CN105543177B - 分泌抗柑橘黄化脉明病毒单抗的杂交瘤细胞株及其单抗应用 - Google Patents
分泌抗柑橘黄化脉明病毒单抗的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柑橘黄化脉明病毒(CYVCV)单抗的杂交瘤细胞株及其单抗的应用。利用原核表达的柑橘黄化脉明病毒的外壳蛋白为抗原免疫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CYVCV单抗的杂交瘤细胞株18H5,其保藏号为CGMCC No.12003。该细胞株分泌单抗体腹水间接ELISA效价达10‑7,抗体类型及亚类为IgG1,kappa轻链,该单抗与CYVCV 32kD的外壳蛋白亚基有特异反应。利用18H5单抗建立检测柑橘树中CYVCV的TAS‑ELISA、dot‑ELISA和Tissue blot‑ELISA检测方法,其中TAS‑ELISA和dot‑ELISA方法检测病叶的灵敏度分别达到1:2560和1:20480倍稀释(w/v,g/mL)。抗CYVCV单抗的制备及其检测方法的建立为该柑橘病毒病的诊断和检测、流行病学分析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柑橘黄化脉明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
柑橘黄化脉明病由柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)引起,该病毒属于Alphaflexiviridae科柑橘病毒属的成员。该病最早于1988年在巴基斯坦被发现,目前在巴基斯坦、印度、土耳其等国的柑橘产区快速扩散,对柑橘产业造成了严重的损失。该病造成柠檬、酸橙等柑橘属植物叶片的叶脉黄化及明脉,并伴有叶片反卷和皱缩等症状,严重时可至叶脉坏死、嫩叶脱落,造成树势衰弱、产量下降。最近,在我国西南地区的云南、重庆相继发现感染CYVCV的柑橘树。研究发现该病毒可以通过摩擦接种和蚜虫介体传播到藜科和豆科植物上。柑橘黄麦明病毒基因组全长为7529个核苷酸,含有6个开放读码框(ORF),其中ORF5编码大小为32KDa的外壳蛋白。通过电镜观察纯化的病毒粒子发现该病毒为直径为13-14nm,长约685nm的卷曲线状病毒。
为了调查我国该柑橘病毒病发病情况、强化我国柑橘黄化脉明病毒病检测和诊断手段、科学指导防控,急需建立检测柑橘中CYVCV的高通量的检测方法。目前,对CYVCV的检测和诊断仅用低效率的电镜观察、RT-PCR方法、核酸电泳等方法进行小样本检测。本发明以原核表达的CYVCV衣壳蛋白(CP)为抗原通过杂交瘤技术制备了1株分泌抗CYVCV的特异性单抗的杂交瘤细胞株,以制备的单抗为核心建立检测CYVCV的高通量的血清学方法及其试剂盒,从而为我国柑橘黄化脉明病毒的检测和诊断、流行病学调查、无毒树苗生产、病毒基因组功能分析及其科学防控体系的建立提供物质和技术支持。
发明内容
本发明的目的是克服现有技术的不足,提供一种分泌抗柑橘黄化脉明病毒单克隆抗体的杂交瘤细胞株及其单抗应用。
分泌抗柑橘黄化脉明病毒单克隆抗体的杂交瘤细胞株18H5,保藏号为CGMCCNo.12003,它能分泌抗柑橘黄化脉明病毒单克隆抗体。
一种所述的杂交瘤细胞株分泌的抗柑橘黄化脉明病毒单克隆抗体,抗柑橘黄化脉明病毒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,该单克隆抗体与柑橘黄化脉明病毒32kD的外壳蛋白有特异性免疫反应,利用TAS-ELISA方法分析发现单抗检测病叶的灵敏度达到1:20480倍稀释。
抗柑橘黄化脉明病毒单克隆抗体能与柑橘黄化脉明病毒有特异性反应,而不与柑橘衰退病毒、柑橘碎叶病毒、苹果茎痘病毒和苹果茎沟病毒和健康的柑橘叶片粗提液反应。
抗柑橘黄化脉明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。
本发明与现有技术相比具有的有益效果:1)提供的杂交瘤细胞株分泌抗柑橘黄化脉明病毒特异性单克隆抗体,以该单抗为核心建立的dot-ELISA、Tissue blot-ELISA、DAS-ELISA和TAS-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、灵敏地检测柑橘黄化脉明病毒;2)利用本发明所制备的单克隆抗体检测柑橘黄化脉明病毒,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,可有效地用于田间柑橘黄化脉明病毒的检测和诊断,也可用于该病毒病的流行病学调查、病毒基因组功能分析、脱毒树苗生产、抗性育种、科学防控等方面。
