CN111139233B - 广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体及应用 - Google Patents
广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体及应用 Download PDFInfo
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Abstract
本发明涉及一种广谱中和抗EV71、CA16、CA10和CA6人‑鼠嵌合IgM型单克隆抗体及应用,属于生物技术领域。该抗体包括重链可变区和轻链可变区,其所述重链可变区的氨基酸序列如序列表中SEQ ID NO.1所示,所述轻链可变区的氨基酸序列如序列表中SEQ ID NO.2所示。本发明获得了一种可以同时抗EV71、CA16、CA 10和CA6的人‑鼠嵌合单克隆抗体,其由鼠源抗体IgM的可变区和人源抗体IgM的恒定区构成。本发明所述嵌合抗体对EV71、CA16、CA10和CA6均具有较高亲和力,对EV71、CA16、CA10和CA6展现广谱高效的中和活力,可用于治疗EV71、CA16、CA10和CA6等A型肠道病毒引起的手足口病。
Description
技术领域
本发明属于生物技术领域,具体涉及一种广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体及其制备方法和应用。
背景技术
肠道病毒71(Enterovirus 71,EV71)与柯萨奇病毒A型16(Coxsackievirus A 16,CA16)已被鉴定是诱发手足口病(hand foot and mouth disease,HFMD)的主要病原体。近年来,研究显示:柯萨奇病毒A型10(Coxsackievirus A 10,CA10)和柯萨奇病毒A型6(Coxsackievirus A 16,CA6)感染导致手足口病病例呈现上升趋势。目前,已经成功上市的灭活EV71疫苗针对EV71感染展现高效的保护效果,但其对CA16、CA10与CA6等其他型别肠道病毒没有交叉保护作用。同时,研究显示:在CA16疫苗免疫恒河猴后,其所产生的中和抗体不能有效保护宿主抵抗病毒感染。由此,开发针对肠道病毒具有广谱高效的中和抗体(broadly neutralizing antibodies,bNAbs)进行被动免疫的病毒防治已成为抗病毒的新策略之一。目前报道的单克隆抗体为IgG型,其仅对单一型别的肠道病毒具有中和治疗效应,且不具有交叉保护作用。因此如何克服现有技术的不足是目前生物技术领域亟需解决的问题。
发明内容
本发明的目的是为了解决现有技术的不足,提供一种广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体及其制备方法和应用,本发明采用杂交瘤法制备得到一株鼠源的单克隆抗体,但由于其可能引起人抗鼠抗体(human anti-mouse antibody,HAMA)反应,影响其在人体内的治疗效果并产生副反应。因此,本发明利用人-鼠嵌合法将该株鼠源抗体进行修饰改造,降低其对人体的免疫原性。人-鼠嵌合抗体是利用基因工程技术将鼠源抗体的可变区与人源抗体的恒定区相连接,构建成嵌合抗体,可以保持原有抗体的亲和力和特异性,减少人抗鼠抗体反应,延长半衰期和改善药代动力学。
为实现上述目的,本发明采用的技术方案如下:
本发明第一方面提供一种广谱中和抗EV71、CA16、CA10和CA6抗体杂交瘤细胞株的制备方法,包括如下步骤:
步骤(1),免疫原的制备:以纯化的EV71和CA16病毒为免疫原;
步骤(2),动物免疫:取6~8周龄Balb/c小鼠,以2周间隔进行四次免疫,程序为:首次免疫,弗氏完全佐剂与等量免疫原充分混合乳化后,背部皮下多点注射;第二次免疫和第三次免疫,佐剂换为弗氏不完全佐剂,方法和计量同首次免疫;第四次采用腹腔注射未加佐剂的免疫原,注射量同首次免疫;3天后,取免疫小鼠脾脏细胞用于制备杂交瘤细胞;
步骤(3),杂交瘤细胞株的制备和筛选:将处于对数生长期的SP2/0细胞与步骤(2)免疫小鼠脾脏细胞通过PEG4000的作用下进行细胞融合,利用1×HAT选择性培养基培养融合后的杂交瘤细胞,细胞融合后10-14天,通过酶联免疫吸附试验检测细胞上清以筛选能特异性分泌广谱中和抗EV71、CA16、CA10和CA6抗体的杂交瘤细胞株。
进一步,优选的是,步骤(2)中,首次免疫注射量为50ul 107TCID50/ml EV71和50ul 107TCID50/ml CA16。
进一步,优选的是,步骤(3)中,将处于对数生长期的SP2/0细胞与步骤(2)免疫小鼠脾脏细胞通过PEG4000的作用下进行细胞融合,利用1×HAT选择性培养基培养融合后的杂交瘤细胞,具体方法为:
选择6~8周龄Balb/c小鼠,取其腹腔巨噬细胞制备饲养层细胞;取处于对数生长期的SP2/0细胞与步骤(2)免疫小鼠脾脏细胞按照1∶10的细胞数量比例进行混和,采用40%聚乙二醇4000进行细胞融合,细胞融合后获得的杂交瘤细胞加入已含有饲养层细胞的培养板中,使用含有1×HAT选择性培养基,在37℃、5%CO2条件下培养。
