CN110885370A - 分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法 - Google Patents
分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法 Download PDFInfo
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- CN110885370A CN110885370A CN201911141647.5A CN201911141647A CN110885370A CN 110885370 A CN110885370 A CN 110885370A CN 201911141647 A CN201911141647 A CN 201911141647A CN 110885370 A CN110885370 A CN 110885370A
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- monoclonal antibody
- antibody
- ringspot virus
- variable region
- tomato ringspot
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Abstract
本发明提供一种分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法,其特征在于:以抗原肽CAFGKGVEEIEQTST免疫BALB/c小鼠,获得杂交瘤细胞株,并从中筛选出能够产生特异性识别该抗原肽单克隆抗体的细胞株,并通过腹水制备法获得特异性识别番茄环斑病毒外壳蛋白AFGKGVEEIEQTST肽段的单克隆抗体,该单克隆抗体重链可变区和轻链可变区长度分别为185个和103个氨基酸;单克隆抗体的产量为500‑5000mg/L腹水,纯度为85%‑99.5%。本发明单克隆抗体具有特异性识别番茄环斑病毒外壳蛋白的特性,在番茄环斑病毒进出口检验检疫方面具有广阔应用前景。
Description
技术领域
本发明涉及一种单克隆抗体,尤其涉及一种分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法。
背景技术
番茄环斑病毒(Tomato ringspot virus,ToRSV)为豇豆花叶病毒科(Comoviridae)线虫传多面体病毒属(Nepovirus)成员,病毒粒子为等径二十面体,直径约30nm。基因组含两条正义单链RNA,分别编码复制酶、运动蛋白(MP)和外壳蛋白(CP)。ToRSV广泛分布于欧美及大洋州,能侵染大豆、葡萄、桃、李、苹果、樱桃、番茄及烟草等150多种作物,引起多种病害,严重时导致绝产。ToRSV有多种传播方式,在田间介体剑线虫(Xiphinema)和嫁接传播是ToRSV传播和流行的重要方式,而远距离传播主要靠种子(苗)调运。种子传毒的植物很多,有的种传率很高,如大豆76%、千日红76%、接骨木11%、蒲公英24%、红三叶草3~7%、番茄3%。该病毒是一种高风险的病毒,为我国禁止入境的一类检疫性有害生物。由于ToRSV是一种可随种子苗木调运进行远距离传播的高风险检疫性有害生物,为防止该病毒进入我国,建立有效的快速检测方法十分重要。目前,国内外针对ToRSV的检疫所使用的手段包括电镜观察、鉴别寄主反应(生物学方法)、血清学试验和分子生物学手段。由于ToRSV是一种直径为28nm的球状病毒,而样品中的病毒含量低,难以在电镜下观察鉴定;接种鉴别寄主则需要专门的隔离温室,花费时间长,更由于病毒的隐症现象而影响了鉴定结果。血清学方法是植物病毒快速检测的必要手段,目前口岸的病毒检测依赖于购置国外的抗血清,价格昂贵,而国内还没有ToRSV血清检测试剂盒用于进境植物种苗的报道。
