒NS2蛋白的单克隆抗体,此抗体与且仅与蓝舌病病毒NS2蛋白发生特异性结合,结合的B细胞表位为氨基酸序列在91~138区间(其氨基酸序列见SEQ ID NO.4)内,此序列与蓝舌病病毒1-24型高度一致,与蓝舌病病毒25-27型仅有3个位点不同,说明本发明的单克隆抗体,可以用于蓝舌病病毒1-27型的检测,其中1-24型的检测高度可靠,25-27型的可以用于参考。
3、提供了一种可靠程度较高的抗蓝舌病病毒NS2蛋白单克隆抗体,本发明提供的抗蓝舌病病毒NS2蛋白单克隆抗体,经试验验证,与蓝舌病病毒的同属病毒(中山病毒CHUV、鹿出血热病毒EHDV-1及EHDV-6、广西环状病毒GXOV)并不发生反应,因此在检测鉴定蓝舌病病毒感染时可靠程度较高,对疫病防治有较好的指导意义。
4、提供了一种使用简便的C-ELISA试剂盒,由于使用了本发明的单克隆抗体及含有特定B细胞表位(91~138AA)的抗原蛋白,因此检测结果可靠准确,操作简单,有利于疫病防治。
附图说明
图1为原核表达的NS2融合蛋白样品及其纯化各阶段样品的PAGE电泳后考马斯亮蓝染色胶图,各样品等体积上样。
1:过柱前的蛋白样品。
2~3:分别为蛋白样品过柱时收集的第3管和第4管流出液样品。
4:蛋白分子量marker(Thermo,#26616)。
5~11:依次为咪唑洗脱液洗脱目的蛋白时收集的1~7管纯化蛋白样品。
12~15:依次为咪唑洗涤液洗涤杂蛋白时收集的1~4管杂蛋白样品。
图2为原核表达多肽x、y、z的WB检测,以鉴定单克隆抗体BTV-2A4对应的NS2抗原表位所在位置,目的多肽分子量在15kDa~25kDa之间。
1:蛋白分子量marker(Thermo,#26616)
2:表达x肽片段的细菌克隆1蛋白样品。
3:表达x肽片段的细菌克隆2蛋白样品。
4:表达y肽片段的细菌克隆3蛋白样品。
5:表达y肽片段的细菌克隆4蛋白样品。
6:表达z肽片段的细菌克隆5蛋白样品。
7:表达z肽片段的细菌克隆6蛋白样品。
图3为WB检验单克隆抗体BTV-2A4对9株特定病毒的蛋白样品的反应性。上图为检测样品中NS2蛋白的结果,下图为检测相同样品中Actin蛋白的结果。M:蛋白分子量marker(Thermo,#26616),图示条带分子量分别为70kDa、55kDa、40kDa。
1:BHK细胞的蛋白样品。
2:BTV-1感染BHK细胞后的总蛋白样品。
3:BTV-8感染BHK细胞后的总蛋白样品。
4:BTV-17感染BHK细胞后的总蛋白样品。
5:BTV-20感染BHK细胞后的总蛋白样品。
6:BTV-21感染BHK细胞后的总蛋白样品。
7:CHUV感染BHK细胞后的总蛋白样品。
8:EHDV-1感染BHK细胞后的总蛋白样品。
9:EHDV-6感染BHK细胞后的总蛋白样品。
10:GXOV感染BHK细胞后的总蛋白样品。
图4为间接ELISA检验单克隆抗体BTV-2A4对9株特定病毒的蛋白样品的反应性。以每孔200ng总蛋白的量包被96孔板(每个样品3孔),用单克隆抗体BTV-2A4及HRP偶联的二抗标记NS2蛋白。底物显色后用酶标仪检测OD450数值并统计。
1:BHK细胞的蛋白样品。
2:BTV-1感染BHK细胞后的总蛋白样品。
3:BTV-8感染BHK细胞后的总蛋白样品。
4:BTV-17感染BHK细胞后的总蛋白样品。
5:BTV-20感染BHK细胞后的总蛋白样品。
6:BTV-21感染BHK细胞后的总蛋白样品。
7:CHUV感染BHK细胞后的总蛋白样品。
8:EHDV-1感染BHK细胞后的总蛋白样品。
9:EHDV-6感染BHK细胞后的总蛋白样品。
10:GXOV感染BHK细胞后的总蛋白样品。
图5为间接免疫荧光(IFA)验证单克隆抗体BTV-2A4的实验。样品分别为BHK细胞及BTV-1感染的BHK细胞(48hpi);以单克隆抗体BTV-2A4及DyLight649偶联的二抗对NS2染色,同时用DAPI对细胞核染色。
1:BHK细胞的NS2染色结果。
2:BHK细胞的细胞核染色结果。
3:BTV-1感染的BHK细胞的NS2染色结果。
4:BTV-1感染的BHK细胞的细胞核染色结果。
具体实施方式
下面结合具体实施实例来进一步阐述本发明,本发明的优点和特点将会随着描述的展开而更加清晰。但这些实施实例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1培养筛选杂交瘤细胞株
1.主要实验材料
1.1主要生物活体材料
BHK细胞、SP2/0细胞、BTV-1毒株由本研究院保存。
8周龄的雌性Balb/c小鼠由昆明医科大学实验动物部提供。
1.2主要试剂及耗材
MEM培养基及RPMI-1640培养基(补充10%FBS及100U/ml青-链霉素,均购自Giboc);杂交瘤筛选试剂HAT及HT、聚乙二醇融合剂(PEG1500)均购自Sigma。RNA提取试剂盒(Takara,#9767)、SuperScript III逆转录试剂盒(Invitrogen,#18080051)、Pfu酶(天根,#EP101-01)、2×PCR Mix(天根,#KT201-02)、DNA marker DL 5 000(天根)、琼脂糖、DNA纯化试剂盒(天根,#DP203-02)、核酸内切酶BamH I与Xho I(Thermo)、T4DNA连接酶(生工,#B600009)、pET-32a质粒、DNA凝胶回收试剂盒(天根,#DP208-02)、Rosetta感受态细胞(天根)、IPTG(BBI)、细胞裂解液(碧云天,#P0013J)、蛋白酶抑制剂(生工,#C500027)、TALON填料(GE,#28957499)、咪唑(BBI)。弗氏不完全佐剂及弗氏完全佐剂(Sigma);抗体亚类鉴定试剂盒(北京博奥龙免疫技术有限公司,#BF16002X)、HRP偶联的羊抗鼠IgG(恩晶)、DyLight649偶联的羊抗鼠IgG(全式金)、WB发光底物(天根)、ELISA底物显色液(碧云天)、DAB底物显色液试剂盒(碧云天)。