进一步,优选的是,还包括杂交瘤细胞的克隆化,具体是:通过有限稀释法对杂交瘤细胞株进行亚克隆培养,连续培养多次,直至克隆孔内抗体检测100%阳性为止,扩大培养并冻存。
本发明第二方面提供一种广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体的制备方法,采用上述广谱中和抗EV71、CA16、CA10和CA6抗体杂交瘤细胞株的制备方法制得的广谱中和抗EV71、CA16、CA10和CA6抗体杂交瘤细胞株;包括如下步骤:
提取抗EV71、CA16、CA10和CA6广谱中和抗体杂交瘤细胞株的RNA,逆转录获取cDNA;然后巢式PCR扩增抗体重链可变区,同时巢式PCR扩增抗体轻链可变区;之后采用PVITRO1-hygro-mcs-IgM/κ连接载体对抗体可变区进行连接,获取IgM型抗体;同时,采用PVITRO1-hygro-mcsIgG/κ连接载体对抗体可变区进行连接,获取IgG型抗体;接着将连接得到的阳性单克隆抗体转染至CHO-S细胞,后纯化,即得广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体和IgG型单克隆抗体;所述的PVITRO1-hygro-mcs-IgM/κ连接载体的DNA序列如SEQ ID NO.7所示;所述的PVITRO1-hygro-mcsIgG/κ连接载体的DNA序列如SEQID NO.8所示。
本发明第三方面提供上述广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体的制备方法制备得到的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体以及其IgG型单克隆抗体。
本发明第四方面提供一种广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体,其特征在于,包括重链可变区和轻链可变区,其所述重链可变区的氨基酸序列如序列表中SEQ ID NO.1所示,所述轻链可变区的氨基酸序列如序列表中SEQ ID NO.2所示。
本发明第五方面提供一种如上述广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体的编码DNA,其特征在于,包括重链可变区和轻链可变区;
所述重链可变区的编码DNA序列如SEQ ID NO.3所示;
所述轻链可变区的编码DNA序列如SEQ ID NO.4所示。
本发明第六方面提供一种表达载体,包含重链恒定区DNA序列和轻链恒定区DNA序列,用于表达如权利要求5所述的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体;
所述重链恒定区(C-Mu)的编码DNA序列如SEQ ID NO.5所示;
所述轻链恒定区(C-kapa)的编码DNA序列如SEQ ID NO.6所示。
本发明第七方面提供一种宿主细胞,包含上述的表达载体。
本发明第八方面提供一种提供药物组合物,包括上述抗PD-1单克隆抗体和药学上可接受的载体。
本发明第八方面提供上述广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体在制备治疗或预防人病毒感染、肿瘤及炎性疾病药物中的应用。
本发明核苷酸分子的制备方法为本领域常规制备方法,较佳地包括以下制备方法:通过基因克隆技术例如PCR方法等,获得编码所述单克隆抗体的核苷酸分子,或者通过人工全序列合成的方法得到编码上述单克隆抗体的核苷酸分子。
本领域技术人员知晓,编码上述单克隆抗体的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、***或增加来提供一个多聚核苷酸的同系物或其保守型变异序列。本发明中多聚核苷酸的同系物或其保守型变异序列,可以通过对编码该单克隆抗体基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明所述表达载体为本领域常规的表达载体,是指包含适当的调控序列,例如
启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当
的序列的表达载体。所述表达载体可以是病毒或质粒,如适当的噬菌体或者噬菌粒,更多技术细节请参见例如Sambrook等,Molecular Cloning:A Laboratory Manual,第二版,ColdSpring Harbor Laboratory Press,1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology,第二版,Ausubel等编著。本发明中的表达载体指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。
