CN114634555B - 一种特异性结合轮状病毒vp6蛋白的抗体及其应用 - Google Patents
一种特异性结合轮状病毒vp6蛋白的抗体及其应用 Download PDFInfo
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Abstract
本发明涉及一种特异性结合轮状病毒VP6蛋白的抗体及其应用,属于生物技术领域。本发明提供了一种抗原,所述抗原由氨基酸序列如SEQ ID NO.1所示的多肽组成;使用所述抗原对动物进行免疫可获得能够与轮状病毒VP6蛋白特异性结合的多克隆抗体,并且,使用所述抗原对动物进行免疫所得血清的效价高达1:320万,在轮状病毒滴定检测以及轮状病毒中和抗体检测极具应用前景。
Description
技术领域
本发明涉及一种特异性结合轮状病毒VP6蛋白的抗体及其应用,属于生物技术领域。
背景技术
轮状病毒(Rotavirus,RV)属于呼肠孤病毒科,轮状病毒属,能够引发包括人类新生儿以及猪、牛、羊等多种幼龄动物急性胃肠道传染病,临床典型症状为呕吐、腹泻脱水,病发严重时导致死亡。据WHO统计,每年因轮状病毒感染而导致的死亡病例高达60万(参见文献:Parashar U D , Gibson C J , Bresee J S , et al. Rotavirus and SevereChildhood Diarrhea[J]. Emerging Infectious Diseases, 2006, 12(2):304-306.),而腹泻病例则高达2亿。
轮状病毒为无囊膜的二十面体粒子,直径60~80nm,其基因组有11股双链RNA组成,分别编码6种结构蛋白(VP1、VP2、VP3、VP4、VP6、VP7)和5种非结构蛋白(NSP1、NSP2、NSP3、NSP4、NSP5)。轮状病毒颗粒呈三层结构,其中,VP1、VP2、VP3为核心蛋白,主要在病毒复制、装配过程发挥作用。VP4、VP7为外衣壳蛋白,主导病毒吸附、侵入细胞过程。VP6蛋白位于轮状病毒中层,是病毒含量最多的蛋白,占病毒蛋白总量的51%,在维持病毒形态、病毒进入细胞、基因组复制和组装过程中作为一个物理受体作用。根据VP6蛋白抗原性差异,可将轮状病毒分为A~G等7个组,其中,A组轮状病毒是引起婴幼儿急性胃肠炎的主要致病病原体。根据VP6蛋白是否存在两个不同的非重叠抗原特异性抗原表位,可将A组轮状病毒分为SGⅠ、SGⅡ亚群、SGⅠ/Ⅱ亚群和非SGⅠ/Ⅱ亚群(参见文献:Thongprachum A , Khamrin P , SaekhowP , et al. Analysis of the VP6 gene of human and porcine group A rotavirusstrains with unusual subgroup specificities[J]. Journal of Medical Virology,2010, 81(1):183-191.)。根据编码框核苷酸序列同源性差异,可以将A组轮状病毒分为至少13个基因型。
VP6蛋白基因全长约1356bp,编码397个氨基酸,蛋白分子量约为45kD。VP6蛋白氨基酸序列高度保守,在A组轮状病毒不同亚群中同源性90~99%,因此,VP6蛋白拥有高度的抗原交叉反应性,可作为轮状病毒抗原诊断检测的靶标蛋白,广泛应用于轮状病毒滴度检测以及轮状病毒中和抗体检测。目前,已经有学者公布了部分VP6蛋白的抗原位点,例如,E.KOHLI等人用单克隆抗体对多个肽段筛选,找出了32~64aa、155~167aa、208~274aa、380~397aa这4个抗原位点(参见文献:Kohli E , Maurice L , Bourgeois C , et al.Epitope Mapping of the Major Inner Capsid Protein of Group A Rotavirus UsingPeptide Synthesis[J]. virology, 1993, 194(1):110-116.)。CHOI等人合成了11条多肽并滴鼻免疫小鼠,最终筛选得到了5条可刺激小鼠产生保护效力的多肽,对应的抗原位点分别为227~244aa、232~261aa、249~277aa、283~307aa、368~397aa(参见文献:Choi A , BasuM , Mcneal M M , et al. Functional Mapping of Protective Domains and Epitopesin the Rotavirus VP6 Protein[J]. Journal of Virology, 2000, 74(24):11574.)。