自行配制的主要试剂包括:1)SOB培养基:20g蛋白胨、5g酵母膏、0.5g NaCl、0.18gKCl加水配制成1L溶液,高压灭菌后加入5ml无菌的2M MgCl2溶液。2)磷酸盐缓冲液:5.9gNaH2PO4-2H2O、58g Na2HPO4-12H2O,补水至1L配制成0.2M磷酸盐缓冲液(pH7.4),高压灭菌。3)咪唑洗液:用磷酸盐缓冲液分别配制5mM及200mM的咪唑溶液,过滤除菌后于4℃储藏,分别用于洗脱杂蛋白及目的蛋白。
主要耗材:蛋白纯化填料柱(生工,#C006169-0005)、管状透析器(G-BIOSCIENCES,#C006616)、一次性注射器、细胞培养用96孔板(Corning)、ELISA用96孔板(Corning),等等。
1.3主要仪器设备
超声破碎仪(Sonic,Uibra cell)、单功能酶标仪(Molecular Devices,VersaMax)、PAGE电泳装置(天能,VE180)、化学发光成像系统(上海勤翔科学仪器有限公司)、倒置荧光显微镜(OLYMPUS,IX71)。
2.单克隆抗体制备(实施例1)
2.1获取BTV基因组cDNA
T75培养瓶培养BHK细胞,接种BTV-1,在48hpi收集细胞,用Trizol法提取总RNA。使用SuperScript III试剂盒(Invitrogen)进行逆转录,获得总cDNA样品。
Trizol法提取RNA步骤:加入1ml Trizol裂解细胞沉淀物,室温静置10min;加入200μl氯仿,震荡后室温静置3min;离心(4℃,12 000rpm,10min)后取上清液,加入等体积异丙醇,混合后室温静置15min;离心(4℃,12 000rpm,10min)后小心除尽上清液,加入1ml冰冷的75%乙醇,再次离心(4℃,10 000rpm,5min);小心除尽上清液,风干RNA沉淀,用20~30μl RNase-free无菌水溶解RNA,-80℃保存。
逆转录合成cDNA步骤:①取9μl总RNA与1μl随机引物及1μl dNTP混合,95℃孵育2min后迅速冰浴降温;②依次加入2μl 10×buffer、4μl 25mM MgCl2、2μl 0.1M DTT、1μlRNaseOut、1μl Super Script III;③孵育程序为25℃/10min,50℃/50min,85℃/5min;④加入1μl RNaseH,37℃孵育20min;⑤短期内-20℃冻存cDNA样品。
2.2NS2蛋白抗原制备
2.2.1BTV-1NS2属性分析及克隆片段的选择
根据BTV-1NS2的氨基酸序列(YP_052952.1),进行蛋白属性(等电点、疏水指数)、结构域、保守区间、抗原性分析。综合考虑高度保守性、抗原性(无规则结构域及loop环优先)、可溶性表达属性(PI值在pH 6.5–7.5以外,疏水指数低于80并尽可能低)三项重要指标,最终选定N端1~228AA片段并插入pET-32a载体,构建TrxA-NS2(1~228)融合蛋白(TrxA可增加目的蛋白在细菌中的溶解性)。
所选定的肽片段及其融合蛋白属性如下:1)比对BTV-1(ACR58465.1)、BTV-4(AAW33677.1)、BTV-16(AFO37733.1)的NS2氨基酸序列,可知NS2的1~170AA、174~195AA、205~228AA、为高保守区间,因此总体上处于高保守区域;2)结构域分析(InterPro网站)显示,8~160AA区间为RNA结合域,是NS2蛋白的重要功能区,而163~199AA区间作为无规则结构域,理论上是适宜的抗原区间;3)ProtParam tool软件分析显示,NS2目的片段PI=5.2,疏水指数=71.55,而其融合蛋白PI=6.03,疏水指数=75.41,因此该蛋白不容易在中性条件下发生沉淀并有较好的可溶性表达性能;4)NS2目的片段本身的理论分子量为26kDa,按照抗原表位出现的概率,理论上可以覆盖2~3个抗原表位。
2.2.2构建BTV-1NS2原核表达载体
根据已知的BTV-1的NS2基因序列(NCBI序列号NC_006007.1)设计PCR引物,并在上、下游引物的5’端分别引入酶切位点BamH I和Xho I。引物序列如下:
引物F:5-AAGGATCCATGGAGCAAAAGCAACGTAGATT
引物R:5-TTCTCGAGCAGATTCCAGTTGATTCCAGCTTC
以总cDNA为模板,使用Pfu高保真酶体系进行PCR,克隆NS2基因编码区(CDS)1~684bp的DNA片段。200μl的PCR反应体系含有:144μl ddH2O、20μl 10×buffer、16μl dNTP、各4μl 10μM引物F及引物R、10μl cDNA、2μl Pfu酶。PCR反应程序为:95℃/2min预变性,35次热循环(95℃/10s,55℃/10s,72℃/2min),72℃/3min延伸反应,4℃终止反应。
使用DNA纯化试剂盒纯化PCR产物,制备高纯度的DNA片段。