本发明所述宿主细胞为本领域常规的宿主细胞,只要能满足使上述重组表达载体稳定地自行复制,且所携带所述的核苷酸可被有效表达即可。本发明中的宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。所述宿主细胞较佳地包括:COS、CHO、HeLa细胞系、骨髓细胞系如SP2/0细胞系、NS0、sf9、sf21、DH5α、BL21(DE3)或E.coliTG1、YB2/0细胞系等以及转化的B-细胞或杂交瘤细胞中的一种或多种的组合。更佳地为E.coli TG1、BL21细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。
将上述表达载体转化至宿主细胞中,即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法,较佳地为化学转化法,热激法或电转法。
本发明中所用的宿主细胞均为现有技术,可通过商业途径直接获取,培养中所用的培养基亦为各种常规培养基,本领域技术人员可根据经验选择适用的培养基,在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的单克隆抗体可以通过本领域任何已知的方式配制药物组合物来使用。这种组合物以所述单克隆抗体为活性成分,加上一种或多种药物可接受的载体、稀释剂、填充剂、结合剂及其它赋形剂,这依赖于给药方式及所设计的剂量形式。本领域枝术人员已知的治疗惰性的无机或有机的载体包括(但不限于)乳糖、玉米淀粉或其衍生物、滑石、植物油、蜡、脂肪、多羟基化合物例如聚乙二醇、水、蔗糖、乙醇、甘油,诸如此类,各种防腐剂、润滑剂、分散剂、矫味剂。保湿剂、抗氧化剂、甜味剂、着色剂、稳定剂、盐、缓冲液诸如此类也可加入其中,这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味,在这种组合物中可以使用抑制剂可是其原始化合物本身的形式,或任选地使用其药物学可接受的盐的形式,本发明的单克隆抗体可以单独给药,或以各种组合给药,以及与其它治疗药剂一起结合形式给药。如此配制的组合物根据需要可选择本领域技术人员已知的任何适当的方式对抑制剂进行给药。
本发明与现有技术相比,其有益效果为:
本发明采用杂交瘤法制备得到一株鼠源的单克隆抗体,但由于其可能引起人抗鼠抗体(humananti-mouse antibody,HAMA)反应,影响其在人体内的治疗效果并产生副反应。因此,本发明利用人-鼠嵌合法将该株鼠源抗体进行修饰改造,降低其对人体的免疫原性。人-鼠嵌合抗体是利用基因工程技术将鼠源抗体的可变区与人源抗体的恒定区相连接,构建成嵌合抗体,可以保持原有抗体的亲和力和特异性,减少人抗鼠抗体反应,延长半衰期和改善药代动力学。
本发明所获人-鼠嵌合抗体:20-IgM,在体外(RD)和体内(一日龄乳鼠)对EV71、CA16、CA10和CA6感染均展现高效广谱中和作用。本发明的抗体可用于预防或治疗EV71、CA16、CA10和CA6感染,更进一步其有可能应用于其他型别的肠道病毒感染所引起的手足口病治疗。
附图说明
图1为抗体重链可变区PCR凝胶电泳图;
图2为抗体轻链可变区PCR凝胶电泳图;
图3为抗体氨基酸序列分析图;其中,A为20-IgM单克隆抗体重链序列及基因打靶结果图;B为20-IgM单克隆抗体轻链序列及基因打靶结果图;C为20-IgM单克隆抗体结构模拟图;
图4为抗体分子大小和纯度鉴定结果图;其中,Ladder:蛋白分子标准;20-IgG(non-reducing):非还原状态20-IgG;20-IgM(non-reducing):非还原状态20-IgM;20-IgG(reducing):还原状态20-IgG;20-IgM(reducing):还原状态20-IgM;
图5为抗体与不同型别肠道病毒交叉识别作用鉴定结果图;
图6为抗体亲和力鉴定结果图;其中,ligant Concentration为配体浓度,Fnorm[‰]为响应值;(a):20-IgM分别于与EV71、CA16、CA10、CA6亲和力测定;(b):20-IgG分别于与EV71、CA16、CA10、CA6亲和力测定。
图7为抗体中和表位鉴定结果图;A:20-IgM与20-IgG识别EV71衣壳蛋白鉴定;B:20-IgM与20-IgG识别CA16衣壳蛋白鉴定;C:20-IgM与20-IgG识别EV71衣壳蛋白鉴定;D:20-IgM与20-IgG识别CA6衣壳蛋白鉴定;E-G:ELISA法鉴定20-IgM与20-IgG识别抗原肽;H-I:抗原肽抑制试验鉴定20-IgM与20-IgG结合抗原肽;J:ELISA法鉴定20-IgM与20-IgG识别关键氨基酸位点;K:20-IgM与20-IgG识别EV71氨基酸位点;L:20-IgM与20-IgG识别CA16氨基酸位点;M:20-IgM与20-IgG识别CA6氨基酸位点;N:20-IgM与20-IgG识别CA10氨基酸位点;
图8为抗体中和效应细胞病变图;
图9为抗体中和效力测定结果图;左上:20-IgM与20-IgG对EV71的IC50与IC80测定;右上:20-IgM与20-IgG对CA16的IC50与IC80测定;左下:20-IgM与20-IgG对CA10的IC50与IC80测定;右下:20-IgM与20-IgG对CA6的IC50与IC80测定。IC50:(half maximalinhibitory concentration,半数有效抑制浓度;