附图说明
图1是dot-ELISA方法检测柑橘黄化脉明病毒(CYVCV)的灵敏度分析;
图2是dot-ELISA方法检测柑橘黄化脉明病毒特异性分析。
图3是38个柑橘检测样品的dot-ELISA检测结果。
图4是RT-PCR对田间疑似发病柑橘样品检测结果。
图5是Tissue blot-ELISA田间检测CYVCV代表性结果。
生物保藏
分泌抗柑橘黄化脉明病毒单克隆抗体的杂交瘤细胞株18H5,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,地址:北京市朝阳区北辰西路1号院3号,邮编:100101,保藏号为CGMCC No.12003,分类命名为分泌抗柑橘黄化脉明病毒(CYVCV)单抗杂交瘤细胞,它能分泌抗柑橘黄化脉明病毒的单克隆抗体。
具体实施方式
一种所述的杂交瘤细胞株分泌的抗柑橘黄化脉明病毒单克隆抗体,抗柑橘黄化脉明病毒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,该单克隆抗体能与柑橘黄化脉明病毒32kDa的外壳蛋白有特异性免疫反应,利用TAS-ELISA方法分析发现单抗检测病叶的灵敏度达到1:20480倍稀释。
抗柑橘黄化脉明病毒单克隆抗体能与柑橘黄化脉明病毒有特异性反应,而不与柑橘衰退病毒、柑橘碎叶病毒、苹果茎痘病毒和苹果茎沟病毒和健康的柑橘叶片粗提液反应。
抗柑橘黄化脉明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能大量分泌抗柑橘黄化脉明病毒单抗,且该分泌单抗特异性强、效价高、稳定性好、灵敏度高。以该单抗为核心建立检测CYVCV的高通量的血清学方法及其试剂盒,可成功应用于田间CYVCV的检测,从而为我国柑橘黄化脉明病毒病检测和诊断、脱毒树苗的生产、科学防控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、杂交瘤细胞获得及其单克隆抗体的制备
1.免疫原及检测抗原的制备
根据GenBank中报道的CYVCV的衣壳蛋白基因(CP)序列(accession no.JX040635)设计一对特异引物:CP-F(5’-ACGGATCCATGAGCTTCGACTAC ACTCA-3’,划线部分为BamH I酶切位点)和CP-R(5’-CGGAGCTCTTAGA TGTTGAAAGGGGTCGG-3’,划线部分为Sac I酶切位点),并由上海英骏生物技术有限公司合成。利用常规TRizol法提取柑橘病叶总RNA(具体操作参照试剂说明书进行),以RNA为模板反转录为cDNA:总RNA变性65℃5min后冰浴5min,然后在一灭菌离心管中加入下列试剂:
反转录参数设置:37℃20min,98℃5min,4℃10min。反转录后用高保真KOD-PCR扩增CYVCV CP,PCR反应体系(50μl):去离子H2O 34.5μl、MgSO4 3μl、KOD plus Neo Buffer 5μl、dNTP 5μl、CP-F和CP-R引物各1μl、反转录cDNA模板0.5μl。PCR反应参数:预变性94℃2min,变性98℃10s,退火52℃35s,延伸68℃45s,35个循环,最后68℃延伸10min。PCR扩增产物于0.8%琼脂糖凝胶进行电泳分析,并用DNA凝胶回收试剂盒(AxyGEN)回收CYVCV CP基因片段,具体操作参照试剂盒说明书进行。将纯化的CYVCV CP基因和pET-32a载体分别用BamHI和Sac I进行双酶切,CYVCV CP基因和pET-32a载体的酶切产物分别进行0.8%琼脂糖凝胶电泳,并用DNA凝胶回收试剂盒(AxyGEN)分别回收酶切片段,酶切回收的PCR产物和pET-32a载体在T4DNA连接酶的作用下构建成重组载体pET-32a-CP,并42℃热击转化入大肠杆菌DH5α的感受态细胞中,用质粒提取试剂盒(AxyGEN)提取重组质粒,对提取的重组质粒进行PCR和双酶切鉴定后通过测序验证重组克隆载体pET-32a-CP中所携带的CP基因序列及读码框的正确性,序列分析软件为DNAstar、
