CN116239682A - 一种识别柯萨奇病毒a2和a4的单克隆抗体及其应用 - Google Patents
一种识别柯萨奇病毒a2和a4的单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明涉及分子生物学和免疫学分析技术领域,具体涉及一种能够识别柯萨奇病毒A2和A4的单克隆抗体及其应用。该单克隆抗体具有SEQ IDNO.9所示的重链互补决定区CDR1、SEQ ID NO.10所示的重链互补决定区CDR2、SEQ ID NO.11所示的重链互补决定区CDR3,以及SEQ ID NO.12所示的轻链互补决定区CDR1、SEQ ID NO.13所示的轻链互补决定区CDR2、SEQ ID NO.14所示的轻链互补决定区CDR3,其能够特异性结合柯萨奇病毒A2和A4的结构蛋白VP1N末端的前15个氨基酸,可以用于临床患者样品检测、病毒分离株实验室抗体鉴定以及生产中间品及产品中柯萨奇病毒A2和A4抗原的定量检测。
Description
技术领域
本发明涉及分子生物学和免疫学分析技术领域,具体涉及一种识别柯萨奇病毒A2和A4(CV-A2和CV-A4)的单克隆抗体7H11E及其应用。
背景技术
手足口病(Hand,foot and mouth disease,HFMD)是由多种人肠道病毒引起的法定丙类传染病,以发热和手、足、口腔等部位出现皮疹等症状,多见于5岁以下儿童,重症患者可出现肺水肿、无菌性脑膜炎等并发症,甚至死亡。近年来,肠道病毒引起的手足口病呈聚集性爆发,多病毒交叉感染等特点,其中肠道病毒A种群病毒如柯萨奇病毒(Coxsackievirus,CV)A2、A4、A5、A6、A10、A16、肠道病毒(Enterovirus,EV)A71等是引起婴幼儿手足口病的主要病原体。
目前,仅有单价的EV-A71全病毒灭活疫苗上市用于预防手足口病,而柯萨奇病毒A种群4型和2型(CV-A4和CV-A2)同属小RNA病毒科,肠道病毒属,也是引起手足口病、疱疹性咽峡炎和急性迟缓性麻痹等疾病的重要病原体,但目前关于其相关的研究则比较少。因此,研究针对CV-A4和CV-A2等其它手足口病肠道相关病毒的特异性或广谱性的单克隆抗体,对临床样品病毒快速检测、实验室病毒鉴别、功能研究和疫苗研发的质量控制及疫苗成品抗原定量等方面具有重要价值。
发明内容
基于此,本发明的目的之一在于提供一种识别柯萨奇病毒A2和A4的单克隆抗体,所述单克隆抗体具有SEQ ID NO.9所示的重链互补决定区CDR1、SEQ ID NO.10所示的重链互补决定区CDR2、SEQ ID NO.11所示的重链互补决定区CDR3,以及SEQ ID NO.12所示的轻链互补决定区CDR1、SEQ ID NO.13所示的轻链互补决定区CDR2、SEQ ID NO.14所示的轻链互补决定区CDR3。
优选的,所述单克隆抗体具有SEQ ID NO.7所示的氨基酸序列的重链;和/或,所述单克隆抗体具有SEQ ID NO.8所示的氨基酸序列的轻链。
优选的,所述单克隆抗体的重链氨基酸序列为SEQ ID NO.7所示的氨基酸序列经替换、缺失或添加一个或多个氨基酸序列后形成的氨基酸序列或与SEQ ID NO.7所示的氨基酸序列具有95%以上同源性的氨基酸序列;和/或,所述单克隆抗体的轻链氨基酸序列为SEQ ID NO.8所示的氨基酸序列经替换、缺失或添加一个或多个氨基酸序列后形成的氨基酸序列或与SEQ IDNo.8所示的氨基酸序列具有95%以上同源性的氨基酸序列。
优选的,所述单克隆抗体为IgG2b亚型。
该单克隆抗体可以特异性识别柯萨奇病毒A2和A4,而不结合其以外的其它肠道病毒,可以进行间接免疫荧光法、免疫印迹法及酶联免疫吸附法等基础实验,对柯萨奇病毒A2和A4引起的手足口病、疱疹性咽峡炎和急性迟缓性麻痹等疾病都具有相同的抗原高效价结合。
本发明的目的之二在于保护编码上述识别柯萨奇病毒A2和A4的单克隆抗体的多核苷酸分子。