但是,32~64aa、155~167aa、208~274aa、380~397aa这4个抗原位点未经免疫动物验证,不清楚是否能刺激动物产生高效价多克隆抗体,而227~244aa、232~261aa、249~277aa、283~307aa、368~397aa这5个抗原位点免疫动物后产生的血清抗体效价均低于1:15万,抗体效价偏低会严重影响轮状病毒滴定检测以及轮状病毒中和抗体检测的检测效果。因此,亟需找到可刺激动物产生高效价多克隆抗体的VP6蛋白抗原位点,以提高轮状病毒滴定检测以及轮状病毒中和抗体检测的检测效果。
发明内容
为解决上述问题,本发明提供了一种抗原,所述抗原由氨基酸序列如SEQ ID NO.1所示的多肽组成。
在本发明的一种实施方式中,所述抗原上偶联有载体蛋白。
在本发明的一种实施方式中,所述载体蛋白与抗原不是来自同一个蛋白,所述载体蛋白包含至少一个T细胞表位,且所述载体蛋白能够增强抗原的免疫原性。
在本发明的一种实施方式中,所述载体蛋白为血蓝蛋白(Keyhole LimpetHemocyanin,KLH)、白喉毒素DT、白喉毒素的跨膜结构域DTT、轮状病毒VP7、利什曼原虫的热休克蛋白、空肠弯曲菌鞭毛蛋白、沙眼衣原体主要外膜蛋白、牛血清白蛋白(Bovine SerumAlbumin,BSA)、鸡卵白蛋白(Ovalbumin,OVA)或纤维蛋白原。
在本发明的一种实施方式中,所述抗原截取自轮状病毒的VP6蛋白。
本发明还提供了一种特异性结合轮状病毒VP6蛋白的抗体,所述抗体能够与上述抗原特异性结合。
在本发明的一种实施方式中,所述抗体为使用上述抗原对动物进行免疫而得。
在本发明的一种实施方式中,所述抗体为多克隆抗体。
在本发明的一种实施方式中,所述动物为兔、鼠、羊或猴。
本发明还提供了一种制备上述抗体的方法,其特征在于,所述方法为:使用上述抗原对动物进行免疫,得到经免疫的动物;从经免疫的动物血清中提取上述抗体。
在本发明的一种实施方式中,所述免疫为:分别于0 d、14 d和21 d共进行3次免疫,第28天采血并分离血清;其中,首次免疫时用0.5mL PBS缓冲液将0.5mg上述抗原溶解,与等体积弗式完全佐剂混合后,于动物背部皮下多点注射,免疫剂量为0.5mg/只,二、三次免疫时除了佐剂改用弗式不完全佐剂外,其余步骤与首次免疫一致。
本发明还提供了一种用于轮状病毒滴度检测的试剂盒,所述试剂盒包含一抗和二抗;所述一抗为以上述抗原对动物进行免疫而得的抗体,所述二抗为能够与一抗特异性结合的抗体。
在本发明的一种实施方式中,所述二抗为HRP-羊抗兔IgG、FITC-羊抗兔IgG或Alexa Fluor 488-羊抗兔IgG。
本发明还提供了一种轮状病毒滴度检测方法,所述方法使用上述试剂盒对待测样本进行检测。
在本发明的一种实施方式中,所述待测样品为轮状病毒收获液或轮状病毒减毒活疫苗。
本发明还提供了上述抗体或上述试剂盒或上述轮状病毒滴度检测方法在定量测定轮状病毒滴度检测中的应用。
本发明还提供了一种用于轮状病毒中和抗体检测的试剂盒,所述试剂盒包含一抗和二抗;所述一抗为以上述抗原对动物进行免疫而得的抗体,所述二抗为能够与一抗特异性结合的抗体。
在本发明的一种实施方式中,所述二抗为HRP-羊抗兔IgG、FITC-羊抗兔IgG或Alexa Fluor 488-羊抗兔IgG。
本发明还提供了一种轮状病毒中和抗体检测方法,所述方法使用上述试剂盒对待测样本进行检测。
在本发明的一种实施方式中,所述待测样品为血清。
本发明还提供了上述抗体或上述试剂盒或上述轮状病毒中和抗体检测方法在定量测定轮状病毒中和抗体检测中的应用。
