CN105671001B - 分泌抗南方菜豆花叶病毒单抗杂交瘤细胞株及其单抗应用 - Google Patents
分泌抗南方菜豆花叶病毒单抗杂交瘤细胞株及其单抗应用 Download PDFInfo
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Abstract
本发明公开了一种分泌抗南方菜豆花叶病毒单抗杂交瘤细胞株及其单抗的应用。用纯化的南方菜豆花叶病毒(SBMV)粒子为抗原免疫BALB/c小鼠,经细胞融合、筛选、克隆,获得1株能稳定传代并分泌抗SBMV单克隆抗体的杂交瘤细胞株19H9,其保藏号为CGMCC No.12004。该杂交瘤细胞株分泌的单抗腹水间接ELISA效价达10‑7,抗体类型及亚类为IgG1、kappa轻链。该单抗与SBMV有特异性反应。利用19H9单抗建立检测豆科植物中SBMV的ACP‑ELISA和dot‑ELISA方法,这两种方法检测病叶的灵敏度分别达到1:81920和1:5120倍稀释(w/v,g/mL)。该杂交瘤细胞株19H9及其单抗的获得和血清学检测方法的建立为该病毒病的诊断、检测及科学防控提供物质和技术支撑。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种分泌抗南方菜豆花叶病毒单克隆抗体的杂交瘤细胞株及其单抗的应用。
背景技术
南方菜豆花叶病毒(Southern bean mosaic virus,SBMV)是南方菜豆花叶病毒属(Sobemovirus)的典型成员,主要包括SBMV-B和SBMV-C两个株系,最早由Zaumeyer和Harter发现报道。该病毒主要分布于热带、亚热带地区,温带地区也有分布,可以感染危害大豆、菜豆、南瓜等植物。南方菜豆花叶病毒病在田间普遍发生,传播途径多,危害严重。自然状况下可引起菜豆、豇豆、黑绿豆和大豆的花叶、斑驳病害,导致花、果、种子数量下降,产量降低。丹麦、澳大利亚、埃及、东非等一些国家和地区将其列为检疫对象。我国具有扎根、繁殖、扩散的条件,一旦传入,后患无穷。目前,已将其列入我国二类检疫性有害生物,并从1992年起依法实施检疫。
SBMV病毒粒子为等轴对称的二十面体(T=3),无包膜,直径约为30nm。其基因组包含一分子线状单链正义RNA,长约4100-4500nt,约占病毒粒子重量的21%。SBMV在CsCl中的浮力密度为1.36g/cm3,沉降系数为115S。病毒的致死温度为90-95℃,稀释限点为10-5-10-6,体外存活期(18-22℃)为1.65-20d。病毒RNA的3’端既无Poly(A),也无类似tRNA结构,其5’端有一个VPg,可能是病毒侵染所必需的,含有4个ORF,ORF1编码一个21kDa的P1蛋白,该蛋白与病毒的胞间运动有关;ORF2编码的105kDa P2蛋白可能是聚合酶;ORF3位于ORF2之内,编码一个18kDa蛋白,其功能还未知;ORF4编码30kDa的外壳蛋白。
该病毒粒子主要存在于寄主植物叶肉细胞中,常分散分布或聚集在细胞质和液泡中。SBMV侵染的植株细胞质内形成大片病毒结晶体,并产生许多含有细的纤维状物的特殊小泡结构。感染细胞呈现囊泡化,叶绿体受挤压变形,但在叶绿体和线粒体中没有发现病毒粒子。SBMV的寄主范围很窄,多为豆科植物,自然寄主为菜豆和豇豆,其中SBMV-B株系能侵染菜豆的大多数品种,而株系SBMV-C只能侵染豇豆。其人工接种寄主相对广泛,可侵染大豆、绿豆、赤小豆、红花菜豆、棉豆、豌豆、蚕豆、草木樨等约12个属23种植物。侵染菜豆后引起的致病症状主要包括斑驳和花叶,但不同品种上的症状差异很大,有的为系统发病,有的是局部发病,有的症状轻微,有的还伴随皱缩和沿脉变色等症状。SBMV在田间主要通过叶甲进行传播,也可通过汁液机械方式和种子传播。