本发明针对我国大量种苗进口可能携带番茄环斑病毒进境的现状,以番茄环斑病毒外壳蛋白为检测靶标,通过番茄环斑病毒特异性通用抗原肽段的选择、合成,以及交联免疫小鼠、细胞融合、抗体活性检测等系列步骤,筛选获得单克隆抗体细胞株。并通过RT-PCR方法获得抗体基因中抗原识别区序列,即重链可变区和轻链可变区序列。最终获得特异性识别目标抗原的序列已知的单克隆抗体、产生该抗体的细胞株和相应抗体生产方法。对比以纯化病毒颗粒免疫小鼠获得单克隆抗体的方法,本发明获得的单克隆抗体有明确的识别表位,而病毒颗粒免疫获得的单克隆抗体识别表位不明确。且本发明首先从番茄环斑病毒外壳蛋白序列中分析出保守区域,继而从保守区域中挑选设计候选抗原肽段,之后采用化学合成方法制备该抗原肽段,因此抗原的获取不受携带病毒样本来源限制,也不违反进口检验检疫物操作许可范围。明确的识别表位也使得本发明单克隆抗体与市场上已有抗体和相关研究报道抗体具有明确易识别的特征。
发明内容
本发明要解决的技术问题是提供一种番茄环斑病毒单克隆抗体,其具有识别并结合番茄环斑病毒外壳蛋白特定肽段的反应特异性,并缺乏对其它病毒和植物组织的结合特异性,为进一步研制开发番茄环斑病毒血清检测试剂盒奠定了基础。
本发明首先公开了一种番茄环斑病毒单克隆抗体,该抗体重链可变区的序列为SEQ ID NO:1所示,轻链可变区序列为SEQ ID NO:2所示。
该单克隆抗体重链可变区和轻链可变区长度分别为185个和103个氨基酸。
作为本发明的优选方案,编码所述重链和轻链可变区的核苷酸序列分别为SEQ IDNO:3所示和SEQ ID NO:4所示。重链可变区185个氨基酸残基序列经BLASTP分析,第一匹配序列相似度为89.77%;轻链103个氨基酸残基序列经BLASTP分析,第一匹配序列相似度为56.14%。
所述的单克隆抗体经腹水诱导法制备,产量为500-5000mg/L,纯度为85%~99.5%。
本发明还公开了一种分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株(小鼠杂交瘤细胞5H9H10),其保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.18320。保藏日期为2019年8月15日,保藏单位地址为北京市朝阳区北辰西路1号院3号。
本发明还公开了所述的番茄环斑病毒单克隆抗体在制备番茄环斑病毒检测试剂或试剂盒中的应用。
所述番茄环斑病毒单克隆抗体由如下步骤制得:
(1)从Genbank中查找获得多个番茄环斑病毒外壳蛋白序列(包括经基因序列翻译后的蛋白质序列),通过序列比对从中选取出保守片段;
(2)针对保守片段进行适宜抗原序列挑选,获得优选抗原肽段CAFGKGVEEIEQTST,如SEQ ID NO:5所示;
(3)通过化学合成法,合成该N端增加1个半胱氨酸残基的抗原肽段CAFGKGVEEIEQTST;
(4)将抗原肽段与KLH交联;
(5)以交联抗原肽段免疫小鼠;
(6)制备杂交瘤细胞并从中筛选产生特异性识别SEQ ID NO:5肽段抗体的细胞株;
(7)采用RT-PCR法克隆测定单克隆抗体细胞株中单克隆抗体重链和轻链可变区序列;
(8)采用腹水诱导法制备能够识别SEQ ID NO:5肽段和对携带番茄环斑病毒样本做出阳性反应的单克隆抗体;
(9)采用硫酸铵沉淀和蛋白G亲和层洗法纯化本发明所述单克隆抗体。
所述的单克隆抗体的腹水诱导法制备过程具体包括如下步骤:
(1)取8-15周BALB/c小鼠腹腔注射灭菌石蜡0.1-0.5mL/只;
(2)5-10天后腹腔接种用PBS悬浮的所述的杂交瘤细胞5×105-5×106/只;
(3)5-10天后采集腹水2-4次;
(4)将腹水1000×g离心10min,吸去最上层的脂肪组织,除去细胞成分和沉淀物,收集上清;
(5)取3mL腹水上清,加入0.06M,pH4.0乙酸钠-乙酸缓冲液3mL,调pH至4.6-5.0;逐滴加入99uL正辛酸,0-10℃下搅拌10-60min,澄清10-60min;4℃下14000×g离心20min,去沉淀,上清加入1/10体积的10×PBS,用1M NaOH调pH至7.2;上清滴加等量饱和硫酸铵溶液,继续搅拌30min,并静置30min;4℃下14000×g离心30min,弃上清,将沉淀溶于1.2mL PBS中,并用0.01M,pH7.4 PBS在0-10℃下透析过夜,再以5%PEG20000浓缩至5mL;