图10为抗体对感染乳鼠保护量效图;其中,Days post-infection为感染后天数;Percent survival为生存百分比;Body weight change为体重变化。A:感染组与抗体组小鼠临床症状;B:EV71感染组与20-IgM治疗组小鼠生存率变化;C:EV71感染组与20-IgM治疗组小鼠体重变化;D::CA16感染组与20-IgM治疗组小鼠生存率变化;E::CA16感染组与20-IgM治疗组小鼠体重变化;
图11为pVITRO1-hygro-mcs-IgM/κ表达载体质粒图谱;
图12为pVITRO1-hygro-mcs-IgG/κ表达载体质粒图谱。
具体实施方式
下面结合实施例对本发明作进一步的详细描述。
本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品。
本发明除非另有说明,否则百分号代表体积百分数,比例代表体积比。
1.抗EV71、CA16、CA10和CA6双效杂交瘤细胞株制备方法
1)免疫原制备:以纯化的EV71和CA16病毒为免疫原;
2)动物免疫:取6~8周龄Balb/c小鼠,以2周间隔进行四次免疫,程序为:首次免疫,弗氏完全佐剂与等量免疫原充分混合乳化后,背部皮下多点注射,100ul 107TCID50/ml;第二次免疫和第三次免疫,佐剂换为弗氏不完全佐剂,方法和计量同首次免疫;第四次采用腹腔注射未加佐剂的免疫原,注射量同首次免疫;3天后,取免疫小鼠脾脏细胞用于制备杂交瘤细胞;
3)杂交瘤细胞株的制备:具体是:细胞融合前2天,取免疫小鼠脾脏细胞用于制备杂交瘤细胞。选择6~8周龄Balb/c小鼠,取其腹腔巨噬细胞制备饲养层细胞,使细胞浓度为105个/mL,加入96孔板,每孔100μL,置于37℃、5%CO2条件下培养备用。取处于对数生长期的SP2/0细胞(购自ATCC)与步骤2)免疫小鼠脾脏细胞按照1∶10的细胞数量比例进行混和,将45%聚乙二醇4000(PEG)(购买自sigma)细胞融合后获得的杂交瘤细胞加入已含有饲养层细胞的96孔板中,使用含有1×HAT选择性培养基[50×HAT(购买自sigma)、20%(V/V)胎牛血清、1640培养基(购买自Corning)],在37℃、5%CO2条件下培养。
4)杂交瘤细胞株的筛选,具体是:将步骤3)获得的杂交瘤细胞在含有1×HAT选择性培养基的条件下培养10-14天,每隔4天更换一半量的1×HAT的完全培养基。培养12天后取杂交瘤细胞上清进行间接ELISA以初次筛选能特异性分泌抗EV71、CA16、CA10和CA6的杂交瘤细胞。具体步骤如下:EV71或CA16包被酶标板,设置空白对照,取培养上清为一抗,SP2/0细胞培养上清为阴性对照,二抗为HRP标记山羊抗小鼠IgG(1:6000)(ThermoScientific),TMB(Solarbio)显色终止后,酶标仪检测450nm吸光度值(A450值),以高于阴性对照A450值2.1倍以上的孔为阳性细胞孔,并进行下一步的亚克隆。
5)杂交瘤细胞的克隆化,具体是:选取步骤4)中筛选得到的阳性细胞孔,采用有限稀释法对阳性孔的细胞进行克隆,将每孔200μL细胞吸取至加样槽,用1×HT完全培养基[(50×HT(购买自sigma)、20%(V/V)胎牛血清、1640培养基(购买自Corning)]稀释至20mL后,均匀加至一个新的96孔板,每孔200μL,37℃、5%CO2条件下培养5天后再次进行间接ELISA检测,再次选取高于阴性对照A450值2.1倍以上且细胞生长状态良好的细胞孔,分克隆至另一个96孔板,并换1×HAT选择性培养基培养,如此重复三至四轮,直至克隆孔内抗体检测100%阳性为止。选取生长状态好的细胞,用普通完全培养基[20%(V/V)胎牛血清、1640培养基(购买自Corning)]逐步扩大培养,并液氮冻存。
在亚克隆培养筛选的过程中,用EV71、CA16、CA10和CA6分别包被酶标板,封闭后取杂交瘤细培养上清进行间接ELISA,设置空白对照,SP2/0细胞培养上清为阴性对照,HRP标记山羊抗小鼠IgG(1:6000)为二抗,根据两块酶标板A450值读数,筛选能够同时识别EV71和CA16的单克隆细胞,这些杂交瘤细胞株进行进一步的亚克隆和扩增培养。
6)杂交瘤抗体型别鉴定:采用Mouse Monoclonal Antibody Isotyping Kit(Roche)鉴定抗体的型别,方法见说明书。
2.人-鼠嵌合单克隆抗体制备
1)RNA提取:采用TRNzol-A+(TIANGEN)提取1中所筛选的同时识别EV71和CA16的杂交瘤细胞的RNA,细胞数为105。
2)逆转录获取cDNA:采用PrimeScript II 1st Strand cDNA Synthesis Kit将2.1获取的RNA逆转录为cDNA,方法参见试剂盒(Takara,Dalian)。
3)巢式PCR扩增抗体重链可变区:巢式PCR扩增体系为共54ul:
I-5TM 2x High-Fidelity Master Mix(Tsingke,China)27ul;
步骤2)获取的cDNA 4ul;
VH Forward(每个引物浓度为2pM):每个1ul,共4个,总共4ul;
VH Reverse(每个引物浓度为2pM):每个1ul,共19个,总共19ul。