优选的,所述多核苷酸分子具有SEQ ID NO.5所示的核苷酸序列;和/或,所述多核苷酸分子具有SEQ ID NO.6所示的核苷酸序列。
本发明的目的之三在于保护一种用于检测柯萨奇病毒A2和A4的试剂盒,所述试剂盒包括上述的单克隆抗体或上述多核苷酸分子编码的单克隆抗体。
本发明的目的之四在于保护上述单克隆抗体或上述多核苷酸分子编码的单克隆抗体在制备检测柯萨奇病毒A2和A4中的单种或两种的试剂或试剂盒中的应用。
本发明的目的之五在于保护上述单克隆抗体或上述多核苷酸分子编码的单克隆抗体在制备抑制、预防、治疗柯萨奇病毒A2和A4的单种或两种药物中的应用。
本发明的目的之六在于保护上述单克隆抗体或上述多核苷酸分子编码的单克隆抗体在柯萨奇病毒A2和A4的VP1蛋白及病毒结构构象改变研究中的应用。如CV-A2和CV-A4病毒颗粒的形态构象转变中间体功能和N末端氨基酸在穿入细胞膜、释放病毒RNA至胞内的基础研究。
本发明所提供的单克隆抗体是以柯萨奇病毒A种群4型(CV-A4)病毒实心颗粒(FP)免疫Balb/c小鼠,制备并筛选杂交瘤细胞后纯化获得,其为能够特异性识别柯萨奇病毒A组2型和4型(CV-A2和CV-A4)的单克隆抗体,为IgG2b亚型非中和抗体,能够特异性结合CV-A2和CV-A4的结构蛋白VP1 N末端第1-15个氨基酸,其中,VP1 N端氨基酸第2-4(DAI)和6(D),为该单克隆抗体的特异性结合CV-A4 VP1 N端氨基酸的足迹。
该单克隆抗体可与偶联物结合(辣根过氧化物酶或异硫氰酸荧光素等)用于直接或间接检测或快速诊断,在临床样本快速经济检测、实验室鉴别、病毒滴定、疫苗制备中间品及产品的抗原定量和结构蛋白VP1功能和抗病毒药物靶点等研发、基于抗原抗体反应的其它应用等方面,具有重要研究和应用价值。
附图说明
图1为单克隆抗体7H11E的IgG轻链、重链还原性SDS-PAGE结果;
图2为单克隆抗体7H11E检测CV-A2和CV-A4感染RD细胞后间接免疫荧光实验结果;
图3为单克隆抗体7H11E识别CV-A2和CV-A4结构蛋白VP1的鉴定图;
图4为单克隆抗体7H11E与不同病毒抗原ELISA结合效价检测结果;
图5为单克隆抗体7H11E线性表位肽库、肽段定位结果,其中A为识别VP1全长(第1~305位)线性表位肽库结果,B为识别VP1线性表位肽段第1~50位氨基酸结果
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
以下各实施例,仅用于说明本发明,但不用来限制本发明的范围。基于本发明中的具体实施例,本领域普通技术人员在没有做出创造性劳动的情况下,所获得的其他所有实施例,都属于本发明的保护范围。
在本发明实施例中,若无特殊说明,所有原料组分均为本领域技术人员熟知的市售产品;在本发明实施例中,若未具体指明,所用的技术手段均为本领域技术人员所熟知的常规手段。
试剂来源:
弗氏完全佐剂和不完全佐剂:采购自Sigma公司;
Isotyping Kit for Mouse Monoclonal Antibody:采购自北京义翘神州科技股份有限公司;
Fast Pure Cell/Tissue Total RNAIsolation Kit:采购自诺唯赞生物科技有限公司;
RD细胞和雌性Balb/c小鼠:由武汉生物制品研究所有限责任公司提供;山羊抗小鼠IgG(H+L)Alexa Fluor 488荧光二抗:采购自赛默飞;
HRP标记羊抗小鼠IgG:采购自武汉博士德生物工程有限公司;
pUC-Kan:采购自南京金斯瑞生物科技有限公司。
实施例1材料制备
CV-A4(毒株为CV-A4/R3179/XY/2017,具体参见文章:田宇璇,汪梦俊和王文辉等.CV-A4单克隆抗体制备及候选疫苗型特异性定量分析方法的建立和验证.现代免疫学,2023,43(01):8-15.)、CV-A2(毒株为CVA2-1388-M14/XY/CHN/2017,NCBI数据库登录号为MW846233)、CV-A5(毒株为CV-A5-3487-M14-XY-CHN-2017,NCBI数据库登录号MW079817)和Ech o 11病毒的培养:取CV-A4、A2、A5和Echo 11病毒分别接种于无血清培养的Vero细胞中,待细胞病变达到90%,收获病毒并分装。