目前,菜豆、豇豆等豆科植物在我国种植面积大、范围广,且随着进出口贸易和种质交换日益增多,此病毒的传入机会也大大增加,为了保护我国豆类作物的生产,防止该病毒自境外传入国内,应建立一套简便、快速、行之有效的检疫检测方法。当前,对于SBMV的口岸检测,一般采用生物学检测、分子生物学检测和电镜检测技术,这几种方法效率低,并不适合大批量样品检测。血清学方法操作简单,特异性好,同时可以检测大规模样品,但必须依赖于特异性的病毒单抗。本发明主要针对南方菜豆花叶病毒单克隆抗体的制备及其检测应用展开工作,利用杂交瘤技术制备了1株能分泌抗SBMV单抗的杂交瘤细胞19H9,并以其分泌的单抗为核心建立了检测该病毒的血清学方法和检测试剂盒,以提高我国对南方菜豆花叶病毒的检测水平和速度,防止该病毒自境外传入国内。
发明内容
本发明的目的是克服现有技术的不足,提供一种分泌抗南方菜豆花叶病毒单克隆抗体的杂交瘤细胞株及其单抗的应用。
分泌抗南方菜豆花叶病毒单克隆抗体的杂交瘤细胞株19H9,于2016年1月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.12004,它能分泌抗南方菜豆花叶病毒的特异性单克隆抗体。
一种所述的杂交瘤细胞株19H9分泌的抗南方菜豆花叶病毒的单克隆抗体,抗南方菜豆花叶病毒的单克隆抗体腹水间接ELISA效价达10-7,抗体类型及亚类为IgG1、kappa轻链。利用ACP-ELISA方法分析发现,该单抗检测病叶的灵敏度达到1:81920倍稀释(w/v,g/mL)。
抗南方菜豆花叶病毒的单克隆抗体仅与感染南方菜豆花叶病毒的病叶组织有特异性免疫反应,而与感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶组织及健康的大豆、毛豆、豇豆、豌豆、蚕豆和菜豆植物组织均不发生任何免疫反应。
抗南方菜豆花叶病毒单克隆抗体在抗南方菜豆花叶病毒检测上的应用是以单克隆抗体为核心建立的各种免疫学检测方法和免疫学试剂盒。
本发明与现有技术相比具有的有益效果:1)提供的杂交瘤细胞株19H9分泌抗南方菜豆花叶病毒特异性单克隆抗体,以该单抗为核心建立的dot-ELISA和ACP-ELISA等免疫学方法及用这些方法建立的试剂盒能高度特异、准确、灵敏地检测南方菜豆花叶病毒;2)利用本发明所制备的单克隆抗体检测南方菜豆花叶病毒不需要昂贵的电子显微镜、PCR仪等设备;3)利用本发明所制备的单克隆抗体可有效地用于口岸及田间作物中的南方菜豆花叶病毒的检测。
附图说明
图1是dot-ELISA方法检测SBMV的灵敏度分析;
图2是dot-ELISA方法检测SBMV的特异性分析;
1列上下2个点为2张感染SBMV的菜豆病叶;2列上下2个点为2张感染菜豆普通花叶病毒的菜豆病叶;3列上下2个点为2张健康菜豆叶片;4列上下2个点为2张健康大豆叶片;5列上下2个点为2张健康毛豆叶片;6列上下2个点分别为感染黄瓜花叶病毒的病叶和健康豇豆叶片;7列上下2个点分别为感染南方豇豆花叶病毒病叶和健康豌豆叶片;8列上下2个点分别为感染烟草花叶病毒病叶和健康蚕豆叶片。
图3是dot-ELISA方法检测口岸豆科植物样品中SBMV的代表性结果;
图4是RT-PCR方法检测口岸豆科植物样品中SBMV的代表性结果。
生物保藏
杂交瘤细胞株19H9于2016年1月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,邮编:100101,保藏号为CGMCCNo.12004。