扩增程序为:98℃预变性1分钟;98℃变性,10秒,65℃复性10秒,72℃延伸10秒,循环35次;72℃延伸5分钟。结果见图1。
4)巢式PCR扩增抗体轻链可变区:巢式PCR扩增体系50ul:
I-5TM 2x High-Fidelity Master Mix(Tsingke,China)25ul;
步骤2)获取的cDNA 4ul;
VL(κ)Forward(每个引物浓度为2pM):每个1ul,共4个,总共4ul;
VL(κ)Reverse(每个引物浓度为2pM):每个1ul,共17个,总共17ul。
扩增方法为:98℃预变性1分钟;98℃变性,10秒,60℃复性10秒,72℃延伸10秒,循环35次;72℃延伸5分钟。结果见图2。
5)琼脂糖凝胶电泳鉴定巢式PCR产物:上样样品:marker(DL2000/DL1000),VH,VL;采用1%琼脂糖凝胶,100mA,20min。结果见图1和图2。
6)抗体可变区测序及序列分析:将步骤5)胶回收产物送Tsingke公司测序,测序结果经IgBLAST比对分析序列。结果显示:20-IgM抗体重链互补决定区3(complementaritydetermining region 3,CDR3)氨基酸序列为:Ala Arg Gly Gly Asn Tyr Tyr Tyr AlaMet Asp Tyr。其余序列结果见图3。
7)连接载体:步骤6)鉴定该株抗体亚型为IgM型,所用载体为PVITRO1-hygro-mcs-IgM/κ;载体由Tsingke公司合成,具体序列见SEQ ID NO.7及图谱见图11。同时,将抗体可变区连接至PVITRO1-hygro-mcsIgG/κ,构建其IgG型单克隆抗体;载体由Tsingke公司合成,具体序列见SEQ ID NO.8及图谱见图12。采用DNA Ligation Kit Ver 2.1(Takara,Dalian)连接。连接体系:SolutionⅠ10ul,PVITRO1-hygro-mcs-IgM/κ(图11)或PVITRO1-hygro-mcsIgG/κ(图12)和VH/VL按摩尔比3:1比例进行连接;即先用VH与PVITRO1-hygro-mcs-IgM/κ按摩尔比3:1比例连接后,再加入VL,VL与PVITRO1-hygro-mcs-IgM/κ的摩尔比也为3:1,这得到了第一个连接产物;同理,先用VH与PVITRO1-hygro-mcsIgG/κ按摩尔比3:1比例连接后,再加入VL,VL与PVITRO1-hygro-mcsIgG/κ的摩尔比也为3:1,这得到了第二个连接产物;
连接方法:16℃,30min。
连接产物加入大肠杆菌DH5α进行转化:冰浴1h,42℃90s,冰浴2min;加入100ul无抗LB[Tryptone(胰蛋白胨):10g,Yeast Extract(酵母提取物):5g,NaCl(氯化钠):10g,加水配制1L],摇床培养1h,180rpm;涂板。
8)阳性单克隆抗体鉴定:挑取单菌落扩大培养后,经Tsingke公司测序,测序结果经IgBLAST比对分析序列。
9)抗体表达:抗体质粒构建成功后,将其转染至FreeStyleTMCHO-S细胞,转染试剂为FreeStyleTMMAX Reagent(Thermo Scientific,USA)。转染方法见说明书。
10)抗体纯化:转染后细胞培养上清采用ProteinG亲和预装柱纯化。
表1 PCR引物
3.抗体特征鉴定
1)分子量及纯度鉴定:采用非还原和还原10%SDS-PAGE凝胶电泳鉴定。还原状态:样品和5×SDS-PAGE蛋白上样缓冲液[250Mm Tris-HCl(pH6.8),10%(W/V)SDS,0.5%(W/V)BPB,50%(V/V)甘油,5%(W/V)β-巯基乙醇]按体积比4:1比例混合,加入样品后缓冲液终浓度为1×,95℃加热10分钟,以充分变性蛋白。非还原状态:样品和5×SDS-PAGE蛋白上样缓冲液[250Mm Tris-HCl(pH6.8)0.5%(W/V)BPB,50%(V/V)甘油]按体积比4:1比例混合,加入样品后缓冲液终浓度为1×。冷却到室温后,把蛋白样品直接上样到SDS-PAGE胶加样孔内即可。80V 30分钟,150V 50分钟电泳。结果显示:未还原状态下:20-IgM分子量大小约为850KDa,20-IgG分子量大小为150KDa;还原状态下:20-IgG可见55KDa的重链和25KDa的轻链;20-IgM可见约80KDa大小的重链和25KDa的轻链。见图4。
2)结合特性鉴定:ELISA法。以EV71、CA16分别包被酶标板,以纯化后抗体为一抗,以HRP标记抗体为二抗(donkey anti-human IgG),TMB显色终止后,酶标仪检测450nm吸光度值(A450值)。结果显示:20-IgM和20-IgG都可以同时识别EV71、CA16、CA10和CA6;见图5。