(6)浓缩液过蛋白质G纯化树脂,以预洗脱液冲洗10-15个柱体积,以5-10个柱体积洗脱缓冲液洗脱单克隆抗体,经浓缩后获得本发明所述单克隆抗体。
优选的,所述的步骤(6)中的预洗脱液包括0.1-0.15M NaCl和10-40mM Na2HPO4,pH为7.0;所述的洗脱缓冲液为0.1-0.3M甘氨酸,pH为3.0。
本发明所用的术语“单克隆抗体(单抗)”指基本均一的细胞群体获得的抗体,即该群体中包含的单个抗体是相同的,除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。且本发明单克隆抗体专一性识别源自番茄环斑病毒外壳蛋白的SEQ ID NO:5所示肽段,识别序列唯一且明确,有别于常规的以番茄环斑病毒免疫小鼠获得的识别序列未知的单克隆抗体,同样明确有别于番茄环斑病毒多克隆抗体。
本发明具有以下有益效果:本发明番茄环斑病毒单克隆抗体经效价测定和酶联免疫试验,结果表明识别SEQ ID NO:5所示肽段的单抗具有与番茄环斑病毒反应的特异性,可作为番茄环斑病毒检测的抗体,可为后续研制番茄环斑病毒血清检测试剂盒奠定了良好的基础,服务出入境检验检疫工作需要。
附图说明
图1为抗原序列及候选抗原肽7在番茄环斑病毒外壳蛋白上的位置。
具体实施方式
以下通过实施例对本发明作进一步的阐述:
实施例1
(1)从Genbank中调取番茄环斑病毒蛋白质序列(序列收录号为:NP_733973),该序列长度为562个氨基酸残基(即562aa),按14个氨基酸残基(14aa)抗原肽段长度要求,理论候选抗原肽段数量为549条。经溶解性和合成难易度以及免疫源性分析预测,获得候选抗原肽7条,候选抗原肽1-6的序列如SEQ ID NO:6-11所示,候选抗原肽7的序列如SEQ ID NO:5所示,其中抗原序列及候选抗原肽7在番茄环斑病毒外壳蛋白上的位置如图1所示。为偶联载体蛋白-血蓝蛋白KLH的需要,在N端或C端增加1个半胱氨酸(C),增加1个半胱氨酸后候选抗原肽如下:
候选抗原肽1:LLRYKEWQRQGFLHC
候选抗原肽2:GPTEIDLTSTPAPNC
候选抗原肽3:KLVDRLSVNVILQEC
候选抗原肽4:GQGAFSLPISTPHAC
候选抗原肽5:CLLRYKEWQRQGFLH
候选抗原肽6:CDDKSEVLLRQHPLS
候选抗原肽7:CAFGKGVEEIEQTST。
(2)委托商业多肽合成公司,以固态合成法制备步骤(1)所述7个抗原多肽,多肽纯度在98%以上。
(3)将候选抗原肽与KLH经两步反应法偶联(本实施例以“候选抗原肽7:CAFGKGVEEIEQTST”为例进行介绍)。偶联反应步骤如下:
a)将20mg SMCC【4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠】溶于2mL DMF(N,N-二甲基甲酰胺。
b)将0.8ml KLH(血蓝蛋白)加入到25ml圆底烧瓶中,补加1×PBS(pH 7.2)使蛋白终浓度为15mg/ml。
c)将溶解好的SMCC溶液缓慢滴加到120mg KLH蛋白体系中,室温搅拌反应1h。
d)用1L1×PBS(PH 7.4)溶液于4℃下透析6小时。
e)将透析后的KLH蛋白倒入50mL离心管中,通过称重法确定其体积(按密度1g/cm3计算),根据反应前加入的KLH蛋白的量来计算透析后蛋白的浓度,然后根据其浓度将2.5mgKLH-SMCC溶液转移到5ml离心管中。
f)将3.0mg“候选抗原肽7:CAFGKGVEEIEQTST”用0.6ml 1×PBS(pH 7.2)溶液溶解。
g)用Ellman试剂检测多肽中的巯基:在96孔板中加入100μl Ellman试剂储备液,再加入10μl多肽溶液,在λ=412nm下测其紫外吸收值,如果OD值>0.15做下一步;OD值<0.15并>0.05补加多肽,直至达到要求;