将克隆到的NS2DNA片段及pET-32a质粒进行BamH I/Xho I双酶切,酶切产物经琼脂糖凝胶电泳及切胶回收后获得纯化的目的DNA。使用T4DNA连接酶体系连接NS2DNA及pET-32a(4℃过夜孵育)。原核表达载体的编码区核苷酸序列见序列表SEQ ID NO.2。
2.2.3NS2融合蛋白的原核表达
通过常规化学转化方法将连接产物(含重组质粒)转化到Rosetta大肠杆菌菌株中,活化后的转化子(37℃,200rpm培养50min)涂抹到Amp+培养平板上,过夜培养。通过菌落PCR鉴定若干个阳性细菌克隆,再提取质粒测序,最终挑选到重组质粒序列正确的单克隆菌株。
重组质粒可表达TrxA-NS2融合蛋白(该蛋白N端为细菌硫氧还蛋白Trx A,C端为NS2的1~228AA片段,蛋白两端有6×His标签),其氨基酸序列见序列表SEQ ID NO.3。
将10μl表达菌种接种到200ml SOB培养基中,37℃、200rpm培养16h。随后加入20ml新鲜的SOB培养基及IPTG(终浓度达到0.2mM),在25℃、150rpm条件下诱导表达5h。最后将菌液离心(3 000rpm,10min),收集菌体沉淀,用20ml RIPA裂解液重悬菌体,在冰浴中超声处理(130W,冲击5s/间歇5s,工作6min)。裂解混合物冰浴30min后,离心(4℃,12 000rpm,10min)。分别取上清液样品及沉淀样品进行PAGE电泳及G-250染色鉴定,确定TrxA-NS2蛋白有较高水平的表达并且为可溶性蛋白。
2.2.4NS2融合蛋白的纯化
将20ml总蛋白样品加入预装1ml TALON树脂的纯化柱,以5ml/管的频率共收集4管流出液;用8ml洗涤液过柱洗涤杂蛋白,以2ml/管的频率收集共4管流出液;再用8ml洗脱液过柱洗脱目的蛋白,以0.5ml/管(E1~E2)及1ml/管(E3~E9)的频率共收集9管纯化蛋白样品。被洗脱的纯化蛋白样品,进行PAGE电泳及G-250染色鉴定(见图1),同时用BCA法测定蛋白浓度,从中选择高纯度、高浓度的纯化蛋白样品(纯度为90%,浓度约1μg/μl)进行透析处理。0.22μm滤膜过滤除菌的蛋白样品置于透析管中,将透析管悬浮于500ml无菌磷酸缓冲液,4℃过夜透析。经过透析,除去咪唑成分的纯化蛋白样品即可用于动物免疫。
2.3小鼠免疫
取制备好的TrxA-NS2融合蛋白(1μg/μl)与等体积弗氏完全佐剂混合,颈背部多点注射方式免疫5只8周龄的Balb/c雌鼠(抗原量100μg/只),之后第14天和第28天以相同方法及剂量(用弗氏不完全佐剂替代弗氏完全佐剂)分别进行第二次和第三次免疫。第三次免疫后10天,眼球后静脉丛采血,通过间接ELISA检测抗体水平,选择抗体水平最高的小鼠,腹腔注射方式进行一次加强免疫(抗原量100μg/只),4天后取脾细胞进行细胞融合。
2.4细胞融合
2.4.1准备SP2/0骨髓瘤细胞
细胞融合前两周,复苏SP2/0细胞株。融合前48h,将SP2/0细胞扩大培养。融合当天,弃细胞培养上清,以RPMI-1640培养基洗涤一次,用10ml RPMI-1640培养基将细胞轻轻吹下,收集于15ml离心管内,1 000rpm离心10min,用RPMI-1640基础培养液重悬后计数备用。
2.4.2准备饲养层细胞
细胞融合前1天,选1只Balb/c小鼠(未被免疫)摘眼球放血,颈椎脱臼处死,75%酒精浸泡10min,仰卧固定于蜡盘,剪开小鼠皮肤,在胸骨处向腹腔中注入约5ml RPMI-1640培养基,用镊子夹酒精棉球轻轻揉压小鼠腹腔,吸出洗液,转入10ml离心管离心(1 000rpm,10min);细胞沉淀用5ml 1%HAT完全培养基悬浮,进行细胞计数并稀释后,加入96孔板培养。
2.4.3脾淋巴细胞准备
颈椎脱臼处死加强免疫的Balb/c小鼠,75%酒精浸泡10min;将小鼠右侧卧固定于蜡盘,剪开皮肤暴露腹膜,取出脾脏置于无菌平皿中,用10ml RPMI-1640培养基轻轻洗涤,尽量去除脂肪及结缔组织;将脾脏移入另一盛有约20ml RPMI-1640培养基的平皿中,用注射器在脾脏一端刺多个小孔,将10ml RPMI-1640基础液培养基从另一端注入脾脏,将脾脏内的细胞冲洗出来,反复冲洗数次,直至脾脏颜色变白为止;将含有分散脾细胞的培养基转入离心管中(避免吸入大的组织碎块)离心(1 000rpm,10min),弃上清,再用10ml RPMI-1640培养基悬浮细胞,重复离心洗涤2次;用10ml RPMI-1640培养基悬浮脾细胞,计数备用。
2.4.4细胞融合
取1×108个脾细胞与1~2×107个对数生长期的骨髓瘤细胞(10:1~5:1)于50ml离心管中混合,离心(1 000rpm,10min),弃上清,将离心管倒倾,尽可能除弃剩余的液体;用手指轻轻弹击管底,使沉淀细胞松散均匀成糊状,置37℃水浴中,缓慢转动离心管,于1min内沿管壁缓缓加入1ml预热至37℃的50%PEG1500,静置1min;然后在5min内加入25ml预热至37℃的RPMI-1640基础培养液终止PEG1500的作用。方法如下:第1min缓慢加入1ml,第2min缓慢加入4ml,第3~5min内缓慢加入剩余的20ml,静置10min;将融合细胞1 000rpm离心10min,弃上清,加入20ml含1%HAT RPMI-1640完全培养基悬浮细胞,分加到已铺好饲养细胞的96孔板中(100μl/孔)。融合后第3天将每孔的培养基吸掉一半,然后添加新鲜的HAT培养基;第10天更换为含1%HT的培养基;融合后第14天吸取细胞上清进行间接ELISA检测抗体效价。
2.5筛选目的杂交瘤细胞株
