CN106701687B - 一种杂交瘤细胞株及其产生的狂犬病毒磷蛋白单克隆抗体 - Google Patents
一种杂交瘤细胞株及其产生的狂犬病毒磷蛋白单克隆抗体 Download PDFInfo
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Abstract
本发明提供了一种杂交瘤细胞株1A4,本发明提供的杂交瘤细胞株可以用于分泌制备高效识别狂犬病毒磷蛋白的单克隆抗体,小鼠腹水抗体采用间接酶联免疫吸附分析法测得的效价为2×105;单克隆抗体与狂犬病病毒的其他蛋白以及其他抗原和病原体无交叉反应,具有高特异性、高敏感性的优点,可以用于对狂犬病毒进行包括ELISA、Western‑blot和免疫荧光在内的生物诊断具有良好的应用前景。
Description
技术领域
本发明属于细胞工程技术领域,具体涉及杂交瘤细胞株1A4及其产生的狂犬病毒磷蛋白单克隆抗体。
背景技术
狂犬病是由狂犬病病毒引起的人兽共患病,发病死亡率几乎100%,全世界每年有6万人死于狂犬病,我国是狂犬病流行较为严重的国家之一。狂犬病是我国法定报告管理的乙类传染病,其病原是狂犬病毒(Rabies virus)。磷蛋白在病毒逃逸、病毒转录复制以及细胞内运动中均发挥着一定的作用,还有可能作为病毒感染的临床血清检测抗原。虽然人源抗狂犬病毒药近年来有了很大的发展,但是目前仍然没有开发出非常有效的治疗性药物,因此,狂犬疫苗仍然是目前防治狂犬病的最有效的手段。
狂犬病毒基因组为不分节段的单股负链RNA,全长约12kb,分别编码酶蛋白(L)、糖蛋白(G)、基质蛋白(M)、磷酸化蛋白(P)、核蛋白(N)五个主要结构蛋白。其中,G蛋白和N蛋白含有多个抗原位点,能够诱导产生抗狂犬病毒抗体。磷蛋白属于病毒RNA聚合酶的辅助因子,与病毒核蛋白和L蛋白构成的复合物紧紧将病毒基因组RNA包裹。磷蛋白是该蛋白复合体中的必要中介,一方面它介导L蛋白聚合酶在N-RNA模板中的正确定位,另一方面它发挥着分子伴侣的功能,形成复合体阻止其结合到细胞RNA上并便于其传递到病毒RNA包装成为新的RNPs。磷蛋白是一种多功能蛋白,除了在病毒RNA合成中具有一定的功能外,还能够与STAT1相互作用,并抑制干扰素信号转导途径,从而克服感染细胞的抗病毒反应,对抗宿主的先天免疫反应,是先天免疫反应的关键调控因子。磷蛋白在病毒逃逸、病毒转录复制以及细胞内运动中均发挥着一定的作用,还有可能作为病毒感染的临床血清检测抗原。
自1978年Wiktor和Koprowski首次制备了狂犬病病毒的单克隆抗体以来,越来越多的实验室建立了狂犬病病毒的单克隆抗体杂交瘤细胞株。但主要集中于病毒核蛋白和糖蛋白上,且狂犬病毒检测试剂盒仅限于检测抗病毒糖蛋白和/或核蛋白抗体的间接ELISA检测方法,在一定程度存在假阳性。而申请人在前期研究中证实在狂犬病毒减毒活疫苗免疫的小鼠中能够与抗狂犬病毒核蛋白抗体一样大量产生抗磷蛋白抗体,推测磷蛋白也可能是一种超抗原,具有一定的临床诊断价值。竞争性ELISA可以有效降低间接ELISA中的非特异性反应。目前狂犬病毒抗体检测试剂盒仅限于狂犬病毒核蛋白和糖蛋白的检测,而基于磷蛋白单克隆抗体的狂犬病毒抗体检测试剂盒尚未见报道。本发明提供的1A4单克隆抗体亚型为IgG1型,能用于竞争性ELISA、Western-blot和免疫荧光检测,从而为基于该蛋白临床检测方法的建立及该蛋白功能的研究奠定坚实的基础。
发明内容
本发明的目的是针对目前狂犬病严重危害人类健康,且防治过程中依然存在各种难处的现状,而提供一种能与狂犬病病毒中的磷蛋白特异性结合的单克隆抗体以及产生该抗体的杂交瘤细胞。本发明的单克隆抗体杂交瘤细胞株产生的单克隆抗体与狂犬病病毒的其他蛋白以及其他抗原和病原体无交叉反应,具有高特异性、高敏感性的优点,可以准确检测狂犬病毒疫苗或感染组织中的磷蛋白成分的含量,具有良好的应用前景。