本发明技术方案,具有如下优点:
1、本发明提供了一种抗原,所述抗原由氨基酸序列如SEQ ID NO.1所示的多肽组成;使用所述抗原对动物进行免疫可获得能够与轮状病毒VP6蛋白特异性结合的多克隆抗体,并且,使用所述抗原对动物进行免疫所得血清的效价高达1:320万,在轮状病毒滴定检测以及轮状病毒中和抗体检测极具应用前景。
2、本发明提供了一种用于轮状病毒滴度检测的试剂盒,所述试剂盒包含一抗和二抗,其中,一抗为以氨基酸序列如SEQ ID NO.1所示的抗原对动物进行免疫而得的抗体,二抗为能够与一抗特异性结合的抗体;使用所述试剂盒对待测样本进行轮状病毒滴度检测具有准确度高的优势。
3、本发明提供了一种用于轮状病毒中和抗体检测的试剂盒,所述试剂盒包含一抗和二抗,其中,一抗为以氨基酸序列如SEQ ID NO.1所示的抗原对动物进行免疫而得的抗体,二抗为能够与一抗特异性结合的抗体;使用所述试剂盒对待测样本进行轮状病毒中和抗体检测具有准确度高的优势。
附图说明
图1:VP6-P1-R血清和VP6-R血清的效价检测结果。
图2:VP6-P1-Rpab抗体和VP6-Rpab抗体的Werstern-Blot结果。
图3:VP6-P1-Rpab抗体和VP6-Rpab抗体的间接免疫荧光(IFA)结果。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明,并不局限于所述最佳实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品,均落在本发明的保护范围之内。
下述实施例中未注明具体实验步骤或条件者,按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
下述实施例中涉及的缓冲液如下:
PBS缓冲液:先取8.0g NaCl、0.2g KCl、1.44g Na2HPO4、0.24g KH2PO4溶于800mL蒸馏水中,然后用HCl调节pH至7.4,最后加蒸馏水定容至1L,即得0.01M、pH 7.4的PBS缓冲液。
CBS缓冲液:先取1.59g Na2CO3、2.93g NaHCO3溶于800mL蒸馏水中,然后用HCl调节pH至9.6,最后加蒸馏水定容至1L,即得0.05M、pH 9.6的CBS缓冲液。
PBST缓冲液:取0.5mL吐温20溶于1000mL 0.01M、pH 7.4的PBS缓冲液中,即得PBST缓冲液。
TBST缓冲液:先取8.8g NaCl溶于800mL蒸馏水中,然后加入20mL 1M、pH 8.0的Tris-HCL和0.5mL吐温20,最后加蒸馏水定容至1L,即得TBST缓冲液。
下述实施例中涉及的轮状病毒病毒收获液样本和轮状病毒灭活原液样品制备过程如下:
单层Vero细胞的制备:将Vero细胞(购自ATCC)复苏至装满含8(v/v)%胎牛血清的DMEM培养基(购自Gibco)的T25细胞瓶内,于37℃培养24h后更换新的含8(v/v)%胎牛血清的DMEM培养基,继续于37℃培养6d,得到长满单层的Vero细胞;用含8(v/v)%胎牛血清的DMEM培养基按照1:8的比例将长满单层的Vero细胞进行传代培养,每隔7天传代1次,直至扩大到10层细胞工厂长满单层Vero细胞,用于轮状病毒接种;
轮状病毒的活化:取轮状病毒CDC-9毒株的病毒液1mL(购自美国CDC),在病毒液中加入终浓度8μg/mL的胰酶(购自Gibco公司)和终浓度为400μg/mL的氯化钙(购自Sigma公司)后,于37℃水浴锅活化1h,得到轮状病毒活化液;
轮状病毒的培养:弃去10层细胞工厂中的细胞上清液,加入DMEM培养基清洗Vero细胞3遍以去除血清;清洗结束后,将轮状病毒活化液以0.001MOI的添加量加入维持液中(含有15μg/mL胰酶的DMEM培养基),得到混合液;将混合液加入细胞工厂,每层加液200mL,于37℃培养4d;培养4d后,将脱落的细胞悬液收获并冻融3次,得到轮状病毒病毒收获液;重复上述步骤6次,得到6个批次的轮状病毒病毒收获液样本,用于轮状病毒滴定检测;