3)亲和力鉴定:微量热泳动法(microscale thermophoresis,MST)。采用MonolithNT Protein Labeling Kit RED分别标记EV71、CA16、CA10和CA6,标记方法见说明书。采用TBST[20mM Tris(pH 7.5),50mM NaCl,5mMβ-mercaptoethanol,0.05%Tween-20and1%DMSO]倍比稀释抗体(稀释倍比为21~216,共16个浓度),分别取10ul稀释的抗体和10ul标记的病毒液混合,加入MST所用标准毛细管(Monolith TM standard-treated capillaries),通过亲和结合分析计算抗体的平衡解离常数Kd。结果显示:20-IgM与EV71、CA16、CA10和CA6均具有将强的结合能力,其亲和力常数(Kd)分别为:0.06nM、0.12nM、3.12nM、0.25nM。20-IgG与EV71、CA16、CA10和CA6的亲和力常数(Kd)分别为:0.6nM、1.2nM、3.12nM、2.5nM。20-IgM与相对应病毒的亲和力显著高于20-IgG(10倍)。见图6。
4)抗体中和表位鉴定:
a)抗体识别病毒表位鉴定:分别以EV71、CA16、CA10和CA6全病毒进行SDS-PAGE电泳后,转PVDF膜进行Western Blot分析。使用半干转膜装置(BIO-RAD),设定转膜电流为100mA,转膜时间为45分钟,按照转膜仪的说明书,将电泳后的蛋白转印至PVDF膜(购买自Millipore)上。转膜完毕后,立即把转膜完成的PVDF膜放置到预先准备好的5%(W/V)脱脂奶粉的TBS[20mMTris-HCl,500mMNaCl(pH7.5)]中,在摇床上缓慢摇动,室温封闭60分钟。纯化的单克隆抗体作为一抗,用TBST洗膜液[20mMTris-HCl,500mMNaCl(pH7.5)0.01%Tween-20(V/V)]将其稀释成1∶1000,加入至封闭完成后的PVDF膜中孵育过夜。弃一抗稀释液。加入TBST洗膜液5ml。在侧摆摇床上摇动,重复5次。随后以HRP标记的山羊抗人IgG为二抗(用TBST稀释1:10000)进行室温孵育1h。弃二抗稀释液,TBST洗膜重复5次,加入ECL(购买自Millipore)试剂,在暗室压片,经显影液定影液进行洗片或者进行机器曝光,检测单克隆抗体的识别效果。结果显示:20-IgM特异性识别EV71、CA16、CA10和CA6的VP1衣壳蛋白(图7A-D)。
抗体识别氨基酸位点鉴定:鉴定结果显示抗体特异性识别EV71、CA16、CA10、CA6的VP1蛋白,为了进一步确定抗体识别的关键氨基酸序列,合成VP1的多肽序列:每个肽段由15个氨基酸组成,如表2,由吉尔生化(上海)有限公司合成。采用ELISA法鉴定:以不同的肽段包被酶标板进行鉴定,结果显示:20-IgM特异性识别VP1蛋白的肽段(Phe Gly Glu His LysGln Glu Lys Asp.Leu Glu Tyr Gly Ala Cys,FGEHKQEKDLEYGAC)。见图7E-7G。并采用抗原肽抑制实验近一步确认20-IgM与FGEHKQEKDLEYGAC肽段结合的特异性:将不同浓度梯度的抗原肽段(15-For,15,15-Back)与20-IgM于37度孵育1小时后,加入微量中和实验,结果显示:15号肽段特异性的影响20-IgM的中和效力,见图7H-7I。以上结果证实:20-IgM特异性的识别VP1衣壳蛋白FGEHKQEKDLEYGAC肽段。20-IgG显示同样结果。
表2抗原肽氨基酸序列
1 | Gly Asp Arg Val Ala Asp Val Ile Glu Ser Ser Ile Gly Asp Ser |
2 | Val Ser Arg Ala Leu Thr His Ala Leu Pro Ala Pro Thr Gly Gln |
3 | Asn Thr Gln Val Ser Ser His Arg Leu Asp Thr Gly Lys Val Pro |
4 | Ala Leu Gln Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala Ser Asp |
5 | Glu Ser Met Ile Glu Thr Arg Cys Val Leu Asn Ser His Ser Thr |
6 | Ala Glu Thr Thr Leu Asp Ser Phe Phe Ser Arg Ala Gly Leu Val |
7 | Gly Glu Ile Asp Leu Pro Leu Glu Gly Thr Thr Asn Pro Asn Gly |
8 | Tyr Ala Asn Trp Asp Ile Asp Ile Thr Gly Tyr Ala Gln Met Arg |
9 | Arg Lys Val Glu Leu Phe Thr Tyr Met Arg Phe Asp Ala Glu |
10 | Thr Phe Val Ala Cys Thr Pro Thr Gly Glu Val Val Pro Gln Leu |
11 | Leu Gln Tyr Met Phe Val Pro Pro Gly Ala Pro Lys Pro Asp Ser |
12 | Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr Asn Pro Ser Val Phe |
13 | Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser Val Pro Phe Met |