h)将“候选抗原肽7”溶液滴加到KLH-SMCC管中,室温下用垂直混匀器混匀反应4小时。
i)用Ellman试剂检测多肽中的巯基:在96孔板中加入100μl Ellman试剂储备液,再加入10μl偶联后的多肽溶液,在λ=412nm下测定紫外吸收值。OD值<0.03说明多肽和KLH蛋白交联率已达到80%以上;OD值>0.03则再补加SMCC活化好的KLH蛋白继续交联。获得偶联抗原“偶联候选抗原肽7-KLH”。
(4)免疫小鼠
a)初次免疫,取1.5mL偶联候选抗原肽7-KLH”(总量为150ug)与等体积福氏完全佐剂混合均匀,按每只小鼠1.5mL量进行皮下多点注射,同时免疫3只小鼠。
b)第二次免疫,初次免疫后3周,进行第二次免疫,剂量途径同上,加福氏不完全佐剂,间隔3周。
c)第三次免疫,剂量同上,不加佐剂,腹腔注射,7天后采血测其效价,检测免疫效果,间隔3周。
d)加强免疫,剂量50ug/只,腹腔注射。
e)3天后,选择效价最高小鼠取脾融合
(5)杂交瘤细胞制备
a)将1×108脾细胞与1×107骨髓瘤细胞SP2/0混合于一支50mL融合管中,补加不完全培养基至30ml,充分混匀。
b)1000r/min离心10分钟,将上清尽量吸净。
c)在手掌上轻击融合管底,使沉淀细胞松散均匀;
d)用1ml吸管在30s内加入预热的50%PEG1 mL,边加边轻轻搅拌。
e)吸入吸管静置1min。
f)加入预热的不完全培养液,终止PEG作用,连续每2min内分别加入1mL,2mL,3mL,4mL,5mL,10mL。
g)800rpm,5分钟;弃去上清。
h)加入5mL完全培养基,轻轻吹吸沉淀细胞,使其悬浮并混匀,然后补加完全培养基至40-50mL。分装96孔细胞培养板,每孔100uL,然后将培养板置37℃,5%CO2培养箱内培养。
i)6h后补加选择培养基。每孔50uL,3天后用选择培养基半换液。
j)经常观察杂交瘤细胞生长情况,待其长至孔底面积1/10以上时吸出上清供抗体检测。
(6)杂交瘤细胞的克隆化(有限稀释法)
a)制备小鼠脾细胞为饲养细胞。
b)制备待克隆的杂交瘤细胞悬液,用含20%血清的HT培养基稀释至每毫升含5、10和20个细胞3种不同的稀释度。
c)按每毫升加入1×105细胞的比例,在上述杂交瘤细胞悬液中分别加入腹腔巨噬细胞。
d)每种杂交瘤细胞分装96孔板一块,每孔量为100ul.
e)37℃、5%CO2培养6天,出现肉眼可见的克隆即可检测抗体;在倒置显微镜下观察,标出只有单个克隆生长的孔,取上清作抗体检测。
f)取抗体检测阳性孔的细胞扩大培养,并冻存。
(7)腹水诱导法制备单克隆抗体
a)取8周BALB/c小鼠腹腔注射灭菌石蜡0.1mL/只;
b)5天后腹腔接种用PBS悬浮的杂交瘤细胞5×105/只;
c)5天后采集腹水2-4次;
d)将腹水1000×g离心10min,吸去最上层的脂肪组织,除去细胞成分和其他的沉淀物,收集上清;
e)取3mL腹水上清,加入0.06M,pH4.0乙酸钠-乙酸缓冲液3mL,调pH至4.6;逐滴加入99uL正辛酸,0℃下搅拌10min,澄清10-60min;4℃下14000×g离心20min,去沉淀,上清加入1/10体积的10×PBS(0.01M,pH7.4),用1M NaOH调pH至7.2;上清滴加等量饱和硫酸铵(pH6.0-8.0)溶液,继续搅拌30min,并静置30min;4℃下14000×g离心30min,弃上清,将沉淀溶于1.2mL PBS(0.01M,pH7.4)中,并用0.01M,pH7.4 PBS在0℃下透析过夜,再以5%PEG20000浓缩至5mL;
f)浓缩液过蛋白质G纯化树脂,以预洗脱液(0.1M NaCl,10mM Na2HPO4,pH7.0)冲洗10个柱体积,以5个柱体积洗脱缓冲液(0.1M甘氨酸,pH 3.0)洗脱单克隆抗体,经浓缩后获得本发明所述单克隆抗体,产量为1200mg/L腹水,纯度为85%,检测携带灭活番茄环斑病毒的番茄叶片,检测信号为阳性。该抗体对未被番茄环斑病毒感染的番茄样本反应为阴性,对烟草花叶病毒,T7噬菌体、南芥菜花叶病毒、菜豆荚斑驳病毒等反应为阴性。
实施例2
实施例2的步骤(1)-(6)与实施例1相同。