轮状病毒的后处理:将第1批次得到的轮状病毒病毒收获液于8000g、4℃下离心15min后,用孔径为0.45μm的滤膜过滤,得到经过滤的轮状病毒病毒收获液;在经过滤的轮状病毒病毒收获液中按体积比1:2000(BPL:经过滤的轮状病毒病毒收获液)添加β-丙内酯(BPL,购自SERVA公司)4℃灭活48h,37℃水解2h,得到轮状病毒灭活液;用100kD超滤膜包将轮状病毒灭活液浓缩为原体积的1/60,得到轮状病毒浓缩液;依次在离心管中添加轮状病毒浓缩液、20%蔗糖溶液、30%蔗糖溶液、40%蔗糖溶液、50%蔗糖溶液和60%(m/v,g/100mL)蔗糖溶液,150000g离心2h,取30~40%和40~50%蔗糖密度交界面蛋白带混合,得到蔗糖液;用100kD超滤膜包将蔗糖液洗滤至蔗糖浓度不超过5%(m/v,g/100mL),得到轮状病毒灭活原液样品,用于Werstern-Blot检测和动物免疫试验。
实施例1:轮状病毒VP6蛋白特异性抗体的制备、纯化和鉴定
1、多肽合成及动物免疫
通过生物信息学手段对轮状病毒的VP6蛋白进行分析,选择肽段332-380aa标记为VP6-P1,VP6-P1多肽和VP6全蛋白的氨基酸序列见表1。
通过体外合成的方式,获得VP6-P1多肽,并于VP6-P1多肽的N端通过交联剂偶联血蓝蛋白。体外合成及偶联均由南京金斯瑞公司完成。
将偶联血蓝蛋白的VP6-P1多肽和VP6全蛋白(购自mybiosource,货号MBS1029122)分别免疫新西兰兔(雌性,体重2~3kg,购自上海杰思捷公司)。具体免疫程序为:分别于0 d、14 d和21 d共进行3次免疫,第28天采血并分离血清。首次免疫时用0.5mL PBS缓冲液将0.5mg多肽溶解,与等体积弗式完全佐剂(购自SIGMA公司)混合后,于新西兰兔背部皮下多点注射,免疫剂量为0.5mg/只。二、三次免疫时除了佐剂改用弗式不完全佐剂外,其余步骤与首次免疫一致。免疫程序结束后分离血清,其中,VP6-P1多肽对应的血清记为VP6-P1-R,VP6全蛋白对应的血清记为VP6-R。通过间接ELISA对VP6-P1-R血清和VP6-R血清的效价进行检测,具体步骤如下:
(1)用CBS缓冲液稀释VP6全蛋白至浓度为10μg/mL,于酶标板中每孔加入100μL,4℃静置16h进行包被;静置结束后,使用PBST缓冲液洗板2次;洗板结束后,每孔加入200μL含3%(v/v)BSA(购自Biofroxx公司)的PBST缓冲液,于37℃封闭2h;
(2)用PBST缓冲液稀释血清,稀释倍数依次为10万、20万、40万、80万、160万、320万、640万、1280万;以PBST缓冲液为阴性对照,将稀释后的血清于步骤(1)获得的酶标板板中每孔加入100μL,37℃孵育1h;孵育结束后,使用PBST缓冲液洗涤4遍;洗涤结束后,每孔加入1:2000(HRP-羊抗兔IgG:PBS=1:2000,v/v)稀释的HRP-羊抗兔IgG(购自博士德生物工程有限公司)100μL,37℃孵育40min;孵育结束后,使用PBST缓冲液洗涤4遍;洗涤结束后,每孔加入TMB显色液(购自碧云天生物技术有限公司)100μL,37℃避光孵育15min;避光孵育结束后,每孔加入浓度为2mol/L的硫酸(购自国药试剂公司)溶液50μL,置于酶标仪450nm读数。Cut off值取阴性对照A450值的2.1倍。
VP6-P1-R血清和VP6-R血清的效价检测结果见图1。由图1可知,阴性孔A450值0.076,Cut off值为0.16。当VP6-P1-R血清稀释至1:320万时A450值为0.219,稀释至1:640万时A450值为0.135。当VP6-R血清稀释至1:160万时A450值0.183,稀释至1:320万时A450值为0.125。因此,VP6-P1-R血清和VP6-R血清的效价分别为1:320万和1:160万。
表1 VP6-P1多肽和VP6全蛋白的氨基酸序列
2、抗体纯化及鉴定
将步骤1获得的VP6-P1-R血清和VP6-R血清分别使用亲和层析法进行纯化,得到VP6-P1-Rpab抗体和VP6-Rpab抗体。纯化由南京金斯瑞公司完成。VP6-P1-Rpab抗体和VP6-Rpab抗体均为多克隆抗体。