14 | Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr Pro Thr |
15 | Phe Gly Glu His Lys Gln Glu Lys Asp.Leu Glu Tyr Gly Ala Cys |
16 | .Ser Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly Thr |
17 | Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr Met Arg Met |
18 | Lys His Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn Gln Asn |
19 | Tyr Leu Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile Lys |
20 | Pro Thr Gly Ala Ser Arg Thr Ala Ile Thr Thr Leu |
15For | Asp Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp.Leu |
15Back | Gln Glu Lys Asp.Leu Glu Tyr Gly Ala Cys Ser Asn Asn Met Met |
b)抗体识别关键氨基酸位点鉴定:鉴定结果显示抗体特异性地识别15号肽段,因此将15号肽段进行氨基酸位点突变,由吉尔生化(上海)有限公司合成(表3)。采用ELISA法鉴定,以点突变肽段包被酶标板。结果显示:20-IgM特异性识别氨基酸位点为:DLEYG(219-223)。结果见图7J。近一步本研究通过比对EV71、CA16、CA10和CA6的VP1衣壳蛋白的氨基酸序列同源性,结果显示:20-IgM结合位点在EV71衣壳蛋白VP1为DLEYG(219-223)(图7K);CA16为DLDYG(219-223)(图7L);CA10为NTTYG(218-222)(图7N);CA6为NLQYG(214-218)(图7M)。20-IgG显示同样结果。
表3抗原肽氨基酸序列
5)中和效力鉴定:微量中和实验。于96孔板中,每孔加入50ul 2000TCID50/ml病毒液(EV71/CA16/CA10/CA6,即4个实验,每组分别为EV71、CA16、CA10、CA6)与50ul梯度稀释(稀释倍比为21~28,共8个浓度)的抗体37℃孵育1h后,加入RD细胞(15000个),37℃,5%CO2培养3天,采用MTT法测量细胞活力。同时,每天观察细胞病变效应。结果显示:1nM 20-IgM抗体可以抑制EV71/CA16/CA10/CA6感染细胞发生病变。相对应,1nM 20-IgG不能完全抑制细胞病变。见图8。
6)MTT法:RD细胞培养3天后,每孔加入20ul噻唑蓝(MTT)溶液(5mg/ml),继续孵育4h,终止培养,小心吸弃孔内培养上清。每孔加入150ul DMSO,振荡10分钟后于490nm波长下在酶联免疫监测仪上测定各孔吸光度值。并于Graphpad绘制生长曲线和计算抗体的半数有效抑制浓度(half maximal inhibitory concentration,IC50)。结果显示:EV71感染组,20-IgM的IC50为0.11nM,IC80为0.35nM;20-IgG的IC50为1.31nM,IC80为4.3nM。CA16感染组,20-IgM IC50为0.45nM,IC80为1.03nM;20-IgG的IC50为6.47nM,IC80为14.14nM。CA10感染组,20-IgM IC50为1.47nM,IC80为3.33nM;20-IgG的IC50为10.85nM,IC80为22.64nM;CA6感染组,20-IgM IC50为0.55nM,IC80为1.39nM;20-IgG的IC50为4.97nM,IC80为11.37nM。见图9。
7)抗体对临床分离病毒株中和效力鉴定:微量中和实验鉴定抗体的结合效力;ELISA鉴定抗体的结合效力。结果显示:20-IgM对分离的5株EV71和5株CA16临床病毒株均具有较高的中和效力和结合效力。相对应,20-IgG同样对分离的5株EV71和5株CA16临床病毒株均具有较高的中和效力和结合效力,但其结合效力和中和效力均低于20-IgM。见表4。
表4抗体对临床分离病毒株的中和效力8)动物保护效力鉴定:
i.以1日龄乳鼠为感染模型来评价抗体在动物体内的保护效应。1日龄乳鼠感染模型:颅腔注射30ul 107TCID50/ml病毒(EV71或CA16)感染1日龄乳鼠(每组15只),每天记录小鼠的生存率以及小鼠的体重变化情况和临床症状。结果显示:感染组小鼠出现体重减轻、四肢麻痹等临床症状直致死亡,感染后10天乳鼠基本全部死亡。见图10A箭头标出。