CV-A4、A2、A5和Echo 11病毒实心颗粒(Full particle,FP)和空心颗粒(Emptyparticle,EP)的获得:取CV-A4、CV-A2、CV-A5和Echo 11(MOI=0.001)分别接种汇合度为100%的Vero细胞十层细胞工厂,37℃培养,细胞病变(Cytopathic effect,CPE)达90%时收获病毒液。病毒收获液反复冻融后离心去除细胞碎片,细胞上清经100KD孔径的超滤浓缩,浓缩10倍,并通过25%(W/V)蔗糖垫底,103745g离心4h,用pH=7.2的PBS缓冲液溶解病毒蛋白,以15%、25%、35%、45%、55%(W/V)的蔗糖密度梯度离心,103745g离心4h,收集病毒获得实心颗粒(Full particle,FP)和空心颗粒(Empty particle,EP);将FP和EP分别进行氯化铯密度梯度离心,离心条件为260000g,4℃离心24h,抽取乳光条带获得CV-A4、CV-A2和CV-A5和Echo 11纯化病毒颗粒。
CV-A2的病毒样颗粒(VLP,Virus-like particles)的制备参见文献禹玉婷,胡岗和罗志宇等人.柯萨奇病毒A2病毒样颗粒的制备、纯化及鉴定.中国生物制品学杂志,2021,34(07):782-787。
CV-A6(EP+FP)、CV-A10(EP+FP)、CV-A16(EP+FP)、EV-A71(EP+FP)病毒的培养及病毒颗粒的获得:取CV-A6、CV-A10、CV-A16和EV-A71(MOI=0.001)分别接种Vero细胞,细胞病变(Cytopathic effect,CPE)达90%时收获病毒液,病毒收获液经100KD孔径的超滤浓缩,浓缩体积,将病毒浓缩液经过两次柱层析,即获得CV-A6、CV-A10、CV-A16和EV-A71的病毒原液,即混合病毒颗粒EP+FP。
CV-A5 DP(DP,致密颗粒):为CV-A5感染病毒后经过氯化铯密度梯度离心时,获得不同于EP和FP的另一个病毒颗粒,该颗粒的VP1蛋白部分被剪切,有不完整的VP1蛋白,即N端前1-40个氨基酸被剪切的部分VP1蛋白。文章Jin WP,Lu J,Zhang XY,et al.Efficacyof Coxsackievirus A5 Vaccine Candidates in an Actively Immunized MouseModel.J Virol.2021;95(6):e01743-20.已公开该颗粒信息。
抗A4 FP鼠血清:将CV-A4 FP抗原在第0天和14天免疫SPF级别6-8周Balb/c鼠,在第28天采取血清,即得抗A4 FP鼠血清。
正常小鼠血清:领取SPF级别6-8周Balb/c鼠,采取血清,即获得正常小鼠血清。
实施例2单克隆抗体的制备
本发明提供了单克隆抗体7H11E的制备方法,具体如下:
S1,将60μg CV-A4 FP颗粒与弗氏完全佐剂混合(V/V=1:1),加强免疫为弗氏不完全佐剂混合抗原,以背部皮下多点注射及腹腔注射免疫雌性6-8周龄Balb/c小鼠,注射量体积为500μL/次,分别于第0天、14天、28天和42天免疫小鼠。通过间接ELISA检测52天小鼠血清效价,将ELISA血清结合效价超过1×106的小鼠进行冲击免疫,免疫第3天采集小鼠脾细胞,使用PEG 1500诱导小鼠脾细胞与SP2/0骨髓瘤细胞融合,融合比例控制为(1:5)~(1:10);通过HAT培养基筛选融合成功的杂交瘤细胞。