以0.05M碳酸盐缓冲液(pH9.5)将TrxA-NS2蛋白稀释至0.2ng/μl,按100μl/孔的量加入96孔板中,4℃过夜孵育;弃包被液,加入200μl/孔含5%脱脂奶的PBST,37℃封闭1h;弃封闭液,每孔加入100μl融合后第14天的细胞上清,37℃孵育1h;PBST洗涤3次,加入2 000倍稀释的HRP标记的羊抗鼠IgG,37℃孵育45min;PBST洗涤5次,每孔加入100μl新鲜配制的0.1%(M/V)3,3’,5,5’四甲基联苯胺(TMB),37℃孵育15min,加入50μl/孔2M硫酸溶液终止反应,测450nm吸收值。RPMI 1640完全培养基作为阴性对照,阳性细胞评定标准为测定值与对照值的比值≥2.0。
将抗体效价较高的阳性孔细胞在24孔细胞培养板内扩大培养,以有限稀释法连续进行3次亚克隆,直到获得稳定分泌抗体的杂交瘤细胞株BTV-2A4。
实施例2单克隆抗体亚类鉴定
使用抗体亚类鉴定试剂盒(北京博奥龙免疫技术有限公司,BF16002X),按照说明书方法对实施例获得的交瘤细胞株BTV-2A4分泌的单克隆抗体进行鉴定。
结果显示,本发明的单克隆抗体BTV-2A4亚类为IgG1,轻链亚型为κ链。
实施例3单克隆抗体的抗原表位鉴定
将抗原肽基因分成x、y、z共3段分别克隆到pET-28a载体中(通过BamHⅠ及XhoⅠ酶切位点插入),进而构建出表达x(理论分子量15kDa,NS2蛋白氨基酸范围1~90AA)、y(理论分子量20kDa,NS2蛋白氨基酸范围46~183AA)、z(理论分子量15kDa,NS2蛋白氨基酸范围139~228AA)肽片段的Rosetta菌。选取重组质粒DNA序列正确的克隆x1、x2、y3、y4、z5、z6。以0.2mM IPTG诱导1ml菌液表达蛋白4h,收集菌体沉淀并用200μl细胞裂解液裂解菌体,冰浴1h后离心(4℃,12 000rpm,10min),收集上清液即蛋白样品。等体积的x、y、z蛋白样品加入1/4体积的5×loading buffer,70℃孵育10min。将等体积x、y、z蛋白样品上样到聚丙烯酰胺凝胶进行电泳,随后转印至PVDF膜,5%脱脂奶室温封闭1h后,分别在室温进行一抗孵育1h(杂交瘤BTV-2A4的上清液,500倍稀释)和二抗孵育1h(HRP偶联的羊抗鼠IgG二抗,5 000倍稀释),每次抗体孵育后用PBST漂洗30min。最后加发光底物,用化学发光成像系统成像。
实验结果(见图2)显示:单克隆抗体BTV-2A4与且仅与y片段发生特异性结合,从而确定抗原表位在NS2蛋白的91~138AA区间内,见SEQ ID NO.4,同时证明该抗体能特异性地识别NS2蛋白的线性抗原位点。
实施例4单克隆抗体BTV-2A4的反应特异性及应用技术鉴定
1、蛋白序列分析
为确定单克隆抗体与27型BTV NS2蛋白的反应性以及特异性,搜索27型BTV毒株的NS2氨基酸序列,对NS2蛋白的关键区间(91~138AA)的氨基酸序列(见SEQ ID NO.4)行比对。结果(见表1)显示:除BTV-17、BTV-20、BTV-21、BTV-25、BTV-26、BTV-27毒株存在1~3个氨基酸位点的差异外,其余21型BTV的对应氨基酸序列与单克隆抗体识别的抗原表位所在区域的序列完全一致。
表1 NS2抗原表位区域(91~138AA)与27型BTV NS2蛋白的对应氨基酸序列比对。
2.WB实验鉴定
为进一步确定该单克隆抗体的反应特异性,取5株BTV参考株(BTV-1、BTV-8、BTV-17、BTV-20、BTV-21)及4株同属病毒(中山病毒CHUV、鹿出血热病毒EHDV-1及EHDV-6、广西环状病毒GXOV)进行Western blot实验。在6孔板中培养BHK细胞,每孔接种50μl病毒储液感染细胞,于细胞病变明显时(48~72hpi)提取细胞蛋白。即吸去培养液后,用PBS润洗细胞,再加入500μl PBS(含蛋白酶抑制剂)刮取细胞,3 000rpm离心1min收集细胞沉淀,除尽PBS,用120μl细胞裂解液裂解细胞,冰浴1h后离心(4℃,12 000rpm,10min),收集上清液备用。用BCA法测定样品蛋白浓度后,配制Western blot上样样品(蛋白浓度调整为0.4μg/μl),70℃变性处理10min后,上样进行聚丙烯酰胺凝胶电泳。通过转膜步骤将凝胶中的蛋白转印至PVDF膜上。5%脱脂奶室温封闭1h后,分别在室温进行一抗孵育1h(杂交瘤BTV-2A4的上清液,500倍稀释)和二抗孵育1h(HRP偶联的羊抗鼠IgG二抗,5 000倍稀释),每次抗体孵育后用PBST漂洗30min。最后加发光底物,在化学发光成像系统下成像。结果如图3所示,BHK细胞样品及CHUV、EHDV、GXOV病毒感染的BHK样品均为阴性结果,而5株BTV感染的BHK样品均为阳性结果。
由此可见,单克隆抗体BTV-2A4能特异性识别1~24型BTV病毒的NS2蛋白(且不排除特异性识别BTV-25~27的NS2蛋白的可能性),因此具有BTV检测的通用性;其次,该抗体不与细胞蛋白及环状病毒属其它种病毒(CHUV、EHDV、GXOV)的蛋白反应,证明单克隆抗体BTV-2A4具有高度种属特异性;其三,单克隆抗体BTV-2A4可应用于WB实验检测至少24型的BTV NS2蛋白。
3.间接ELISA实验鉴定
