CN109970852B - 分泌抗狂犬病毒m蛋白单克隆抗体的杂交瘤细胞株及应用 - Google Patents
分泌抗狂犬病毒m蛋白单克隆抗体的杂交瘤细胞株及应用 Download PDFInfo
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Abstract
本发明公开了一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株及应用,属于生物技术领域。所述杂交瘤细胞株的分类命名为杂交瘤细胞株4A1,于2019年4月1日保藏于中国典型培养物保藏中心,地址:中国武汉,武汉大学,保藏编号为CCTCC NO:C201947。该杂交瘤细胞株制备的单克隆抗体效价高,特异性好,生物学特性优良;鉴定了其识别RABV M蛋白的精细抗原变异表位,能够用于区分疫苗Flury毒株和其它RABV毒株;将其应用于制备检测狂犬病毒RABV的试剂盒,可以检测RABV感染以及鉴别诊断疫苗Flury毒株和其它RABV毒株。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株及应用。
背景技术
狂犬病(Rabies)是由狂犬病毒(RABV)引起的一种高度致死性人畜共患传染病。RABV感染发病后,致死率几乎为100%,是迄今为止人类历史上死亡率最高的急性传染病。目前仍无任何治疗措施,只能通过疫苗免疫接种预防RABV感染。在全球范围内有150多个国家均有狂犬病病例报道,每年约有5~7万人死于狂犬病,其中95%的病例发生在非洲和亚洲。中国的人狂犬病感染病例仅次于印度,位于亚洲第二位。犬是RABV最常见的病毒储藏库,99%的人感染狂犬病是由犬传播介导的。
RABV在病毒分类学上属于弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus)的成员,是单股副链非节段RNA病毒,其基因组主要编码核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)和RNA依赖的RNA聚合酶蛋白(L)。M蛋白,由202个氨基酸组成,约23kDa,是RABV病毒粒子最小、含量最丰富的一种非糖基化蛋白,其位于病毒囊膜和核衣壳之间,起到连接G蛋白和核衣壳的作用。M蛋白具有多种功能:参与病毒粒子的组装和出芽,对病毒粒子形态呈长杆状起到决定因素,调控病毒的转录和翻译过程,参与决定病毒的致病力,诱导细胞凋亡,抑制宿主相关基因的表达等。
目前已制备出的抗RABV蛋白的单克隆抗体主要是NP蛋白和G蛋白。NP蛋白单克隆抗体可用来区分狂犬病毒属不同血清型;G蛋白单克隆抗体则表现出较高的中和活性。RABVM蛋白的单克隆抗体报道很少,而且M蛋白单克隆抗体识别的精细表位还不清楚,缺乏对这些单克隆抗体生物学特性的了解,因此也限制了抗M蛋白单克隆抗体在病毒诊断中的应用,以及对M蛋白结构和抗原特性的研究。
发明内容
本发明的目的在于提供一种能分泌特异性识别RABV M蛋白的单克隆单体的杂交瘤细胞株,其分泌的单克隆抗体能与RABV毒株发生特异性反应,不能与疫苗Flury毒株发生反应。
为实现上述目的,本发明采用如下技术方案:
一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株,所述杂交瘤细胞株的分类命名为杂交瘤细胞株4A1,于2019年4月1日保藏于中国典型培养物保藏中心,地址:中国武汉、武汉大学,保藏编号为CCTCC NO:C201947。
本发明首先从建立的杂交瘤细胞库中得到12株稳定分泌抗狂犬病毒RABV M蛋白单克隆抗体的杂交瘤细胞,并最终筛选出杂交瘤细胞4A1株,其生物学特性优良。采用间接酶联免疫吸附分析法测得制备的小鼠腹水单克隆抗体4A1的效价为1×105。
本发明还提供了由杂交瘤细胞4A1株制备得到的抗狂犬病毒M蛋白的单克隆抗体。