NCBI-BLAST、GenBank数据库。把原核表达质粒pET-32a-CP 42℃热击转化入大肠杆菌表达菌株BL21(DE3)中,挑取单菌落接种到含氨苄青霉素抗性的LB液体培养基,37℃培养过夜,第二天早上按1:100的比例将培养物接种于含氨苄青霉素抗性的新鲜LB培养基中,振荡培养至OD600≈0.5,加入终浓度为1mM IPTG诱导表达6h,离心收集菌体。部分菌体加入2×SDS-PAGE上样缓冲液悬浮,沸水中处理5-l0min,12 000rpm离心后取上清10μ1进行12.5%SDS-PAGE电泳分析,其余菌体经超声波破碎,收集上清按照产品说明书用Ni2+-NTAagarose(Qiagen)纯化目的蛋白,重组表达蛋白经纯化后得到大小为55kDa的重组表达CYVCV CP蛋白。以纯化的重组CP蛋白作为免疫原及检测抗原制备杂交瘤细胞和单抗。
2.免疫动物
用纯化的CYVCV CP蛋白免疫7周龄体重18-20g BALB/C雌性小鼠:1mg/mL免疫原CYVCV CP 0.5mL与等体积弗氏完全佐剂混合,充分乳化后,经腹部皮下多点注射0.2mL每只,间隔3周,取与一免等量抗原和等体积的弗氏不完全佐剂充分乳化后,第二次腹腔注射0.2mL每只,过3周后用加倍剂量的抗原进行腹腔注射,3d后取脾细胞进行融合。
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9:1的比例,在无血清的RPMI-1640(Gibco)培养基中混匀,1500rpm离心5min后去除培养基,含有细胞的离心管在37℃水浴中加入1ml 50%PEG(Sigma,分子量1500)融合剂,融合2min,用无血清的RPMI-1640培养基终止融合后1500rpm离心5min,吸去上清后沉淀用HAT培养基悬浮,分装到96孔细胞板中,37℃,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5d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5%-20%时,以表达纯化的CYVCV CP蛋白为包被抗原用常规间接ELISA方法筛选阳性孔,共获63个阳性孔。对63孔进行特异性分析,筛选出10个呈特异性的细胞株,进行有限稀释法克隆,获得1株能分泌抗CYVCV的特异性单抗的杂交瘤细胞株18H5。经4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7×105个杂交瘤细胞,注射杂交瘤细胞后7-10天可见小鼠腹部明显膨大,用针头采取腹水,3000rpm离心3min,收集上清液即为单克隆抗体腹水。
取1倍体积腹水加2倍体积0.06M PH4.8醋酸缓冲液稀释,室温下边加边搅拌加辛酸(30μl/mL腹水),4℃澄清1h,12000rpm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,3000rpm离心20min,沉淀用2倍体积的PBS溶液溶解,在4℃透析24h后即获纯化的单抗,-70℃保存。6.单克隆抗体的亚类鉴定和腹水效价测定
将纯化的单抗腹水与Sigma公司的标准抗BALB/C小鼠IgG1、IgG2a、IgG2b、IgG3、IgM抗体作双向琼脂扩散试验,结果分析18H5单抗亚类为IgG1、kappa轻链。以表达的纯化CP蛋白为包被抗原,用间接ELISA方法检测单抗腹水效价,分析结果表明单抗腹水效价达10-7。
7.单克隆抗体的特异性检测
用感染柑橘衰退病毒(CTV)、柑橘碎叶病毒(CTLV)、苹果茎痘病毒(ASPV)和苹果茎沟病毒(ASGV)病叶粗提液包被ELISA板,以相应的健叶粗提液作阴性对照,以感染CYVCV的柑橘病叶粗提液为阳性对照,用间接ELISA法测定单抗的特异性反应。间接ELISA方法的步骤:上述病毒感染的病叶和健康叶片分别用液氮研磨成粉末,按1:20(w/v,g/mL)加入ELISA包被液匀浆,5000rpm离心3min后100μl/孔包被ELISA板,4℃过夜或37℃2h使其吸附于聚苯乙烯板孔;PBST洗涤三次后用3%的脱脂奶粉封闭30-60min;加入1:5000倍稀释的单抗100μl/孔,37℃1-2h;PBST洗涤三次后加:1:10000倍稀释的碱性磷酸脂酶(AP)标记兔抗鼠IgG二抗(Sigma公司)100μl/孔,37℃1-2h;PBST洗涤四次后用PNPP底物显色30-60min,2mol/L氢氧化钠终止反应后,用酶标仪读取OD405的值,与阴性OD值比值大于2.1的样品为阳性。结果发现,18H5单抗对CYVCV有特异性反应,而与感染CTV、CTLV、ASPV、ASGV的病叶和健康柑橘叶片组织粗提液均无任何反应。
二、检测CYVCV免疫学方法及其试剂盒的建立
1.检测CYVCV的TAS-ELISA检测方法