(7)腹水诱导法制备单克隆抗体
a)取15周BALB/c小鼠腹腔注射灭菌石蜡0.5mL/只;
b)10天后腹腔接种用PBS悬浮的杂交瘤细胞5×106/只;
c)10天后采集腹水4次;
d)将腹水1000×g离心10min,吸去最上层的脂肪组织,除去细胞成分和其他的沉淀物,收集上清;
e)取3mL腹水上清,加入0.06M,pH4.0乙酸钠-乙酸缓冲液3mL,调pH至5.0;逐滴加入99uL正辛酸,0-10℃下搅拌60min,澄清60min;4℃下14000×g离心20min,去沉淀,上清加入1/10体积的10×PBS(0.01M,pH7.4),用1M NaOH调pH至7.2;上清滴加等量饱和硫酸铵(pH8.0)溶液,继续搅拌30min,并静置30min;4℃下14000×g离心30min,弃上清,将沉淀溶于1.2mL PBS(0.01M,pH7.4)中,并用0.01M,pH7.4 PBS在10℃下透析过夜,再以5%PEG20000浓缩至5mL;
f)浓缩液过蛋白质G纯化树脂,以预洗脱液(00.15M NaCl,40mM Na2HPO4,pH7.0)冲洗15个柱体积,以10个柱体积洗脱缓冲液(0.3M甘氨酸,pH 3.0)洗脱单克隆抗体,经浓缩后获得本发明所述单克隆抗体,产量为500mg/L腹水,纯度为87.2%,检测携带灭活番茄环斑病毒的番茄叶片,检测信号为阳性。该抗体对未被番茄环斑病毒感染的番茄样本反应为阴性,对烟草花叶病毒,T7噬菌体、南芥菜花叶病毒、菜豆荚斑驳病毒等反应为阴性。
实施例3
实施例3的步骤(1)-(6)与实施例1相同。
(7)腹水诱导法制备单克隆抗体
a)取11.5周BALB/c小鼠腹腔注射灭菌石蜡0.3mL/只;
b)7天后腹腔接种用PBS悬浮的杂交瘤细胞1.25×106/只;
c)7天后采集腹水3次;
d)将腹水1000×g离心10min,吸去最上层的脂肪组织,除去细胞成分和其他的沉淀物,收集上清;
e)取3mL腹水上清,加入0.06M,pH4.0乙酸钠-乙酸缓冲液3mL,调pH至4.8;逐滴加入99uL正辛酸,5℃下搅拌10-60min,澄清35min;4℃下14000×g离心20min,去沉淀,上清加入1/10体积的10×PBS(0.01M,pH7.4),用1M NaOH调pH至7.2;上清滴加等量饱和硫酸铵(pH7.0)溶液,继续搅拌30min,并静置30min;4℃下14000×g离心30min,弃上清,将沉淀溶于1.2mL PBS(0.01M,pH7.4)中,并用0.01M,pH7.4 PBS在5℃下透析过夜,再以5%PEG20000浓缩至5mL;
a)浓缩液过蛋白质G纯化树脂,以预洗脱液(0.125M NaCl,25mM Na2HPO4,pH7.0)冲洗13个柱体积,以7个柱体积洗脱缓冲液(0.2M甘氨酸,pH 3.0)洗脱单克隆抗体,经浓缩后获得本发明所述单克隆抗体,产量为500-5000mg/L腹水,纯度为93.5%,检测携带灭活番茄环斑病毒的番茄叶片,检测信号为阳性。该抗体对未被番茄环斑病毒感染的番茄样本反应为阴性,对烟草花叶病毒,T7噬菌体、南芥菜花叶病毒、菜豆荚斑驳病毒等反应为阴性。
实施例4
实施例2的步骤(1)-(6)与实施例1相同。
(7)腹水诱导法制备单克隆抗体
b)取12周BALB/c小鼠腹腔注射灭菌石蜡0.3mL/只;
c)7天后腹腔接种用PBS悬浮的杂交瘤细胞1×106/只;
d)7天后采集腹水3次;
e)将腹水1000×g离心10min,吸去最上层的脂肪组织,除去细胞成分和其他的沉淀物,收集上清;
f)取3mL腹水上清,加入0.06M,pH4.0乙酸钠-乙酸缓冲液3mL,调pH至4.8;逐滴加入99uL正辛酸,4℃下搅拌20min,澄清40min;4℃下14000×g离心20min,去沉淀,上清加入1/10体积的10×PBS(0.01M,pH7.4),用1M NaOH调pH至7.2;上清滴加等量饱和硫酸铵(pH7.3)溶液,继续搅拌30min,并静置30min;4℃下14000×g离心30min,弃上清,将沉淀溶于1.2mL PBS(0.01M,pH7.4)中,并用0.01M,pH7.4 PBS在4℃下透析过夜,再以5%PEG20000浓缩至5mL;