本发明首次鉴定了该单克隆抗体所识别的变异抗原表位,其氨基酸序列如SEQ IDNO:1和SEQ ID NO:2所示。该抗原表位在不同的RABV毒株M蛋白上相对保守,但在疫苗Flury毒株上存在抗原变异(Flury毒株氨基酸序列如SEQ ID NO:3所示),导致单克隆抗体不能识别。因此,本发明提供的单克隆抗体能与狂犬病毒毒株发生特异性反应,但是不能与疫苗Flury毒株反应。
本发明提供了杂交瘤细胞4A1株在制备检测狂犬病毒RABV的试剂盒中的应用。所述杂交瘤细胞4A1株可以应用于ELISA、免疫荧光、Western Blot、免疫组化等方法,可以在制备临床诊断RABV感染试剂中得到应用,或在不以诊断为目的的RABV的病原检测中得到应用。
本发明提供了所述的单克隆抗体在制备检测狂犬病毒RABV的试剂盒中的应用。所述单克隆抗体可以应用于ELISA、免疫荧光、Western Blot、免疫组化等方法,可以在制备临床诊断RABV感染试剂中得到应用,或在不以诊断为目的的RABV的病原检测中得到应用。
本发明还提供了一种检测狂犬病毒RABV的试剂盒,包括所述的抗狂犬病毒M蛋白的单克隆抗体。
本发明具备的有益效果:
本发明提供的杂交瘤细胞4A1株生物学特性优良,其分泌的单克隆抗体4A1能与RABV毒株发生特异性反应,但是不能与Flury毒株反应;本发明提供的单克隆抗体可应用于制备ELISA、免疫荧光、Western Blot、免疫组化等方法的试剂盒,可用于RABV的临床检测或者是Flury毒株和其他毒株的鉴别诊断。
附图说明
图1为单克隆抗体的特异性实验。
图2为单克隆抗体识别表位在不同RABV毒株的比对。
图3为单克隆抗体与不同毒株的反应性。
图4为单克隆抗体与不同突变表位的反应性。
图5为单克隆抗体的免疫组化实验。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1
(一)分泌单克隆抗体杂交瘤细胞株的建立
1.CVS-11 M蛋白的原核表达及纯化
根据GenBank中注册的CVS-11株(Liu Juan,et al.BECN1-dependent CASP2incomplete autophagy induction by binding to rabies virusphosphoprotein.Autophagy,2017,13(4):739-753)M蛋白基因(序列号:ADJ29910.1)的核苷酸序列设计特异引物,以提取的病毒RNA反转录产物为模板,进行M蛋白基因的扩增,然后将M蛋白的基因克隆至pET-28a(+)载体,测序验证正确后转化感受态细胞E.coli BL21(DE3),在37℃条件下用IPTG(1mM)诱导约5h后,用Ni-NTA柱(购自QiAGEN)纯化重组M蛋白。重组M蛋白的表达和纯化用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blot)分析。
2.动物免疫
用以上纯化的M蛋白作为免疫原,背部皮下注射免疫6~8周龄雌性BALB/c小鼠(购自上海西普尔-必凯实验动物有限公司),100μg/只。首免用等体积弗氏完全佐剂(购自Sigma)乳化抗原,以后每隔14d用弗氏不完全佐剂(购自Sigma)乳化抗原,加强免疫2次。第3次免疫一周后断尾采血,测定小鼠血清抗体效价。选取血清抗体效价>106的小鼠,细胞融合前3d用不加佐剂的抗原以腹腔注射的方式再次加强免疫(200μg/只)。
3.细胞融合