S2,对融合成功的杂交瘤细胞采用间接ELISA法检测培养上清抗体效价,筛选出ELISA结合效价大于1×104的阳性杂交瘤细胞,三次克隆纯化细胞,并扩大培养制备腹水。采用三步饱和硫酸铵法纯化腹水,从而获得单克隆抗体,并利用Isotyping Kit for MouseMonoclonal Antibody试剂盒鉴定抗体亚型。
其中,间接ELISA筛选杂交瘤细胞的方法为:
将CV-A4 FP用pH=9.6的碳酸盐缓冲液分别配制1μg/mL的包被液加入96孔酶标板,100μL/孔,4℃过夜孵育,孵育结束后用pH=7.4的PBST洗板5次;用pH=7.4的含1%(W/V)牛血清白蛋白(Bovine serum albumin,BSA)的PBST于37℃封闭1h,弃去封闭液;将待检测杂交瘤细胞上清进行十倍倍比(V/V)稀释,加入酶标板,100μL/孔,37℃孵育1h,PBST洗板5次;将0.1μg/mL的HRP标记羊抗小鼠IgG(博士德)抗体加入酶标板,100μL/孔,37℃孵育1h,PBST洗板5次;加入TMB显色液于37℃避光显色30min,用2M硫酸终止反应,用酶标仪检测450nm吸光值;效价计算方式:大于阴性孔OD值的2.1倍时的最大稀释倍数;阴性孔和杂交瘤细胞培养基作为阴性对照。
实施例3单克隆抗体序列分析
将实施例2中筛选出来的杂交瘤细胞(分泌7H11E单克隆抗体)接种于含10%胎牛血清的RPMI 1640培养基(Gibco),37℃、5% CO2培养。待细胞数量约为2-5×106个,收获杂交瘤细胞,采用Fast Pure Cell/Tissue Total RNAIsolation Kit提取总RNA。使用TaKaRaRimeScrip II 1st Strand cDNA Synthesis Kit中的Oligo dT引物逆转录总RNA获得单链cDNA,通过与克隆载体pUC-Kan同源序列的引物扩增单克隆抗体7H11E的重链和轻链可变区基因,并分别将纯化后的PCR产物克隆至pUC-Kan载体(购自南京金斯瑞生物科技有限公司)中,筛选阳性克隆后测序,通过比对将正确的可变区氨基酸序列,在Kabat数据库中序列分析。其中,重链通用正向引物VH-F的序列SEQ ID NO.1:acggccagtgaattcmarctgcagsagtcwgg,反向引物VH-R的序列SEQ ID NO.2:gattacgccaagctttgaggagacggtgaccg;轻链通用正向引物VL-F的序列SEQ ID NO.3:acggccagtgaattccgattgtkctsacycartctcca,反向引物VL-R的序列SEQ ID NO.4:gattacgccaagcttcgttggatctccagcttg。
测出的单克隆抗体7H11E的7H11E的重链可变区的核苷酸序列如下:TCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTACCCAGCTATGATGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACACATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCGGGGCTACGGGCCTTTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO.5);
轻链可变区的核苷酸序列如下:CCGATTGTGCTCACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAACATACACTGGTATCAGCAAGGAGCAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAGAGTAATAGCTGGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGATCCAACGAGCT(SEQ ID No.6);