取上述WB实验鉴定中的10份蛋白样品进行间接ELISA检测:1)以200ng/孔的蛋白量进行包被(每个样品加3孔),4℃过夜孵育;2)弃包被液,加入200μl/孔含5%脱脂奶的PBST,37℃孵育1h;3)弃封闭液,加入100μl/孔150倍稀释的单克隆抗体BTV-2A4,37℃孵育1h;4)PBST洗涤3次,加入2 000倍稀释的HRP偶联的羊抗鼠IgG,37℃孵育30min;5)PBST洗涤5次,每孔加入100μl新鲜配制的0.1%(M/V)3,3’,5,5’四甲基联苯胺(TMB),37℃孵育15min后每孔加入50μl 2M硫酸溶液终止反应,测450nm吸收值。统计实验结果的OD450并做成柱状图(图4)。
结果(见图4)说明:单克隆抗体BTV-2A4可应用于间接ELISA技术检测蛋白质样品中BTV NS2蛋白。
4.间接免疫荧光(IFA)实验鉴定
取BHK细胞在12孔板培养,待长满单层之后接种BTV-1参考毒,在48hpi固定细胞。吸弃培养液,用PBS洗涤3次。甲醇-丙酮(1:1)固定液(提前预冷)室温固定20min,PBS洗涤3次;用含5%BSA的封闭液37℃封闭1h,PBS洗涤3次;加入250倍稀释的单克隆抗体BTV-2A4,37℃孵育1h;用PBST洗涤3次,每次5min,然后加入1 000倍稀释的DyLight 649偶联的羊抗鼠IgG二抗,37℃孵育45min后,PBST洗4次,每次10min。在倒置荧光显微镜下观察荧光并拍照。
实验结果(图5)说明:单克隆抗体BTV-2A4可应用于间接免疫荧光法检测细胞中的BTV NS2蛋白。
实施例5BTV-NS2抗体C-ELISA参数的优化
将纯化的TrxA-NS2蛋白分别以25ng/孔、50ng/孔、100ng/孔、200ng/孔包被,在4℃条件下包被过夜;再将单克隆抗体BTV-2A4(杂交瘤细胞上清液)用稀释液稀释1:80、1:120、1:160、1:200、1:240倍,将HRP标记的山羊抗鼠IgG用稀释液稀释1:1000、1:1500、1:2000、1:2500倍;将自然感染蓝舌病病毒1型的BTV抗体阳性血清,蓝舌病病毒抗体阴性血清用稀释液进行1:2、1:4、1:8倍稀释选择最佳待检样品加入量,进行矩阵交叉BTV C-ELISA方法检测,确定最佳稀释倍数。同时,对反应时间分别设置0.5h、1h、2h,温度为37℃,确定各步骤最佳反应时间。TMB显色温度设置为37℃,显色反应时间分别为5min、10min、15min、20min,确定最佳显色时间。根据优化反应剂量和时间的结果,确定该检测方法最优反应参数。
经矩阵交叉法试验,确定BTV-NS2抗体C-ELISA检测的最优反应参数如表2:
表2 BTV-NS2抗体C-ELISA检测的反应条件优化结果
实施例6单克隆抗体BTV-2A4在鉴定野生型BTV感染方面的应用
在开发BTV灭活疫苗的研究中需要评估疫苗对动物的免疫效果,该抗体BTV-2A4可应用于C-ELISA技术来鉴定动物是否存在天然感染的情况,对疫苗效果评估具有重要辅助作用。因为灭活疫苗本身不携带非结构蛋白NS2,也不能在动物体内合成NS2蛋白,所以灭活疫苗所免疫的动物不会产生抗NS2蛋白的抗体。而天然感染BTV的动物能在体内合成NS2蛋白从而有产生NS2抗体的可能性。我们对动物血清样品的C-ELISA检测证明了这种设想的可行性。
实验共使用75份动物血清,其中7份来自BTV灭活苗(5份BTV-1和2份BTV-8)免疫的绵羊;另外68份来自未接种BTV疫苗的黄牛,且经“BTV抗体C-ELISA检测试剂”鉴定,其中60份牛血清为抗体阳性,8份牛血清为抗体阴性(见表3)。
C-ELISA测BTV抗体实验步骤:1)取预先包被BTV的96孔板,以每孔10μl的量加入待检血清、阴性对照血清、阳性对照血清、强阳性对照血清,留4孔各加10μl PBST作为抗原对照;2)加入50μl/孔用PBST稀释的兔抗BTV血清(100稀释),37℃孵育40min;3)PBST洗板1次,加入50μl/孔1 000倍稀释的绵羊抗兔酶标二抗,37℃孵育30min;4)用PBST洗板5次,拍干后每孔加入100μl新鲜配制的0.1%(M/V)3,3’,5,5’四甲基联苯胺(TMB),37℃孵育10min后加入50μl 2M硫酸溶液终止反应,测450nm吸收值(阴性对照组结果即最高读数为1.0)并计算吸光度抑制率。抑制率=(阴性对照OD450-样品OD450)÷阴性对照OD450。评定标准:OD450抑制率≥50%为阳性结果,OD450抑制率<40%为阴性结果。
C-ELISA测NS2抗体实验步骤:1)以0.05M碳酸盐缓冲液(pH9.5)将纯化的TrxA-NS2蛋白分别以100ng/孔的量进行包被,在4℃条件下包被过夜;2)弃包被液,每孔加入200μl含5%脱脂奶的PBST,37℃封闭1h;3)弃封闭液,每孔加入1:2稀释的待检动物血清50μl,设置阴性和阳性血清对照,37℃孵育30min;4)加入50μl/孔160倍稀释的单克隆抗体BTV-2A4(杂交瘤细胞上清液),37℃孵育1h;5)PBST洗涤3次,加入100μl/孔2 000倍稀释的HRP偶联的羊抗鼠IgG,37℃孵育1h;6)PBST洗涤5次,每孔加入100μl新鲜配制的0.1%(M/V)3,3’,5,5’四甲基联苯胺(TMB),37℃孵育10min后每孔加入50μl 2M硫酸溶液终止反应,测450nm吸收值(阴性对照组结果即最高读数为0.8)并计算吸光度抑制率(方法同上)。评定标准:OD450抑制率≥35%为阳性结果,OD450抑制率<30%为阴性结果。
表3单克隆抗体BTV-2A4应用于C-ELISA技术测血清样品中的NS2抗体
实验结果如表3所示:1)对于疫苗免疫的绵羊血清样品,可检测到BTV抗体,而检测不到BTV NS2抗体,符合率100%;2)对于60份BTV抗体阳性牛血清样品,有58份能检测到BTVNS2抗体,符合率97%;3)8份BTV抗体阴性牛血清样品未被检出BTV NS2抗体,符合率100%。