1.1.TAS-ELISA方法的步骤:
1)抗CYVCV的兔抗血清适当稀释后(用表达纯化的CYVCV CP蛋白免疫兔子获得)100μl/孔包被聚苯乙烯板,37℃,2-4h或4℃过夜;
2)PBST洗涤三次后加含3%的脱脂奶粉PBST 250μl/孔,37℃封闭30-60min;
3)加入1:30倍稀释的(w/v,g/mL)检测样品粗提液100μl/孔。以CYVCV病叶为阳性对照,以健康柑橘叶片作阴性对照,37℃1-2h;
4)PBST洗涤3次后用封闭液适当稀释的单抗腹水100μl/孔,37℃1-2h;
5)PBST洗涤3次后加入适当稀释的AP标记兔抗鼠IgG二抗(Sigma)100μl/孔,37℃1-2h;
6)PBST洗涤4次后加PNPP底物于室温显色30-60min,肉眼观察底物颜色变成黄绿色的孔为阳性,2M氢氧化钠终止反应后,用680型酶联免疫检测仪测405nm的OD值,以P/N>2.1作为阳性判断标准。
1.2.TAS-ELISA检测方法的建立:
采用TAS-ELISA方阵试验进行,即横向分别加用包被缓冲液从1:100至1:102400倍比稀释的兔抗CYVCV血清;加入1:30倍稀释的(w/v,g/mL)CYVCV病叶粗提液;纵向分别加用封闭液从1:5至1:2048000倍比稀释单抗腹水;AP标记的兔抗鼠IgG二抗按1:7000倍稀释,按TAS-ELISA方法流程进行分析,确定抗血清和单抗的最适工作浓度分别为1:4000和1:6000倍稀释。在抗血清和单抗的最适工作浓度下,用封闭液从1:5至1:2048000倍比稀释AP标记的兔抗鼠IgG二抗,用TAS-ELISA分析二抗最适稀释度,结果发现,二抗的最适工作浓度为1:7000倍稀释。根据抗血清、单抗和AP标记的兔抗鼠IgG二抗的最适工作浓度建立检测CYVCV的TAS-ELISA方法。
1.3.TAS-ELISA方法检测灵敏度和特异性的确定
在抗体最适工作浓度下,将CYVCV病叶粗提液用PBS缓冲液倍比稀释后进行TAS-ELISA测定,结果表明,TAS-ELISA检测病叶的灵敏度达到20480倍稀释(w/v,g/mL),说明制备的单抗和建立的方法具有很好的灵敏度。该方法检测CYVCV病叶粗提液呈强阳性反应,检测感染CTV、CTLV、ASPV、ASGV的果树组织和健康柑橘叶片组织呈阴性反应,且对比差异极显著,说明该方法和单抗的特异性好。
2.dot-ELISA方法的建立及田间检测应用
2.1dot-ELISA检测柑橘中CYVCV方法的建立及其田间样品检测
dot-ELISA检测柑橘中CYVCV方法的步骤:将柑橘叶片称重后用液氮研磨成粉末,按1:10-30(w/v,g/mL)加入0.01M PBS(pH7.4)后匀浆;匀浆液5000rpm离心3min;取3μl上清点到NC上,同时设置健康和感病柑橘叶片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33μL BCIP底物(Promega)加入到10ml底物缓冲液(0.1M Tris Cl、0.1MNaCl、0.025M MgCl、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自来水漂洗膜终止反应,拍照记录结果。
用常规方阵试验确定检测柑橘病叶的dot-ELISA单抗和酶标二抗的最适工作浓度,试验表明18H5单抗及酶标二抗的最适工作浓度分别为1:5000和1:8000倍稀释。以上述抗体的最适工作浓度建立检测CYVCV病叶的dot-ELISA方法。特异性分析表明,该方法检测CYVCV呈特异性阳性反应,而检测感染CTV、CTLV、ASPV、ASGV病叶和健康柑橘叶片均呈阴性反应(图1)。灵敏度分析表明,当柑橘病叶粗提液稀释到1:2560倍(w/v,g/mL)时,用建立的dot-ELISA检测仍呈现紫色的阳性斑点,即其检测病叶的灵敏度达到1:2560倍稀释(图2)。
用建立的dot-ELISA方法对2014年采自重庆、云南等田间疑似发病柑橘样品进行检测,结果发现,38个柑橘检测样品中有16个样品产生紫色的阳性斑点(图3),阳性样品进一步用RT-PCR分析,结果表明所有的dot-ELISA阳性样品均检测到CYVCV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CYVCV。说明该dot-ELISA方法能准确、可靠地用于柑橘样品中柑橘黄化脉明病毒的检测。
3.检测CYVCV的Tissue blot-ELISA方法的建立
a.Tissue blot-ELISA操作步骤:
1)样品准备:取柑橘叶片的刚展开不久的嫩叶和嫩茎,将嫩叶卷成圆筒状用刀片切一个横断面,嫩茎直接切成横断面;
2)点样:将横断面在NC膜上按压3sec,同时将健康和感病组织分别作为阴性和阳性对照,37℃烘箱干燥5min;
3)封闭: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1h;
4)一抗孵育:NC膜放入适当稀释的CYVCV单抗中室温孵育1h;
5)二抗孵育:用PBST洗膜3次后NC膜放入适当稀释的AP酶标记羊抗鼠IgG中室温孵育1h;
6)PBST洗膜4-5次,每次3min;
7)加底物显色:10ml显色缓冲液(0.1M Tris Cl、0.1M NaCl、0.025M MgCl、pH9.5)中加入66μl NBT和33μl BCIP底物(Promega)混匀,将膜浸入显色液中进行显色反应,直至特异性条带显色明显、阴性对照没有背景时将膜在去离子水中漂洗终止反应,记录显色结果。
b.Tissue blot-ELISA最适抗体工作浓度的确定
通过常规方阵实验确定单抗和酶标二抗的最适工作浓度。选择检测灵敏度和特异性最高时一抗、酶标二抗稀释度组合为Tissue blot-ELISA的最适工作浓度。结果发现单抗18H5和AP标记的羊抗鼠二抗分别稀释1:5000和1:8000倍时Tissue blot-ELISA方法的检测灵敏度和特异性最佳,根据抗体最适工作浓度建立检测柑橘病叶中CYVCV的Tissue blot-ELISA方法。
4.柑橘黄化脉明病毒dot-ELISA检测试剂盒
1)试剂盒主要成分:
以上试剂均保存于4℃
硝酸纤维素膜(NC)10张
2)检测柑橘样品的操作步骤:
a.将柑橘叶片称重后用液氮研磨成粉末,按1:10-30(w/v,g/mL)加入0.01M PBS(pH7.4)后匀浆;
b.匀浆液5000rpm离心3min;
c.取3μl上清点到NC上,同时设置健康和感染CYVCV柑橘叶片分别作为阴性和阳性对照,室温干燥10-20min;
d.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;
e.NC膜放入1:2000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1:3000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;
h.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 MgCl、pH9.5)中,混匀后膜放入底物液中反应,肉眼观察结果;
i.待阳性对照显色明显(紫色),而阴性没有任何显色时自来水漂洗膜终止反应,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(0.01M PBS,pH7.4):
加蒸馏水950溶解后调pH至7.4,定容至1000mL
ELISA洗涤液(0.01M PBST):1000mL 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,g/mL)。
Claims (5)
1.一种分泌抗柑橘黄化脉明病毒单克隆抗体的杂交瘤细胞株18H5,其特征在于能分泌抗柑橘黄化脉明病毒单克隆抗体,杂交瘤细胞株18H5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.12003。
2.一种如权利要求1所述的杂交瘤细胞株18H5分泌的抗柑橘黄化脉明病毒单克隆抗体,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,该单克隆抗体与柑橘黄化脉明病毒32kDa的外壳蛋白有特异性免疫反应,利用TAS-ELISA方法分析发现单抗检测病叶的灵敏度达到1:20480倍稀释。
3.如权利要求2所述的抗柑橘黄化脉明病毒单克隆抗体,其特征在于所述的单克隆抗体能与柑橘黄化脉明病毒有特异性反应,而不与柑橘衰退病毒、柑橘碎叶病毒、苹果茎痘病毒和苹果茎沟病毒和健康的柑橘叶片粗提液反应。
4.一种如权利要求2所述的抗柑橘黄化脉明病毒单克隆抗体在该病毒检测上的应用,其特征在于以所述单克隆抗体为核心建立免疫学检测方法检测该病毒。
5.一种如权利要求2所述的抗柑橘黄化脉明病毒单克隆抗体在制备免疫学检测柑橘黄化脉明病毒的试剂盒中的应用。
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