重链可变区的核苷酸序列编码的氨基酸序列如下:SGPGLVAPSQSLSITCTVSGFSLPSYDVHWVRQPPGKGLEWLGVIWAGGSTHYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAGLRAFAMDYWGQGTSVTVSS(SEQ ID No.7);
轻链可变区的核苷酸序列编码的氨基酸序列如下:PIVLTQSPAILSVSPGERVSFSCRASQSIGTNIHWYQQGANGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTKLEIQRA(SEQ ID No.8)。
分析出的六个CDR(互补决定区)的序列分别如下:
重链的互补决定区VHCDR1的氨基酸序列:SYDVH(SEQ ID No.9);
重链的互补决定区VHCDR2的氨基酸序列:VIWAGGSTHYNSALMS(SEQ ID No.10);
重链的互补决定区VHCDR3的氨基酸序列:LRAFAMDY(SEQ ID No.11);轻链的互补决定区VLCDR1的氨基酸序列:RASQSIGTNIH(SEQ ID No.12);轻链的互补决定区VLCDR2的氨基酸序列:YASESIS(SEQ ID No.13);
轻链的互补决定区VLCDR3的氨基酸序列:QQSNSWPWT(SEQ ID No.14)。
实施例4单克隆抗体7H11E的亚型鉴定
取实施例2重纯化后的单克隆抗体7H11E,进行4~20%SDS-PAGE。还原性SDS-PAGE鉴定结果如图1所示,从图1中可知7H11E单克隆抗体的重链和轻链所对应的分别为50kDa和25kDa的蛋白条带,亚型为IgG 2b,与预期结果符合。
实施例5单克隆抗体7H11E的功能分析
分别采用免疫荧光实验和免疫印迹实验等方法对筛选出的单克隆抗体7H11E进行分析,具体如下:
(1)间接免疫荧光实验
将CV-A2和CV-A4病毒接种于汇合度为95%的RD细胞的6孔板,未接种病毒的RD细胞为阴性对照,放置于37℃,5% CO2孵箱;将6孔板培养24h后弃掉细胞上清,加入2mL/孔4%多聚甲醛室温固定1h,0.01M PBS洗涤5次/5min;加入2mL/孔2% BSA-PBST(W/V)溶液(含0.5% Triton-X 100,V/V),室温通透30min,0.01M PBS洗涤5次/5min;用2% BSA-PBST溶液于室温封闭1h,弃封闭液;分别加入1mL/孔2μg/mL的单克隆抗体7H11E于室温孵育1h,0.01M PBS洗涤5次/5min;加入1mL/孔,2μg/mL山羊抗小鼠荧光抗体IgG(H+L)(赛默飞),避光室温孵育1h,0.01M PBS洗涤5次/5min,并加入1mL/孔5μg/mL DAPI溶液(碧云天);用荧光显微镜观察并拍照,结果如图2所示。
从图2中可以看出,单克隆抗体7H11E可用于间接免疫荧光实验识别CV-A2和CV-A4。
(2)免疫印迹实验
单克隆抗体7H11E识别CV-A2、CV-A4结构蛋白区域鉴定。肠道病毒结构蛋白为VP1、VP2、VP3和VP4,可通过WB实验确定7H11E识别的CV-A2和CV-A4结构蛋白区域,具体步骤如下:
分别取相同抗原量的CV-A4 FP、CV-A2 FP、CV-A2 VLP、CV-A5 FP、CV-A5 DP(DP,致密颗粒)和Vero细胞裂解液加入4×SDS-PAGE的上样缓冲液(Bio-rad),100℃加热10min进行SDS-PAGE检测,电泳结束,使用快速湿转仪(金斯瑞)将抗原转至0.45μm的硝酸纤维素膜;转膜结束,用2%BSA(W/V)封闭30min;封闭结束,分别加入10mL 0.3μg/mL的7H11E,37℃孵育1h并洗涤;洗涤结束,加入10mL HRP标记羊抗小鼠IgG,稀释度为1:10,000,37℃孵育45min并洗涤;使用显色液进行用化学曝光成像(Gene),抗体识别结果如图3所示。
由图3可知,7H11E单克隆抗体可特异性结合CV-A2和CV-A4病毒的结构蛋白VP1区。