采用PEG细胞融合方法。取骨髓瘤细胞SP2/0与完成免疫的BALB/c小鼠脾细胞按1:10的比例充分混匀,在1min内加完预热至37℃的50%PEG-4000(购自Sigma)1mL,边加边轻轻振荡,1min内加完,再静置1min。沿管壁加入已预热至37℃的无血清RPMI-1640(购自Hyclone)培养基,终止融合反应。要求在第1min内加完4mL,前30s内加入1mL,后30s内加入3mL;在第2min内加完11mL,最后3min内加至总体积40mL。然后1000rpm低速离心5min,弃去上清,再沿管壁加入30mLRPMI-1640不完全培养基,再次离心、弃上清,以尽可能地去除残留的融合剂PEG。沿50mL离心管壁加入40mL预热至37℃的含HAT(购自Sigma)和10%FBS(购自Gibco)完全培养基,轻柔地吹打以重悬细胞。将重悬的细胞转移至10cm细胞培养皿内,将混匀的细胞以100μL/孔接种至已铺有饲养层细胞的96孔细胞培养板中,然后将细胞培养板放置于37℃、5%CO2的细胞培养箱(购自Thermo)中静置培养。5d后改用含HT(购自Sigma)和10%FBS(购自Gibco)的RPMI-1640(购自Hyclone)培养基,10d后换成含10%FBS(购自Gibco)的RPMI-1640(购自Hyclone)培养基培养,当融合的细胞生长至96孔板孔底1/10-1/5时,取培养上清检测抗体。
4.杂交瘤细胞的筛选
用0.05mol/L碳酸盐缓冲液(pH9.6)对纯化的M蛋白进行稀释,并以0.02mg/mL蛋白浓度包被ELISA板(100μL/孔),置4℃包被过夜(不少于12h),PBST洗涤3次,每次5min,第3次后拍干;以含10%脱脂奶粉的PBS进行封闭(200μL/孔),37℃放置1h,PBST洗涤3次,每次5min,第3次后拍干;将融合后14d的细胞上清、1:1000稀释的免疫小鼠阳性血清和1:1000稀释的小鼠阴性血清分别加入相应孔内(100μL/孔),37℃作用1h,PBST洗涤3次,每次5min,第3次后拍干;加入1:10000稀释的辣根过氧化物酶(HRP)标记的羊抗鼠IgG(购自KPL)的脱脂奶粉(100μL/孔),37℃作用1h,PBST洗涤3次,每次5min,第3次后拍干;加入底物TMB-H2O2(100μL/孔),37℃避光作用10min;每孔加入50μL硫酸(2mol/L)终止反应。以空白对照调零,酶标仪测定各孔的OD450nm值,P为各待测孔中样品的OD450nm值,N为阴性参考血清的OD450nm值,将N≤0.1,阳性参考血清的OD450nm值与阴性参考血清的OD450nm值的比值≥2.1,即阴、阳性对照成立的前提下,将P/N≥2.1的检测孔判为阳性,2.1>P/N≥1.5的检测孔杂交瘤细胞株判为可疑,P/N<1.5的检测孔判为阴性。隔3d后再检测一次,选择两次检测结果均为阳性的杂交瘤细胞株进行克隆化。
5.杂交瘤细胞的克隆化
首先将阳性孔活细胞用台盼兰进行染色和计数,用含10%FBS(购自Gibco)的RPMI-1640培养基(购自Hyclone)稀释成100个细胞/10mL培养基的细胞悬液,将稀释的细胞悬液加入已铺有饲养层细胞的96孔细胞培养板(100μL/孔),置于37℃、5%CO2培养箱中培养,每天于光学显微镜下观察克隆细胞的形成情况,记录只有单个克隆的生长孔,当细胞生长至96孔板孔底1/10-1/5时取细胞上清用于ELISA检测。选择检测为阳性的单克隆细胞再进行同样的克隆,直至克隆后所有细胞孔上清检测均为阳性,取OD450nm值最大的孔进行扩大培养,最终从杂交瘤细胞库中筛选到了12株稳定分泌RABV特异性单克隆抗体的杂交瘤细胞。
6.单克隆抗体腹水的制备
将灭菌的液体石蜡腹腔注射6~8周龄的雌性BALB/c小鼠(购自上海西普尔-必凯实验动物有限公司),0.5mL/只,7d后小鼠腹腔注射杂交瘤细胞株(0.5mL/只,含2×106~5×106个杂交瘤细胞),7~10d后采取腹部明显鼓起小鼠的腹水,5000rpm离心10min,收集上清,即为RABV单克隆抗体,分装后于-20℃保存备用。
(二)单克隆抗体4A1的生物学特性
生物保藏
杂交瘤细胞4A1株,于2019年4月1日保藏于中国典型培养物保藏中心,地址:中国武汉武汉大学,保藏编号为CCTCC NO:C201947,分类命名:杂交瘤细胞株4A1。
1.单克隆抗体的类型测定