g)浓缩液过蛋白质G纯化树脂,以预洗脱液(0.12M NaCl,30mM Na2HPO4,pH7.0)冲洗15个柱体积,以7个柱体积洗脱缓冲液(0.15M甘氨酸,pH 3.0)洗脱单克隆抗体,经浓缩后获得本发明所述单克隆抗体,产量为5000mg/L腹水,纯度为99.5%,所制抗体经间接ELISA法检测携带灭活番茄环斑病毒的番茄叶片,检测信号为阳性。该抗体对未被番茄环斑病毒感染的番茄样本反应为阴性,对烟草花叶病毒,T7噬菌体、南芥菜花叶病毒、菜豆荚斑驳病毒等反应为阴性。
实施例5
实施步骤同实施例4,抗原肽为“候选抗原肽1:LLRYKEWQRQGFLHC”,杂交瘤细胞产生单克隆抗体经间接ELISA法检测,对未偶联“候选抗原肽1”阳性,对携带灭活番茄环斑病毒的番茄叶片阴性。未获得能够用于天然病毒颗粒检测单克隆抗体。
实施例6
实施步骤同实施例4,抗原肽为“候选抗原肽2:GPTEIDLTSTPAPNC”,杂交瘤细胞产生单克隆抗体经间接ELISA法检测,对未偶联“候选抗原肽2”阳性,对携带灭活番茄环斑病毒的番茄叶片阴性。未获得能够用于天然病毒颗粒检测单克隆抗体。
实施例7
实施步骤同实施例4,抗原肽为“候选抗原肽3:KLVDRLSVNVILQEC”,杂交瘤细胞产生单克隆抗体经间接ELISA法检测,对未偶联“候选抗原肽3”阳性,,对携带灭活番茄环斑病毒的番茄叶片阴性。未获得能够用于天然病毒颗粒检测单克隆抗体。
实施例8
实施步骤同实施例4,抗原肽为“候选抗原肽4:GQGAFSLPISTPHAC”,杂交瘤细胞产生单克隆抗体经间接ELISA法检测,对未偶联“候选抗原肽4”阳性,对携带灭活番茄环斑病毒的番茄叶片阴性。未获得能够用于天然病毒颗粒检测单克隆抗体。
实施例9
实施步骤同实施例4,抗原肽为“候选抗原肽5:CLLRYKEWQRQGFLH”,杂交瘤细胞产生单克隆抗体经间接ELISA法检测,对未偶联“候选抗原肽5”阳性,对携带灭活番茄环斑病毒的番茄叶片阴性。未获得能够用于天然病毒颗粒检测单克隆抗体。
实施例10
实施步骤同实施例4,抗原肽为“候选抗原肽6:CDDKSEVLLRQHPLS”,杂交瘤细胞产生单克隆抗体经间接ELISA法检测,对未偶联“候选抗原肽6”阳性,对携带灭活番茄环斑病毒的番茄叶片阴性。未获得能够用于天然病毒颗粒检测单克隆抗体。
序列表
<110> 中国计量大学
<120> 分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株及其抗体和抗体制备方法
<160> 11
<170> SIPOSequenceListing 1.0
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Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asn Tyr
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Gly Gly Trp Gly Ser Gly Ser Leu Leu Leu Leu Ser Arg Phe Thr Cys
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Ser Ala His Val Arg Cys Trp Asp Gln Ala Gly Asn Lys Gln Leu Glu
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gatactaact acaaccagaa gttcaagggc aaggccaaac tgactgcagt cacatccgcc 240
agcactgcct acatggagct cagcagcctg acaaatgagg actctgcggt ctattactgt 300
acaagattaa gtatgctggg acgctcgtac tactttgact actggggcca aggcaccact 360