(3)单克隆抗体7H11E结合效价检测
采用ELISA测定单克隆抗体7H11E与肠道病毒A种群病毒CV-A2(FP)、CV-A4(EP,FP)、CV-A5(FP)、CV-A6(EP+FP)、CV-A10(EP+FP)、CV-A16(EP+FP)、EV-A71(EP+FP)的结合能力,肠道病毒B种群病毒Echo11(FP)纯化颗粒用pH=9.6的碳酸盐缓冲液分别配制1μg/mL的包被液加入酶标板,100μL/孔,4℃过夜孵育,孵育结束后洗涤3次;用1%(W/V)BSA的PBST于37℃封闭1h,弃去封闭液;
加入一抗:将1mg/mL单克隆抗体7H11E按十倍倍比(体积比)稀释,取稀释后的抗体100μL/孔加入酶标板,37℃孵育1h,用pH=7.4的PBST洗板5次后;
加入二抗:将0.1μg/mL的HRP标记羊抗小鼠IgG抗体加入酶标板,100μL/孔,37℃孵育1h,用pH=7.4的PBST洗板5次;
加入TMB显色液37℃避光显色30min,2M硫酸终止反应后检测450nm吸光值;
效价计算方式:大于阴性孔OD值的2.1倍时的最大稀释倍数。阴性对照为未加7H11E,只加二抗,阳性对照为分别特异性识别病毒CV-A2、CV-A4,CV-A5、CV-A6、CV-A10、CV-A16和EV-A71颗粒的鼠血清。
检测结果如图4所示,根据图4可以计算出单克隆抗体对CV-A4和CV-A2的结合效价相当。
实施例6单克隆抗体7H11E的线性表位研究
(1)单克隆抗体7H11E线性表位初步定位
通过ELISA初步鉴定7H11E单克隆抗体线性表位氨基酸序列,方法如下:合成CV-A4结构蛋白VP1区重叠多肽,覆盖VP1全长305个氨基酸,多肽序列见下表1:
表1CV-A4结构蛋白VP1区重叠多肽序列表
使用间接ELISA实验初步定位:将多肽以4μg/mL浓度用pH=9.6的碳酸盐缓冲液分别配制1μg/mL的包被液加入酶标板,100μL/孔,4℃过夜孵育后PBST洗板3次;用pH=7.4的含1%(W/V)BSA的PBST于37℃封闭1h,弃去封闭液;按十倍倍比(V/V)稀释单克隆抗体7H11E,取1μg/ml的单克隆抗体,100μL/孔加入酶标板,37℃孵育1h,PBST洗板5次;将0.1μg/mL的HRP标记羊抗小鼠IgG抗体加入酶标板,100μL/孔,37℃孵育1h,PBST洗板5次;加入TMB(3,3',5,5'-四甲基联苯胺)显色液于37℃避光显色30min,用2M硫酸终止反应后检测450nm吸光值。
效价计算方式:大于阴性孔OD值的2.1倍时的最大稀释倍数;正常小鼠血清为阴性对照,阳性对照为抗A4 FP鼠血清。
单克隆抗体线性表位初步定位结果见图5A和5B,从图5A和5B中可以看出,单克隆抗体能结合CV-A4结构蛋白VP1的1号肽段(即VP1前1-15氨基酸)。
(2)单克隆抗体7H11E线性表位精确定位
为了进一步缩小7H11E单克隆抗体结合肽的定位范围,将单个肽包被在96孔板的孔中以鉴定特异性结合肽,为了进一步确定结合残基,合成一组X(除G外的任何氨基酸)到G的单一氨基酸替代物,共14单位点突变合成肽,如表2所示,覆盖VP1 N端前15个氨基酸,用于进一步定位负责单个单克隆抗体结合的残基,实验测定包被浓度范围为5μg-10μg/ml,100μl/孔合成肽溶液,后续ELISA的步骤与上述初步定位方法相同,结果如表2:
表2单克隆抗体7H11E的关键结合位点精确定位
VP1氨基酸位置 | 突变氨基酸 | ELISA结果 |
2 | D-G | - |
3 | A-G | - |
4 | I-G | - |
5 | A-G | + |
6 | D-G | - |
7 | A-G | + |
8 | I-G | + |
9 | Q-G | + |
10 | N-G | + |
11 | T-G | + |
12 | V-G | + |
13 | T-G | + |
14 | T-G | + |
15 | T-G | + |