亚类测定按照单克隆抗体亚类鉴定试剂盒(购自Thermo)操作说明书进行,结果显示,单克隆抗体4A1亚型为IgG2bκ。
2.单克隆抗体效价和稳定性测定
将分泌单克隆抗体的杂交瘤细胞4A1株进行连续培养传代20次、液氮冻存与复苏试验,以纯化的原核表达的RABV M蛋白为检测抗原,通过间接ELISA方法连续检测单克隆抗体4A1的效价,结果各代次杂交瘤细胞分泌的抗体ELISA效价基本一致,达到103-104。
3.单克隆抗体的特异性鉴定
采用间接免疫荧光试验验证单克隆抗体4A1与RABV的反应性。用CVS-11毒株感染BHK-21细胞(购自ATCC),培养48h后吸弃细胞培养液,加入-20℃预冷的甲醇-丙酮(1:1)固定液,200uL/孔,-20℃固定15min后,弃去固定液,用PBS洗涤3次后拍干;加入5%脱脂奶粉(200uL/孔),37℃封闭1h后,PBST洗涤3次,加入单克隆抗体4A1(1:500),37℃孵育1h,PBS洗涤3次,拍干后加入1:500稀释的FITC标记的羊抗鼠IgG抗体(购自KPL),37℃孵育1h,PBS洗涤3次,拍干后每孔加入50uL PBS,置于荧光显微镜下观察。单克隆抗体4A1与CVS-11感染的BHK-21细胞反应,产生绿色荧光,而正常未感染CVS-11毒株的BHK-21细胞无绿色荧光(见图1)。
4.单克隆抗体识别抗原表位的鉴定
将M蛋白基因609bp序列(aa1-aa203)分为相互重叠的片段,设计引物(表1),上、下游分别引入HindⅢ/KpnⅠ酶切位点,将扩增纯化的目的片段克隆于表达载体pET-28a(+)中构建重组质粒,转化感受态细胞E.coli BL21(DE3),诱导表达的重组His标签多肽与单克隆抗体进行Western blot验证,对阳性区段逐步进行截短表达,完成抗原表位初步定位。为精确鉴定抗原表位,从初步定位的肽段的N端和C端分别以1个氨基酸残基为单位逐步递减设计引物,上、下游分别引入BamHⅠ/XhoⅠ酶切位点(表2),通过上、下游引物退火得到目的片段,将目的片段克隆于表达载体pGEX-4T-1中构建重组质粒,转化感受态细胞E.coli BL21(DE3),用Western blot检测诱导表达的重组GST多肽与单克隆抗体的反应性,确定单克隆抗体识别的抗原表位为25PPYDDD30。
表1鉴定单克隆抗体抗原表位所在区段引物
表2鉴定单克隆抗体精细表位引物
5.单克隆抗体表位序列分析
将鉴定的抗原表位序列25PPYDDD30与NCBI注册的RABV毒株M蛋白氨基酸序列进行比对分析(图2),结果表明该抗原表位在RABV毒株中主要是25PPDDDD30序列。对氨基酸P26L和Y27D分别突变后进行Western blot检测,结果发现P26L和Y27D突变后,该单克隆抗体仍然能够与之反应,说明单克隆抗体4A1识别CVS-11毒株的抗原表位发生Y27D置换,但是并不影响表位的抗原性(图4)。同时,Western blot实验结果发现本发明的M蛋白单克隆抗体可与CVS-11、WT型毒株反应,不能与HEP-Flury毒株(购自中国兽医微生物菌种保藏管理中心,保藏号:CVCC AV2013)反应(图3),同时RABV毒株的N蛋白单克隆抗体可分别与3个毒株发生特异性反应,说明病毒感染细胞是成功的。RABV序列比对发现HEP-Flury毒株的抗原表位序列是25PPDGDD30,进一步将25PPYDDD30表位进行氨基酸D28G突变,Western blot实验发现单克隆抗体4A1无法与之识别(图4)。然而本发明鉴定的抗原表位与Flury及变体毒株(图2:HEP-Flury、rHEP5.0-CVSG、LEP-Flury、Flury-LEP-C)发生氨基酸D28G突变,影响了表位的抗原性,因此本发明的单克隆抗体也可用于将Flury及变体毒株与其它流行RABV毒株进行区分。
6.单克隆抗体的免疫组化实验