ctcacagtct cctcagccaa aacaacaccc ccatcagtct atccactggc ccctgggtgt 420
ggagatacaa ctggttcctc cgtgactctg ggatgcctgg tcaggggcta cttccctgag 480
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tttcacactc aagatcagca gagtggaggc tggggatctg ggagtttatt actgctttca 180
aggttcacat gttccgctca cgttcggtgc tgggaccaag ctggaaataa acaattagaa 240
gggcgacacg cgcacgttcg gtgctgggaa caagctggag atccaacgaa gcttgtaagg 300
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Leu Leu Arg Tyr Lys Glu Trp Gln Arg Gln Gly Phe Leu His
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Claims (7)
1.一种番茄环斑病毒单克隆抗体,其特征在于:该抗体重链可变区的序列为SEQ IDNO:1所示,轻链可变区序列为SEQ ID NO:2所示。
2.根据权利要求1所述的单克隆抗体,其中,该单克隆抗体重链可变区和轻链可变区长度分别为185个和103个氨基酸。
3.一种编码权利要求1所述单克隆抗体的基因,其特征在于包含编码轻链可变区的核苷酸序列和编码重链可变区的核苷酸序列,编码所述重链可变区的核苷酸序列如SEQ IDNO:3所示,编码所述轻链可变区的核苷酸序列如SEQ ID NO:4所示。
4.一种分泌番茄环斑病毒单克隆抗体的杂交瘤细胞株,其特征在于,其保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.18320。
重链可变区185个氨基酸残基序列经BLASTP分析,第一匹配序列相似度为89.77%;轻链103个氨基酸残基序列经BLASTP分析,第一匹配序列相似度为56.14%。
5.一种权利要求1所述的单克隆抗体的制备方法,其特征在于包括如下步骤:
(1)取8-15周BALB/c小鼠腹腔注射灭菌石蜡0.1-0.5mL/只;
(2)5-10天后腹腔接种用PBS悬浮的权利要求4所述的杂交瘤细胞5×105-5×106/只;
(3)5-10天后采集腹水2-4次;
(4)将腹水1000×g离心10min,吸去最上层的脂肪组织,除去细胞成分和沉淀物,收集上清;
(5)取3mL腹水上清,加入0.06M,pH4.0乙酸钠-乙酸缓冲液3mL,调pH至4.6-5.0;逐滴加入99uL正辛酸,0-10℃下搅拌10-60min,澄清10-60min;4℃下14000×g离心20min,去沉淀,上清加入1/10体积的10×PBS,用1M NaOH调pH至7.2;上清滴加等量饱和硫酸铵溶液,继续搅拌30min,并静置30min;4℃下14000×g离心30min,弃上清,将沉淀溶于1.2mL PBS中,并用0.01M,pH7.4 PBS在0-10℃下透析过夜,再以5%PEG20000浓缩至5mL;
(6)浓缩液过蛋白质G纯化树脂,以预洗脱液冲洗10-15个柱体积,以5-10 个柱体积洗脱缓冲液洗脱单克隆抗体,经浓缩后获得本发明所述单克隆抗体。
6.根据权利要求5所述的制备方法,其特征在于所述的步骤(6)中的预洗脱液包括0.1-0.15M NaCl和10-40mM Na2HPO4,pH为7.0;所述的洗脱缓冲液为0.1-0.3M甘氨酸,pH为3.0。
7.权利要求1所述的番茄环斑病毒单克隆抗体在制备番茄环斑病毒检测试剂或试剂盒中的应用。
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