分别使用Werstern-Blot和间接免疫荧光(IFA)对纯化后的VP6-P1-Rpab抗体和VP6-Rpab抗体进行鉴定。
其中,Werstern-Blot方法为:将轮状病毒灭活原液样品煮沸变性后进行SDS-PAGE电泳;转膜后用5%(w/v,g/100mL)脱脂奶粉(购自内蒙古伊利实业集团股份有限公司)水溶液封闭2h;封闭结束后,将膜分别转移到1:4000稀释的VP6-P1-Rpab抗体(VP6-P1-Rpab:TBST=1:4000,v/v)和VP6-Rpab抗体(VP6-Rpab:TBST=1:4000,v/v)中,37°孵育1h;孵育结束后,使用TBST缓冲液洗膜4次;洗膜结束后,将膜转移到1:3000稀释的HRP-羊抗兔IgG(HRP-羊抗兔IgG:TBST=1:2000,v/v)中,37°孵育40min;孵育结束后,使用TBST缓冲液洗膜4次;洗膜结束后,使用显色液(购自碧云天生物工程有限公司)显色并拍照。Werstern-Blot结果如图2所示。
间接免疫荧光(IFA)方法为:将浓度为1*105cell/mL的MA104细胞悬液(MA104细胞悬液为将MA104细胞分散于培养基中所得,MA104细胞购自CCTCC,培养基为购自Gibco的DMEM培养基)以每孔100µL的接种量提前铺板至96孔细胞培养板后,于37℃培养3d至细胞长满单层;培养结束后,将轮状病毒SA11毒株的病毒液(轮状病毒SA11毒株购自美国CDC)以MOI=0.01的接种量接种至MA104细胞中进行培养;培养18h后,弃掉96孔细胞培养板中的上清,加入80(v/v)%丙酮水溶液(购自国药试剂公司)于-20℃固定30min;固定结束后,吸弃丙酮并吹干;吹干后,每孔分别加入1:500稀释的VP6-P1-Rpab抗体(VP6-P1-Rpab:PBS=1:500,v/v,一抗)和VP6-Rpab抗体(VP6-Rpab:PBS=1:500,v/v,一抗)100μL,37℃孵育60min;孵育结束后,使用PBS缓冲液洗板3次;洗板结束后,每孔加入1:500(Alexa 488-羊抗兔IgG:PBS=1:500,v/v)稀释的Alexa 488-羊抗兔IgG(购自invitrogen公司,二抗)100μL,37℃避光孵育40min;孵育结束后,使用PBS缓冲液洗板3次;洗板结束后,荧光显微镜观察。间接免疫荧光(IFA)结果如图3所示(图3中的阴性对照为未接毒的MA104细胞)。
由图2可知,VP6-P1-Rpab抗体和VP6-Rpab抗体均识别约45kd大小的条带,与轮状病毒VP6蛋白单体大小基本一致,说明VP6-P1-Rpab抗体和VP6-Rpab抗体均特异性识别VP6蛋白。
由图3可知,VP6-P1-Rpab抗体和VP6-Rpab抗体均可在接毒的MA104细胞中见病毒荧光灶,而未接毒的MA104细胞中无特异性荧光灶,阴性对照成立,表明VP6-P1-Rpab抗体和VP6-Rpab抗体均可与轮状病毒SA11毒株特异性结合。同时,VP6-P1-Rpab组的荧光强度明显比VP6-Rpab组高,表明同稀释度下VP6-P1-Rpab的结合效果更好。
实施例2:轮状病毒VP6蛋白特异性抗体VP6-P1-Rpab在轮状病毒滴度检测中的应用
1、轮状病毒滴度检测方法的建立
以实施例1制得的VP6-P1-Rpab抗体作为一抗对待测样本的轮状病毒滴度进行检测,检测方法(间接免疫荧光)如下:
将待测样本用含2μg/mL胰酶和200μg/mL CaCl2的DMEM培养基进行10倍稀释至10-4、10-5、10-6、10-7,得到不同稀释倍数的待测样本;将浓度为1*105cell/mL的MA104细胞悬液(MA104细胞悬液为将MA104细胞分散于培养基中所得,MA104细胞购自ATCC,培养基为购自Gibco的DMEM培养基)以每孔100µL的接种量提前铺板至96孔细胞培养板后,于37℃培养3d至细胞长满单层;培养结束后,用DMEM培养基将96孔细胞培养板内的MA104细胞洗涤2遍,并将不同稀释倍数的待测样本分别以每孔100μL的接种量接种至MA104细胞中,于37℃进行培养;培养18h后,弃掉96孔细胞培养板中的上清,加入80(v/v)%丙酮水溶液(购自国药试剂公司)于-20℃固定20min;固定结束后,吸弃丙酮并吹干;吹干后,每孔加入1:800稀释的VP6-P1-Rpab抗体(VP6-P1-Rpab:PBS=1:800,v/v,一抗)100μL,37℃孵育60min;孵育结束后,使用PBS缓冲液洗板3次;洗板结束后,每孔加入1:500(Alexa 