此实验证明:该C-ELISA方法可用于鉴定动物血清中是否含有NS2抗体,从而鉴定被测试动物是否发生过BTV天然感染,并辅助判断被免疫动物的抗病毒抗体来自疫苗免疫还是野生型BTV感染。
需要说明的是,在本文中,诸如术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 云南省畜牧兽医科学院
<120> 特异性检测蓝舌病病毒的单抗及其杂交瘤细胞株和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 228
<212> PRT
<213> 蓝舌病病毒 (Bluetongue virus)
<400> 1
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Ala Asn Gly Lys Thr Leu Cys Gly Ala Ile Ala Lys Leu Ser Ser Gln
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Lys Asn Pro Glu Pro Lys Gly Tyr Val Leu Asn Val Pro Gly Pro Gly
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Ala Tyr Arg Ile Gln Asp Gly Gln Asp Ile Ile Ser Leu Met Leu Thr
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Pro His Gly Val Glu Ala Thr Thr Glu Arg Trp Glu Glu Trp Lys Phe
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Glu Gly Val Ser Val Thr Pro Met Ala Thr Arg Val Gln His Asn Gly
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Val Met Val Asp Ala Glu Ile Lys Tyr Cys Lys Gly Met Gly Ile Val
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Gln Pro Tyr Met Arg Asn Asp Phe Asp Arg Asn Glu Met Pro Asp Leu
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Pro Gly Val Met Arg Ser Asn Tyr Asp Val Arg Glu Leu Arg Gln Lys
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Ile Lys Asn Glu Arg Glu Ser Ala Pro Arg Leu Gln Val Gln Ser Val
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Ala Pro Arg Glu Glu Ser Arg Trp Met Asp Asp Asp Glu Ala Lys Val
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Asp Glu Glu Ala Lys Glu Met Ile Pro Gly Thr Ser Arg Leu Glu Lys
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<211> 1206
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<213> 大肠杆菌(Escherichia coli)
<400> 2
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60
gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120
ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180
atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240
ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300
aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360
catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420
ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480
gctgatatcg gatccatgga gcaaaagcaa cgtagattta ctaaaaacat ttttgttttg 540
gatgcaaatg gcaaaacatt atgcggagcg atcgcaaagt tgagttcgca accgtattgt 600