其中,+、-表示ELISA阳性或阴性结果。
如表2所示,VP1 N末端的第2至第15个氨基酸(VP1-2至15)从原始残基突变为甘氨酸,即VP1-2D-G表示位置2的D突变为G,第一个甘氨酸被保留下来,这些合成肽包含单个氨基酸的改变,如表所示携带单点突变(G),包被到孔板中用于检测。7H11E与CV-A4和CV-A2结合的突变残基分别用绿色表示,也即2-4(DAI)和6(D)。
发明人经过实验进一步发现,该单克隆抗体7H11E的重链的氨基酸序列SEQ IDNo.7所示的氨基酸序列经替换、缺失或添加一个或多个氨基酸后形成的新的序列或与SEQID No.7所示的氨基酸序列具有95%以上同源性的氨基酸序列具有同SEQ ID No.7所示序列的同等功能;轻链的氨基酸序列SEQID No.8所示的氨基酸序列经替换、缺失或添加一个或多个氨基酸后形成的新的序列或与SEQ ID No.8所示的氨基酸序列具有95%以上同源性的氨基酸序列具有同SEQ ID No.8所示序列的同等功能。
在此有必要指出的是,以上实施例仅限于对本发明的技术方案做进一步的阐述和说明,并不是对本发明的技术方案的进一步的限制,本发明的方法仅为较佳的实施方案,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种识别柯萨奇病毒A2和A4的单克隆抗体,其特征在于,所述单克隆抗体具有SEQID NO.9所示的重链互补决定区CDR1、SEQ ID NO.10所示的重链互补决定区CDR2、SEQ IDNO.11所示的重链互补决定区CDR3,以及
SEQ ID NO.12所示的轻链互补决定区CDR1、SEQ ID NO.13所示的轻链互补决定区CDR2、SEQ ID NO.14所示的轻链互补决定区CDR3。
2.根据权利要求1所述的识别柯萨奇病毒A2和A4的单克隆抗体,其特征在于,所述单克隆抗体具有SEQ ID NO.7所示的氨基酸序列的重链;和/或,所述单克隆抗体具有SEQ IDNO.8所示的氨基酸序列的轻链。
3.根据权利要求2所述的识别柯萨奇病毒A2和A4的单克隆抗体,其特征在于,所述单克隆抗体的重链氨基酸序列为SEQ ID NO.7所示的氨基酸序列经替换、缺失或添加一个或多个氨基酸序列后形成的氨基酸序列或与SEQ ID NO.7所示的氨基酸序列具有95%以上同源性的氨基酸序列;和/或,所述单克隆抗体的轻链氨基酸序列为SEQ ID NO.8所示的氨基酸序列经替换、缺失或添加一个或多个氨基酸序列后形成的氨基酸序列或与SEQ ID No.8所示的氨基酸序列具有95%以上同源性的氨基酸序列。
4.根据权利要求1~3任一项所述的识别柯萨奇病毒A2和A4的单克隆抗体,其特征在于,所述单克隆抗体为IgG2b亚型。
5.编码权利要求1或2或3所述的识别柯萨奇病毒A2和A4的单克隆抗体的多核苷酸分子。
6.根据权利要求5所述的多核苷酸分子,其特征在于,所述多核苷酸分子具有SEQ IDNO.5所示的核苷酸序列;和/或,所述多核苷酸分子具有SEQ ID NO.6所示的核苷酸序列。
7.用于检测柯萨奇病毒A2和A4的试剂盒,其特征在于,所述试剂盒包括权利要求1或2或3或4所述的单克隆抗体或权利要求5或6所述的多核苷酸分子编码的单克隆抗体。
8.权利要求1或2或3或4所述的单克隆抗体或权利要求5或6所述的多核苷酸分子编码的单克隆抗体在制备检测柯萨奇病毒A2和A4中的单种或两种的试剂或试剂盒中的应用。
9.权利要求1或2或3或4所述的单克隆抗体或权利要求5或6所述的多核苷酸分子编码的单克隆抗体在制备抑制、预防、治疗柯萨奇病毒A2和A4的单种或两种药物中的应用。
10.权利要求1或2或3或4所述的单克隆抗体或权利要求5或6所述的多核苷酸分子编码的单克隆抗体在柯萨奇病毒A2和A4的VP1蛋白及病毒结构构象改变研究中的应用。
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