488-羊抗兔IgG:PBS=1:500,v/v)稀释的Alexa 488羊抗兔IgG(购自invitrogen公司,二抗)100μL,37℃避光孵育40min;孵育结束后,使用PBS缓冲液洗板3次;洗板结束后,荧光显微镜观察;根据观察结果,利用Karber法(参见文献:Hamilton MA, Russo RC, Thurston RV (1977) Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicitybioassays. Environ Sci Technol 11: 714–719.)计算待测样本的轮状病毒滴度。
、轮状病毒滴度检测方法的验证
将步骤1中的一抗替换为市售VP6单抗(购自abcam公司,货号ab252728)作为对照,使用步骤1的轮状病毒滴度检测方法对6个批次的轮状病毒病毒收获液样本进行轮状病毒滴度检测,检测结果见表2。
由表2可知,使用VP6-P1-Rpab抗体对6个批次的轮状病毒病毒收获液样本进行轮状病毒滴度检测的检测结果与市售VP6单抗一致,表明VP6-P1-Rpab抗体可用于轮状病毒滴度检测,且准确性高。
表2 使用VP6-P1-Rpab抗体及市售VP6单抗对样品进行轮状病毒滴度检测的结果
实施例3:轮状病毒VP6蛋白特异性抗体VP6-P1-Rpab在轮状病毒中和抗体检测中的应用
1、轮状病毒中和抗体检测方法的建立
以实施例1制得的VP6-P1-Rpab抗体作为一抗对待测样本的轮状病毒中和抗体进行检测,检测方法如下:
将待测样本用DMEM培养基进行2倍稀释至4、8、16、32、64、128倍,得到不同稀释倍数的待测样本;将轮状病毒Wa毒株的病毒液(购自美国CDC公司)用DMEM培养基稀释至浓度为3*104FFU/mL,得到病毒稀释液;将病毒稀释液以每孔25µL的接种量接种至96孔细胞培养板中后,将不同稀释倍数的待测血清样本以每孔25µL的接种量提前铺板至96孔细胞培养板中与病毒37℃中和1h;中和结束后,在96孔细胞培养板中每孔加入2*104cell的MA104细胞于37℃培养3d;培养结束后,弃掉96孔细胞培养板中的上清,加入80(v/v)%丙酮水溶液(购自国药试剂公司)于-20℃固定20min;固定结束后,吸弃丙酮并吹干;吹干后,每孔加入1:800稀释的VP6-P1-Rpab抗体(VP6-P1-Rpab:PBS=1:800,v/v,一抗)100μL,37℃孵育60min;孵育结束后,使用PBS缓冲液洗板3次;洗板结束后,每孔加入1:500(Alexa 488-羊抗兔IgG:PBS=1:500,v/v)稀释的Alexa 488-羊抗兔IgG(购自invitrogen公司公司,二抗)100μL,37℃避光孵育40min;孵育结束后,使用PBS缓冲液洗板3次;洗板结束后,荧光显微镜观察,根据观察结果利用Reed-Muench法(参见文献:Reed LJ, Muench H. A simple method ofestimating fifty percent end points. The American Journal of Hygiene, 1938,27: 493-497.)计算待测样本的轮状病毒中和抗体效价。
、轮状病毒中和抗体检测方法的验证
将步骤1中的一抗替换为市售VP6单抗(购自abcam公司,货号ab252728)作为对照,使用步骤1的轮状病毒中和抗体检测方法对4个血清样本进行轮状病毒滴度检测,检测结果见表3;
其中,4个血清样本分别来源于4组经轮状灭活疫苗免疫的小鼠;小鼠的免疫过程为:取40只小鼠(雌性,体重17~21g,购自维通利华)分为4组,每组10只,分别于0、14天以每只10µg的剂量免疫4个批次的轮状病毒灭活原液,第21天采血分离血清,各组小鼠血清分别混合得到4个小鼠血清样本。