具体实施方式
分泌抗南方菜豆花叶病毒单克隆抗体的杂交瘤细胞株19H9于2016年1月7日保藏于中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.12004,它能分泌抗南方菜豆花叶病毒的单克隆抗体。
一种所述的杂交瘤细胞株19H9分泌的抗南方菜豆花叶病毒的单克隆抗体,抗南方菜豆花叶病毒的单克隆抗体腹水间接ELISA效价达10-7,抗体类型及亚类为IgG1、kappa轻链。利用ACP-ELISA方法分析发现,该单抗检测病叶的灵敏度达到1:81,920倍稀释(w/v,g/mL)。
抗南方菜豆花叶病毒的单克隆抗体仅与感染南方菜豆花叶病毒的病叶组织有特异性免疫反应,而与感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶组织及健康的大豆、毛豆、豇豆、豌豆、蚕豆和菜豆植物组织均不发生任何免疫反应。
抗南方菜豆花叶病毒单克隆抗体在该病毒检测上的应用是以单克隆抗体为核心建立的各种免疫学检测方法和免疫学试剂盒。
本发明提供的杂交瘤细胞株19H9能大量分泌抗南方菜豆花叶病毒单抗,且其分泌的单抗特异性强、效价高、稳定性好。以该单抗为核心建立的检测SBMV的高通量的血清学方法及其检测试剂盒可成功应用于口岸SBMV的检测,从而为提高我国对南方菜豆花叶病毒的检测水平,防止该病毒自境外传入国内提供物质和技术支持。
下面结合实施例和附图对本发明作进一步说明。
一、杂交瘤细胞的获得及其单克隆抗体的制备
1.免疫原及检测抗原的制备
用下面的操作步骤提纯病毒粒子:
1)将大研钵和组织搅拌器预冷;
2)称取500g感染SMBV菜豆病叶,每100g病叶中加入pH 7.5的0.5M磷酸盐缓冲液(即PB缓冲液)200mL(含0.01M Na-EDTA和0.1%巯基乙醇),匀浆2min后用双层棉纱布过滤,滤液6,000rpm离心20min,去植物组织残渣;
3)所得上清液边搅拌边滴加至终浓度2.5%Triton X-100,4%PEG(分子量6,000)和0.1M NaCl,4℃搅拌4h以上;
4)11,000r/min离心15min得沉淀;
5)沉淀用pH 7.5的0.5M PB(含0.01M MgCl2和0.5M脲)充分悬浮,6,000rpm离心15min后吸出上清置于离心管,沉淀再悬浮、离心重复3次;
6)合并上清液,33,000rpm超速离心100min,所得沉淀用0.5M PB悬浮后8,000rpm离心15min,收集上清,沉淀再悬浮、离心重复3次;
7)合并上清液后加到底部已有30%蔗糖垫的离心管中,33,000rpm超速离心100min;
8)所得沉淀用pH 7.5的0.5M PB(含0.01M MgCl2)悬浮,悬浮液即为病毒提纯液;
9)用电子显微镜观察提纯样品,发现大量高纯度的南方菜豆花叶病毒粒子。2.免疫动物
用SBMV提纯病毒免疫六周龄体重18-20g BALB/c雌性小鼠。即SBMV提纯病毒粒子100μL/只与等体积福氏完全佐剂混合,充分乳化后,经背腹部皮下多点注射0.2mL/只,间隔3周,取与一免等量抗原和等体积的福氏不完全佐剂充分乳化后,第二次腹腔注射0.2mL/只,过3周后用加倍剂量的抗原进行腹腔注射,3天后取脾细胞进行细胞融合。
3.细胞融合