本发明的杂交瘤细胞株1A4已于2016年12年12日保藏于中国典型培养物保藏中心(CCTCC),保藏地址是:中国,武汉,武汉大学,保藏编号为CCTCC NO:C2016194。
本发明另一目的是提供一种由上述杂交瘤细胞株1A4产生的狂犬病毒磷蛋白单克隆抗体,其轻链可变区的氨基酸序列如SEQ ID NO:1所示,重链可变区的氨基酸序列如SEQID NO:2所示。
本发明所述的单克隆抗体可经生物标记或化学标记得到的标记复合物;所述的生物标记为本领域常规,如可以是以酶、生物素等标记抗体,优选的,所述的生物标记为酶标记,更优选地,所述的酶为辣根过氧化物酶或碱性磷酸酶。
本发明所述的单克隆抗体用于制备检测狂犬病毒的试剂盒中。
本发明提供的技术方案之一是:如前所述的单克隆抗体在狂犬病毒疫苗生产毒株活力质检中的应用。
本发明提供的技术方案之二是:特异性检测狂犬病毒病毒抗体的ELISA检测试剂盒,其是以狂犬病病毒重组磷蛋白作为包被抗原,以酶标记的单克隆抗体作为检测竞争抗体。其中,所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO:1所示, 重链可变区的氨基酸序列如SEQ ID NO:2所示。
本发明提供的能够识别狂犬病毒磷蛋白的单克隆抗体具有良好的特异性,实验表明,各克隆间识别没有交叉反应。间接ELISA表明这个抗体具有较高的效价,因此可用于狂犬病病毒及疫苗组分的检测。
本发明所用试剂盒原料均市售可得。
本发明的积极进步效果在于:本发明提供的单克隆抗体与狂犬病病毒其他蛋白以及其他抗原和病原体无交叉反应,用于检测具有高特异性、高敏感性的优点,可以准确检测样品中狂犬病疫苗有效成分或感染组织样品中磷蛋白的含量,在疫苗和临床检测中将会得到广泛应用。
附图说明
图1是狂犬病病毒磷蛋白重组表达的SDS-PAGE电泳图;其中,M是蛋白分子量标准(kDa),泳道1为未诱导菌体蛋白,泳道2为诱导菌体蛋白,泳道3为纯化的狂犬病病毒磷蛋白;
图2为单克隆抗体的Western blot结果图;泳道1为未感染病毒N2a细胞裂解液;泳道2为感染狂犬病毒的N2a细胞裂解液;
图3为单克隆抗体的免疫荧光结果图;其中A图为没有感染狂犬病毒的细胞,添加1A4抗体;B图是感染了狂犬病毒的细胞,添加1A4抗体;C图是没有感染狂犬病毒的细胞,添加阳性对照血清;D图是感染了狂犬病毒的细胞,添加阳性对照血清;
图4为检测狂犬病病毒磷蛋白的ELISA法拟合曲线。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中方法如无特殊说明的采用常规方法,使用的试剂如无特殊说明的使用常规市售试剂或按常规方法配制的试剂。
实施例1:狂犬病毒磷蛋白的表达与纯化
一、基因克隆与表达载体构建
狂犬病毒Flury-HEP毒株由本实验室保存,如下所示设计引物扩增目的片段:上游引物 RV-PF:5' -CGCGAATTCATGAGCAAGATCTTTGTTAATCCGAG- 3';下游引物RV-PR:5'-GTCGACGCCGCTTAGCATGATGTGTAGCGATCCAAGT- 3';引物两端分别添加EcoRI和SalI酶切位点;目的片段大小为894bp;将狂犬病毒感染N2a细胞48h后,提抽RNA并反转录获得cDNA,作为基因克隆的模板。PCR产物用试剂盒纯化,克隆至pMD19-T,挑选出阳性克隆并测序。将序列正确的阳性克隆双酶切,克隆到pET-32a表达载体,转化到Rosetta(DE3)感受态。
二、表达与纯化重组蛋白