caaattaaaa ttggaagagt aatagctttt aaacctgtca aaaatccgga acctaaggga 660
tacgtgctga atgttccagg acctggtgcg tacagaattc aggatgggca ggatatcatc 720
agcctgatgt tgacaccaca tggggttgaa gcgacaacgg aaaggtggga agagtggaag 780
tttgagggtg tcagtgtaac gccaatggct actagggtac aacataatgg tgtaatggtt 840
gatgctgaga ttaagtattg taaaggaatg ggaatagtgc aaccatatat gcggaatgat 900
tttgatcgga acgagatgcc cgatttacca ggtgtgatga ggtcaaacta cgatgttcgt 960
gaactgcggc aaaagatcaa aaatgaacga gaatcagcgc cacggcttca agttcaaagc 1020
gtggcgccaa gggaagagtc acgctggatg gatgatgatg aagcaaaggt ggacgaagag 1080
gctaaagaga tgattccggg aaccagcaga ttggagaagc tgcgtgaagc gagaagcaat 1140
gttttcaagg aggtggaagc tggaatcaac tggaatctgc tcgagcacca ccaccaccac 1200
cactga 1206
<210> 3
<211> 401
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 3
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
115 120 125
Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
130 135 140
His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
145 150 155 160
Ala Asp Ile Gly Ser Met Glu Gln Lys Gln Arg Arg Phe Thr Lys Asn
165 170 175
Ile Phe Val Leu Asp Ala Asn Gly Lys Thr Leu Cys Gly Ala Ile Ala
180 185 190
Lys Leu Ser Ser Gln Pro Tyr Cys Gln Ile Lys Ile Gly Arg Val Ile
195 200 205
Ala Phe Lys Pro Val Lys Asn Pro Glu Pro Lys Gly Tyr Val Leu Asn
210 215 220
Val Pro Gly Pro Gly Ala Tyr Arg Ile Gln Asp Gly Gln Asp Ile Ile
225 230 235 240
Ser Leu Met Leu Thr Pro His Gly Val Glu Ala Thr Thr Glu Arg Trp
245 250 255
Glu Glu Trp Lys Phe Glu Gly Val Ser Val Thr Pro Met Ala Thr Arg
260 265 270
Val Gln His Asn Gly Val Met Val Asp Ala Glu Ile Lys Tyr Cys Lys
275 280 285
Gly Met Gly Ile Val Gln Pro Tyr Met Arg Asn Asp Phe Asp Arg Asn
290 295 300
Glu Met Pro Asp Leu Pro Gly Val Met Arg Ser Asn Tyr Asp Val Arg
305 310 315 320
Glu Leu Arg Gln Lys Ile Lys Asn Glu Arg Glu Ser Ala Pro Arg Leu
325 330 335
Gln Val Gln Ser Val Ala Pro Arg Glu Glu Ser Arg Trp Met Asp Asp
340 345 350
Asp Glu Ala Lys Val Asp Glu Glu Ala Lys Glu Met Ile Pro Gly Thr
355 360 365
Ser Arg Leu Glu Lys Leu Arg Glu Ala Arg Ser Asn Val Phe Lys Glu
370 375 380
Val Glu Ala Gly Ile Asn Trp Asn Leu Leu Glu His His His His His
385 390 395 400
His
<210> 4
<211> 48
<212> PRT
<213> 蓝舌病病毒(Bluetongue virus)
<400> 4
Trp Glu Glu Trp Lys Phe Glu Gly Val Ser Val Thr Pro Met Ala Thr
1 5 10 15
Arg Val Gln His Asn Gly Val Met Val Asp Ala Glu Ile Lys Tyr Cys