取上述免疫小鼠脾细胞与小鼠骨髓瘤细胞(SP2/0)按7:1的比例在无血清的RPMI-1640(Gibco)培养基中混匀,1,500rpm离心5min,去除培养基,在37℃水浴中加入1mL 50%PEG(分子量1500)并融合2min,用无血清的RPMI-1640培养基终止融合后1,500rpm离心5min,沉淀用HAT培养基悬浮,分装到96孔含有饲养细胞的细胞板中,并置于37℃,含5%CO2的细胞培养箱中培养。
4.杂交瘤细胞、阳性孔的筛选及其克隆
细胞培养箱中培养5天后,用HAT培养基换液一次,第10天用HT培养基换液,等到融合细胞覆盖孔底10-30%时,以感染SBMV病毒的菜豆叶片和提纯病毒为抗原包被,用常规间接ELISA方法筛选分泌单抗的阳性孔,共获137个阳性孔。选择5个呈强阳性反应的细胞孔,进行有限稀释法克隆,获得1株能分泌抗SBMV的特异性单抗的杂交瘤细胞株19H9。经6个月以上体外传代和多次冻存复苏后,细胞株均能良好生长,并稳定分泌抗体。经扩大培养后,用于腹水制备和液氮保存。
5.单克隆抗体腹水制备及纯化
取8周龄左右BALB/c小鼠,腹腔注射0.3-0.5mL降植烷(Sigma),7-10天后腹腔注入8×105个杂交瘤细胞,注射后7-10天可见小鼠腹部明显膨大,注射针头采取腹水,8,000rpm离心3min,收集上清液即为单抗腹水。取1倍体积腹水加2倍体积0.06M pH 4.8醋酸缓冲液稀释,室温下边搅拌边加辛酸(30uL/mL腹水),4℃澄清1h,12,000rpm离心20min,收集上清,再用50%饱和硫酸铵沉淀免疫球蛋白,4℃放置2h,3,000rpm离心20min,沉淀用2倍体积的PBS溶液溶解,在4℃流动透析24h后即获纯化的腹水抗体,-70℃保存。
6.单克隆抗体的类型及亚类鉴定和腹水效价测定
用Sigma公司的用于鉴定单抗类型的DAS-ELISA试剂盒鉴定单抗的类型及亚类,结果显示,19H9单抗亚类为IgG1、kappa轻链。用常规间接ELISA方法检测单抗腹水效价,结果表明19H9单抗腹水效价达到10-7。
7.单克隆抗体的特异性检测
用感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶组织及健康的大豆、毛豆、豇豆、豌豆和蚕豆的植物组织粗提液包被ELISA板,以健康菜豆叶片提取液作阴性对照,以感染南方菜豆花叶病毒的菜豆汁液为阳性对照,用ACP-ELISA法测定单抗的特异性反应。ACP-ELISA方法具体为:上述病毒感染的病叶按1:30(w/v,g/mL)加入ELISA包被液匀浆后100uL/孔包被ELISA板,4℃过夜或37℃2h,使其吸附于ELISA聚苯乙烯板孔;PBST洗涤三次后用3%的脱脂奶粉封闭30-60min;加入适当稀释的单抗100uL/孔,37℃1-2h;PBST洗涤三次后加入适当稀释的碱性磷酸脂酶(AP)标记羊抗鼠IgG二抗(Sigma公司)100uL/孔,37℃1-2h;PBST洗涤四次后,用PNPP底物显色30-60min后用2M氢氧化钠终止反应,用酶标仪读取OD405的值,以与阴性OD值比值大于2.1为阳性。结果发现,19H9单抗对感染SBMV病叶有特异性反应,而与感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶及健康的大豆、毛豆、豇豆、豌豆、蚕豆和菜豆植物组织均无任何特异性免疫反应。
二、检测SBMV免疫学方法及其试剂盒的建立
1.检测SBMV的ACP-ELISA方法
1.1ACP-ELISA方法的操作步骤:
(1)病叶按1:30(w/v,g/mL)比例加入0.05M碳酸盐缓冲液(pH 9.6)匀浆,5,000rpm离心3min,离心上清100uL/孔包被ELISA板,以SBMV病叶为阳性对照,健叶为阴性对照,37℃2h,或4℃过夜;