将菌液以1:100的体积比扩大培养至OD为0.6 - 1,加入1mM IPTG诱导蛋白,收集菌体后用含有8mol/L尿素的磷酸盐缓冲液重悬菌体并超声破碎;离心上清与菌体破碎沉淀,目的蛋白为不可溶性蛋白;用HiTrap TALON crude(GE)纯化目的蛋白并用SDS-PAGE检测(图1)。
实施例2:杂交瘤细胞系的建立
一、实验材料
1、免疫原:以狂犬病病毒重组磷蛋白作为免疫原;
2、培养基:RPMI-1640培养基购于HyClone公司;HAT购于Gibco公司;HT购于Gibco公司;
3、实验动物:Balb/c小鼠,8-12周龄,雌性,SPF级动物培养;
4、其他实验材料:弗氏完全佐剂、弗氏不完全佐剂购于SIGMA公司;PEG4000购于SIGMA公司;HRP-山羊抗小鼠IgG抗体购于abcam公司;其余试剂均为国产分析纯产品。
二、杂交瘤细胞系的建立
1、动物免疫
(1)基础免疫:将免疫原与弗氏完全佐剂等体积混合并充分乳化,小鼠背部皮下多点注射,每只Balb/c小鼠每次注射量为20μg;
(2)持续免疫:基础免疫后两周进行持续免疫(持续免疫两周一次)。将免疫原与弗氏不完全佐剂等体积混合并充分乳化,小鼠背部皮下多点注射,每只Balb/c小鼠每次注射量为20μg;
(3)加强免疫:在进行细胞融合前3天,经腹腔注射含20μg抗原的磷酸盐缓冲液。
2、杂交瘤细胞的制备
按照常规方法收集小鼠的脾细胞与SP2/0细胞。细胞计数后,小鼠的脾细胞与SP2/0细胞个数比以5:1的比例用PEG4000进行融合。融合细胞用HAT培养基悬浮后,铺96孔板,于37℃条件培养。融合后两周,采用间接ELISA实验检测细胞培养上清,筛选识别狂犬病病毒重组磷蛋白的杂交瘤细胞株。对所得阳性克隆株采用有限稀释法进行亚克隆。间接ELISA法的操作步骤如下:以2μg /mL的狂犬病病毒重组磷蛋白进行包板,每孔100μl,37℃培养3h后,PBST洗3次。5%脱脂奶每孔200μL,封闭2h,PBST洗3次。1:1000倍稀释免疫小鼠血清,作为阳性对照,无克隆生长的培养基上清和正常小鼠血清作为阴性对照,每孔100μL,37℃培养2h后,PBST洗3次。1:5000 倍稀释HRP-山羊抗小鼠IgG,每孔100μL,37℃培养1h后,PBST洗3次。最后测定450nm OD值。凡OD450大于阴性对照2倍以上者,即可初步判定为阳性克隆。
3、杂交瘤细胞系的建立
经过3次亚克隆和间接ELISA筛选,得到1株针对狂犬病病毒重组磷蛋白的,稳定分泌单克隆抗体的杂交瘤细胞系;筛选得到的1株杂交瘤细胞命名为1A4。
4、应用上述杂交瘤细胞系注射小鼠腹腔,收集纯化腹水后,将所得的腹水进行效价检测,结果如表1所示;
表1狂犬病病毒重组磷蛋白抗体效价检测(腹水)
(1)1A4细胞培养上清中单克隆抗体的效价测定:间接ELISA法检测上述杂交瘤细胞培养上清液中单克隆抗体效价为1:50000-1:100000;
(2)小鼠腹水效价的测定:间接ELISA法检测上述杂交瘤细胞制备的腹水效价为1:100000-1:200000。
5、杂交瘤细胞系的传代培养
将上述稳定分泌1A4的杂交瘤细胞系在含有10%胎牛血清的DPMI-1640培养基中继续进行培养、传代,培养到10代后,杂交瘤细胞系仍然能够生长良好、稳定传代,培养液上清效价仍然可以达到1×106以上。
以上结果表明,所得杂交瘤细胞系能够稳定传代,可以持续、稳定地分泌抗狂犬病病毒磷蛋白的单克隆抗体。
实施例3:抗狂犬病病毒磷蛋白单克隆抗体的大量制备及鉴定
一、抗体制备
选择6-8周龄Balb/c小鼠,腹腔注射0.5mL液体石蜡,每只小鼠0.5mL。一周后腹腔接种杂交瘤细胞细胞,每只小鼠接种细胞数为1×105-1×106个。间隔5天后,待腹部明显膨大,以手触摸时,皮肤有紧张感,即可用9号针头采集腹水。