颅内注射感染6-8周龄C57BL/6小鼠(购自上海西普尔-必凯实验动物有限公司)30uL CVS-11毒株(105TCID50/mL)和HEP-Flury毒株,待小鼠发病症状明显时,用2.5%avertin(购自sigma)麻醉小鼠,取脑。以颅内注射等体积的DMEM为空白对照。
(1)固定与包埋:4%多聚甲醛固定24h,经酒精脱水与透明,浸石蜡包埋,切片。
(2)石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min-二甲苯Ⅱ15min-二甲苯III 15min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-85%酒精5min-75%酒精5min-蒸馏水洗。
(3)抗原修复:组织切片置于盛满柠檬酸抗原修复缓冲液(pH6.0)(购自福州迈新生物技术开发公司)的修复盒中于微波炉内中火5min,停火5min,转低火10min进行抗原修复,此过程中应防止缓冲液过度蒸发,切勿干片。自然冷却后将玻片置于PBS(pH7.4)中洗涤3次,每次5min。
(4)阻断内源性过氧化物酶:切片放入3%双氧水溶液,室温避光孵育25min,将玻片置于PBS(pH7.4)中洗涤3次,每次5min。
(5)血清封闭:在组化圈内滴加3%BSA(购自Servicebio)均匀覆盖组织,室温封闭30min。
(6)加一抗:轻轻甩掉封闭液,在CVS-11毒株、HEP-Flury毒株和DMEM感染脑组织切片上滴加1:500稀释的一抗(本发明单克隆抗体),切片平放于湿盒内4℃孵育过夜(湿盒内加少量水防止抗体蒸发)。其中HEP-Flury毒株感染脑组织切片再做一个阳性对照,该切片以HEP-Flury感染小鼠的血清为一抗。
(7)加二抗:玻片置于PBS(pH7.4)中洗涤3次,每次5min。切片稍甩干后在圈内滴加HRP标记的羊抗鼠IgG(购自KPL)覆盖组织,室温孵育50min。
(8)DAB显色:玻片置于PBS(pH7.4)中洗涤3次,每次5min。切片稍甩干后在圈内滴加新鲜配制的DAB显色液(购自福州迈新生物技术开发公司),显微镜下控制显色时间,阳性为棕黄色,自来水冲洗切片终止显色。
(9)复染细胞核:苏木素复染3min左右,自来水洗,苏木素分化液分化数秒,自来水冲洗,苏木素返蓝液返蓝,流水冲洗。
(10)脱水封片:将切片依次放入75%酒精5min-85%酒精5min--无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片。
(11)显微镜镜检,图像采集分析显示苏木素染细胞核为蓝色,DAB显出的阳性表达为棕黄色,即该单克隆抗体能对感染的脑组织中的RABV病毒进行标记(图5)。
序列表
<110> 浙江大学
<120> 分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株及应用
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Claims (6)
1.一种分泌抗狂犬病毒M蛋白单克隆抗体的杂交瘤细胞株,其特征在于,所述杂交瘤细胞株的分类命名为杂交瘤细胞株4A1,其保藏编号为CCTCC NO:C201947。
2.如权利要求1所述的杂交瘤细胞株在制备检测狂犬病毒RABV的试剂盒中的应用。
3.一种抗狂犬病毒M蛋白的单克隆抗体,其特征在于,所述单克隆抗体由权利要求1所述的杂交瘤细胞株制备得到。
4.如权利要求3所述的抗狂犬病毒M蛋白的单克隆抗体,其特征在于,所述单克隆抗体识别的狂犬病毒RABV抗原表位的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2所示。
5.如权利要求3所述的单克隆抗体在制备检测狂犬病毒RABV的试剂盒中的应用。
6.一种检测狂犬病毒RABV的试剂盒,其特征在于,包括如权利要求3所述的抗狂犬病毒M蛋白的单克隆抗体。
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