由表3可知,使用VP6-P1-Rpab抗体对4个血清样本进行轮状病毒中和抗体检测的检测结果与市售VP6单抗一致,表明VP6-P1-Rpab抗体可用于轮状病毒中和抗体检测,且准确性高。
表3 使用VP6-P1-Rpab抗体及市售VP6单抗对样品进行轮状病毒中和抗体检测的结果
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
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<110> 北京赛尔富森生物科技有限公司
<120> 一种特异性结合轮状病毒VP6蛋白的抗体及其应用
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Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly
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Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr
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Phe Asn Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg
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Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Phe Pro Tyr
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Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Met His Asp Asn Leu
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Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly
180 185 190
Phe Asp Tyr Ser Cys Ala Leu Asn Ala Pro Ala Asn Ile Gln Gln Phe
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Glu His Ile Val Gln Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr
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Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val Ile Asn Ser
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Claims (6)
1.一种抗原,其特征在于,所述抗原由氨基酸序列如SEQ ID NO.1所示的多肽组成。
2.一种偶联物,其特征在于,所述偶联物为偶联有载体蛋白的权利要求1所述的抗原。
3.如权利要求2所述的偶联物,其特征在于,所述载体蛋白为血蓝蛋白、白喉毒素DT、白喉毒素的跨膜结构域DTT、轮状病毒VP7、利什曼原虫的热休克蛋白、空肠弯曲菌鞭毛蛋白、沙眼衣原体主要外膜蛋白、牛血清白蛋白、鸡卵白蛋白或纤维蛋白原。
4.权利要求1所述的抗原或权利要求2或3所述的偶联物在制备特异性结合轮状病毒VP6蛋白的抗体中的应用。
5.权利要求1所述的抗原或权利要求2或3所述的偶联物在制备轮状病毒滴度检测试剂盒中的应用。
6.权利要求1所述的抗原或权利要求2或3所述的偶联物在制备轮状病毒中和抗体检测试剂盒中的应用。
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