(2)PBST洗涤后用3%脱脂奶粉封闭30min;
(3)加入适当稀释的单抗腹水,100uL/孔,37℃1h;
(4)PBST洗涤后加入适当稀释的碱性磷酸脂酶(AP)标记的羊抗鼠IgG二抗(Sigma),100uL/孔,37℃1h;
(5)用PBST洗涤后加入PNPP硝基磷酸盐底物,100uL/孔,室温30min;
(6)用肉眼观察,底物颜色变成黄绿色的孔为阳性,或用2M氢氧化钠终止反应后,用酶联免疫检测仪测OD405,以P/N>2.1作为阳性判断标准。
1.2ACP-ELISA方法检测灵敏度和特异性的确定
用常规方阵试验确定ACP-ELISA检测方法中单抗和酶标二抗的最适工作浓度,试验表明19H9单抗及酶标二抗的最适工作浓度分别为1:5,000和1:8,000倍稀释。以抗体的最适工作浓度建立检测SBMV的ACP-ELISA方法对病叶粗提液进行1:20-1:655,360倍比稀释(w/v,g/mL),并分别以相应稀释度的健叶粗提液作阴性对照,用上述ACP-ELISA方法进行检测。结果表明ACP-ELISA方法对1:81,920倍稀释(w/v,g/mL)的病叶粗提液仍呈阳性反应,即对病叶的检测灵敏度达到1:81,920倍稀释(w/v,g/mL),表明该单抗及建立的ACP-ELISA方法具有高度的灵敏性和可靠性。该方法检测SBMV病叶粗提液呈强阳性反应,检测感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶及健康的大豆、毛豆、豇豆、豌豆、蚕豆和菜豆植物组织呈阴性反应,且阴阳性对比差异极显著,说明该方法和单抗的特异性均很好。
2.dot-ELISA方法的建立及田间检测应用
2.1检测豆科植物中SBMV的dot-ELISA方法的操作步骤
将菜豆叶片称重后用按1:30(w/v,g/mL)的比例加入0.01M PBS(pH 7.4)后匀浆;匀浆液5,000rpm离心3min;取3μL离心上清点到硝酸纤维素(NC)膜上,同时设置健康和感染SBMV菜豆叶片组织粗提液分别作为阴性和阳性对照;室温干燥10-20min;NC膜浸入到含5%脱脂奶粉的PBST(含0.05%Tween-20的0.01M PBS)封闭液中室温封闭30min;NC膜放入适度稀释的单抗中室温孵育30-60min;用PBST洗膜3-4次,每次3min;NC膜放入适度稀释的AP酶标记羊抗鼠IgG二抗中室温孵育30-60min;PBST洗膜4-5次,每次3min;66μL NBT和33μLBCIP底物(Promega)加入到10mL底物缓冲液(0.1M Tris Cl、0.1M NaCl、0.025M MgCl2、pH9.5)混匀,膜放入底物液中反应,肉眼观察结果;待阳性对照显色明显而阴性没有任何显色时在自来水中漂洗终止反应,拍照记录结果。
2.2dot-ELISA方法检测灵敏度和特异性的确定
用常规的方阵试验确定dot-ELISA检测方法中单抗和酶标二抗的最适工作浓度,试验结果表明,19H9单抗及酶标二抗的最适工作浓度分别为1:5,000和1:8,000倍稀释。以上述抗体的最适工作浓度建立检测SBMV的dot-ELISA方法。灵敏度分析表明,当菜豆病叶稀释到1:5,120倍(w/v,g/mL)时,以19H9单抗为核心建立的dot-ELISA方法检测仍呈现紫色的阳性斑点,即其检测病叶的灵敏度达到1:5,120倍稀释(w/v,g/mL)(图1)。特异性分析表明,该方法检测SBMV感病植株有强烈的特异性阳性反应,而检测感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶及健康的大豆、毛豆、豇豆、豌豆、蚕豆和菜豆植物组织时均呈阴性反应(图2)。