将腹水离心(13000r/min 30分钟),除去细胞成分和其他的沉淀物,收集上清。用Protein-Sepharose CL-4B进行纯化,上柱液为20mM的PBS缓冲液,柱层析洗脱液为pH2 .7,20mM的甘氨酸缓冲液,得到抗狂犬病病毒重组磷蛋白的单克隆抗体。
二、抗体的鉴定
1、单克隆抗体的Western验证
狂犬病病毒裂解液用12%的SDS-PAGE分离,160mA处理90min后将蛋白醋酸纤维膜(NC膜)上。NC膜用5%脱脂奶室温封闭一小时。PBST洗3次之后,加入单克隆抗体作为一抗,室温孵育1h。PBST洗三次之后,用标记HRP的羊抗鼠IgG Fc二抗(1:5000)室温孵育1h,最后用EasySee Western Blot Kit (TRAN)检测免疫反应(图2)。
2、单克隆抗体的免疫荧光验证
应用1A4抗体做免疫荧光检测,将感染狂犬病病毒的N2a细胞和正常的N2a细胞用预冷的细胞固定液(甲醇/丙酮=1:1)固定,室温风干后,进行5%脱脂奶封闭。加入单克隆抗体1A4作为一抗,再加入用异硫氰酸荧光素(FITC)偶联的羊抗鼠IgG Fc作为二抗,用PBST洗三次后用荧光显微镜观察,检测结果显示(见图3),狂犬病毒阳性血清和1A4抗体均在未感染的阴性NA细胞无荧光,在狂犬病病毒HEP-Flury感染的N2a细胞(Flury-N2a)有荧光信号,且有明显的细胞轮廓,该结果验证1A4抗体为特异性识别狂犬病病毒磷蛋白的抗体。
3、识别狂犬病病毒磷蛋白1A4可变区序列测定
将克隆的细胞提取mRNA,反转录为cDNA,使用可变区通用引物进行高保真PCR扩增,将PCR产物片段插入到T载体内进行DNA序列测定,并将获得的序列翻译成蛋白质的氨基酸序列。识别磷蛋白的1A4的抗体的可变区氨基酸序列:轻链如SEQ ID NO:1所示,重链如SEQ ID NO:2所示。
实施例4:应用纯化抗体制备抗狂犬病病毒检测试剂
ELISA竞争法:确定以狂犬病病毒重组磷蛋白为包被抗原,用HRP标记1A4作为检测抗体,确定了ELISA检测方法,试剂盒检测灵敏度可达0.1IU/mL,具体的结果如表2所示,根据结果制定的拟合曲线如图4所示。
表2 :ELISA法检测狂犬病病毒磷蛋白的检测结果
检测方法:包被抗原用pH 9.6 0.05mol/L的碳酸盐缓冲溶液稀释成5μg/mL,每孔加100μL,37℃包被3h,倾去包被液,用PBST洗涤3次,拍干。加入200μL 5%脱脂奶,37℃ 封闭2小时,用PBST洗涤3次。分别加入HRP-1A4、等体积混匀的HRP-1A4和待测血清100μL/孔,37℃孵育1小时,用PBST洗涤3次。加入显色剂100μL/孔,37℃避光反应15分钟后,2M H2SO4终止液100μL/孔终止显色反应,读取OD450数值。
检测结果见表2,说明本申请的抗狂犬病病毒磷蛋白单克隆抗体具有良好的灵敏性。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明的基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 昆明理工大学
<120> 一种杂交瘤细胞株及其产生的狂犬病毒磷蛋白单克隆抗体
<160> 4
<170> PatentIn version 3.3
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Claims (2)
1.一种杂交瘤细胞株1A4,其在中国典型培养物保藏中心的保藏编号为CCTCC NO:C2016194。
2.一种由权利要求1所述杂交瘤细胞株1A4产生的狂犬病毒磷蛋白单克隆抗体,其特征在于:其轻链可变区的氨基酸序列如SEQ ID NO:1所示,重链可变区的氨基酸序列如SEQ IDNO:2所示。
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