20 25 30
Lys Gly Met Gly Ile Val Gln Pro Tyr Met Arg Asn Asp Phe Asp Arg
35 40 45
Claims (10)
1.一种分泌抗蓝舌病病毒NS2蛋白单克隆抗体的杂交瘤细胞株,命名为BTV-2A4,保藏在中国典型培养物保藏中心,菌种保藏编号:CCTCC NO:C2018232。
2.一种权利要求1所述杂交瘤细胞株所分泌的抗蓝舌病病毒NS2蛋白的单克隆抗体。
3.一个与权利要求2所述单克隆抗体发生特异性结合的蓝舌病病毒NS2蛋白的B细胞表位多肽,其特征在于:所述表位多肽的氨基酸序列在91 ~ 138区间内,所述氨基酸序列如SEQ ID NO.4所示。
4.权利要求2所述抗蓝舌病病毒NS2蛋白的单克隆抗体在制备蓝舌病病毒诊断试剂或检测试剂中的应用。
5.权利要求3所述蓝舌病病毒NS2蛋白B细胞表位多肽在制备诊断或检测蓝舌病病毒感染的试剂中的应用。
6.一种以权利要求2所述单克隆抗体制备的特异性检测蓝舌病病毒NS2蛋白的C-ELISA试剂盒,其特征在于,所述C-ELISA试剂盒包括酶标板,还包括如权利要求2所述的抗蓝舌病病毒NS2蛋白的单克隆抗体,及抗鼠的酶标二抗、阳性对照、阴性对照、稀释液、TMB显色液、洗涤液和终止液。
7.根据权利要求6所述C-ELISA试剂盒,其特征在于,所述酶标板由抗原蛋白包被,所述抗原蛋白为细菌Trx A蛋白与蓝舌病病毒NS2蛋白片段的融合蛋白,其序列如SEQ ID NO.3所示;所述蓝舌病病毒NS2蛋白片段的序列为1型蓝舌病病毒Seg8基因编码的1 ~ 228位氨基酸序列,其氨基酸序列如SEQ ID NO.1所示。
8.根据权利要求6所述特异性检测蓝舌病病毒NS2蛋白的C-ELISA试剂盒,其特征在于,所述抗鼠的酶标二抗为HRP标记的羊抗鼠IgG,所述所述阳性对照为自然感染蓝舌病病毒1型的BTV抗体阳性血清;所述阴性对照为蓝舌病病毒抗体阴性的血清;所述稀释液为含有5%(W/V)脱脂奶的PBS缓冲液;所述洗涤液为PBS缓冲液。
9.根据权利要求7所述特异性检测蓝舌病病毒NS2蛋白的C-ELISA试剂盒,其特征在于,所述包被酶标板的TrxA-NS2抗原蛋白为经过钴离子树脂纯化的多肽,其包被量为100 ng/孔;所述单克隆抗体的稀释倍数为160倍;所述酶标二抗的稀释倍数为2000倍;孵育时间均为1 h,TMB显色时间为10 min。
10.根据权利要求7所述特异性检测蓝舌病病毒NS2蛋白的C-ELISA试剂盒,其特征在于,待测血清用量为25μl/孔。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4873189A (en) * | 1984-01-12 | 1989-10-10 | The United States Of America As Represented By The Secretary Of Argriculture | Monoclonal antibodies to bluetongue virus antigen |
CN103642757A (zh) * | 2013-10-14 | 2014-03-19 | 中国农业科学院哈尔滨兽医研究所 | 蓝舌病病毒ns2蛋白单克隆抗体btv-4d4及其识别的b细胞表位和应用 |
CN104582724A (zh) * | 2012-06-13 | 2015-04-29 | 梅里亚有限公司 | 重配btv和ahsv疫苗 |
-
2019
- 2019-05-21 CN CN201910421549.0A patent/CN110016466B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4873189A (en) * | 1984-01-12 | 1989-10-10 | The United States Of America As Represented By The Secretary Of Argriculture | Monoclonal antibodies to bluetongue virus antigen |
CN104582724A (zh) * | 2012-06-13 | 2015-04-29 | 梅里亚有限公司 | 重配btv和ahsv疫苗 |
CN103642757A (zh) * | 2013-10-14 | 2014-03-19 | 中国农业科学院哈尔滨兽医研究所 | 蓝舌病病毒ns2蛋白单克隆抗体btv-4d4及其识别的b细胞表位和应用 |
Non-Patent Citations (2)
Title |
---|
Identification of a linear B-cell epitope within the Bluetongue virus serotype 8 NS2 protein using a phage-displayed random peptide library;Yong-LiQin等;《Veterinary Immunology and Immunopathology》;20130815;第154卷(第3-4期);第93-101页 * |
抗蓝舌病病毒VP7和NS2蛋白单克隆抗体的制备及鉴定;秦永丽等;《中国预防兽医学报》;20111031;第33卷(第10期);第816-819页 * |
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