2.3dot-ELISA检测方法的田间应用
用建立的dot-ELISA方法对2015年由上海检验检疫局提供的疑似发病豆科样品进行检测,结果发现,50个检测样品中有23个样品产生紫色的阳性斑点(图3),样品同时进一步用RT-PCR检测,结果表明所有dot-ELISA检测阳性样品中均扩增到SBMV特异性的PCR产物(图4),PCR产物核酸测序表明阳性样品确实感染SBMV,而dot-ELISA检测阴性的样品中RT-PCR没有扩增到任何基因片段。说明该建立的dot-ELISA方法能准确、可靠地用于豆科样品中南方菜豆花叶病毒的检测。
3.南方菜豆花叶病毒dot-ELISA检测试剂盒
1)试剂盒主要成分:
以上试剂均保存于4℃下
硝酸纤维素膜(NC)10张2)检测豆科植物样品的操作步骤:
a.将豆科样品叶片称重后按1:30(w/v,g/mL)比例加入0.01M PBS(pH 7.4)后匀浆;
b.匀浆液5,000rpm离心3min;
c.取3μL上清点到NC膜上,同时设置健康和感染SBMV菜豆叶片粗提液分别作为阴性和阳性对照,室温干燥10-20min;
d.NC膜浸入到含3%脱脂奶粉的PBST(含0.05%Tween-20的0.01M PBS)封闭液中室温封闭30min;
e.NC膜放入1:2,000倍稀释的单抗中室温孵育30-60min;
f.用PBST洗膜3-4次,每次3min;NC膜放入1:3,000稀释的AP酶标记羊抗鼠IgG二抗中室温孵育30-60min;
g.PBST洗膜4-5次,每次3min;66μL NBT和33μL BCIP底物加入到10mL底物缓冲液(0.1M Tris Cl、0.1M NaCl、0.025M MgCl2、pH 9.5)混匀,膜放入底物液中显色10-20min;
h.肉眼观察结果,待阳性对照显色明显(紫色),而阴性没有任何显色时用自来水漂洗终止反应,拍照记录结果。
3)保存及有效期
于2-8℃避光保存,有效期12个月。
4)缓冲液配方:
磷酸盐缓冲液(PBS,0.01M,pH 7.4):
加蒸馏水950溶解后调pH至7.4,定容至1,000mL
ELISA洗涤液(0.01M PBST):
1,000mL 0.01M PBS中加0.5mL Tween-20
ELISA封闭液:
0.01M PBST中加入脱脂奶粉至终浓度5%(w/v)。
Claims (3)
1.一种分泌抗南方菜豆花叶病毒单克隆抗体的杂交瘤细胞株19H9,其特征在于能分泌抗南方菜豆花叶病毒的特异性单克隆抗体,杂交瘤细胞株19H9于2016年1月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.12004。
2.一种如权利要求1所述的杂交瘤细胞株19H9分泌的抗南方菜豆花叶病毒的单克隆抗体,其特征在于该单克隆抗体腹水间接ELISA效价达10-7,抗体类型及亚类为IgG1、kappa轻链;利用ACP-ELISA方法分析发现,该单抗检测病叶的灵敏度达到1:81920倍稀释;该单克隆抗体仅与感染南方菜豆花叶病毒的病叶组织有特异性免疫反应,而与感染菜豆普通花叶病毒、南方豇豆花叶病毒、烟草花叶病毒、黄瓜花叶病毒的病叶组织及健康的大豆、毛豆、豇豆、豌豆、蚕豆和菜豆植物组织均不发生任何免疫反应。
3.一种如权利要求2所述的抗南方菜豆花叶病毒单克隆抗体在南方菜豆花叶病毒检测中 的应用。
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