ii.抗体保护效果评价:乳鼠感染病毒1天后,腹腔注射抗体(5mg/kg),每天记录小鼠的生存率以及小鼠的体重变化情况和临床症状。结果显示:20-IgM治疗组小鼠临床症状减轻,体重增长恢复趋近于对照组(PBS)(图10C和图10E);生存率统计结果显示:5mg/kg20-IgM对EV71/CA16感染小鼠的保护率达到100%(图10B和图10D)。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 中国医学科学院医学生物学研究所
<120> 广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体及应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 123
<212> PRT
<213> 人工序列()
<400> 1
Ile Pro Gly Ser Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
1 5 10 15
Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr
20 25 30
Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu
35 40 45
Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln
50 55 60
Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
65 70 75 80
Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Gly Gly Asn Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser
115 120
<210> 2
<211> 98
<212> PRT
<213> 人工序列()
<400> 2
Glu Phe Asp His Cys Ser Gln Ser Ser Gln Ser Leu Leu Asn Ser Arg
1 5 10 15
Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
20 25 30
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro
35 40 45
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
50 55 60
Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln Ser
65 70 75 80
Tyr Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
85 90 95
Gln Leu
<210> 3
<211> 359
<212> DNA
<213> 人工序列()
<400> 3
caggttccaa gctgcagcag tctggacctg agctggtgaa gcctggggct tcagtgaaga 60
tatcctgcaa gacttctgga tacacattca ctgaatacac catgcactgg gtgaagcaga 120
gccatggaaa gagccttgag tggattggag gtattaatcc taacaatggt ggtactagct 180
acaaccagaa gttcaagggc aaggccacat tgactgtaga caagtcctcc agcacagcct 240
acatggagct ccgcagcctg acatctgagg attctgcagt ctattactgt gcaagaggtg 300
gtaactacta ctatgctatg gactactggg gtcaaggaac ctcagtcacc gtctcctcg 359
<210> 4
<211> 282
<212> DNA
<213> 人工序列()
<400> 4
gatcattgtt ctcaatccag tcagagtctg ctcaacagta gaacccgaaa gaactacttg 60
gcttggtacc agcagaaacc agggcagtct cctaaactgc tgatctactg ggcatccact 120
agggaatctg gggtccctga tcgcttcaca ggcagtggat ctgggacaga tttcactctc 180
accatcagca gtgtgcaggc tgaagacctg gcagtttatt actgcaagca atcttataat 240
cttcggacgt tcggtggagg caccaagctg gaaataaaac gt 282
<210> 5
<211> 1362
<212> DNA
<213> 人工序列()
<400> 5
ggatctgcat ccgctccaac cctgttcccc cttgtgagct gcgaaaattc tccatccgac 60
acaagcagcg tcgcggtcgg ctgcctggct caagactttc tccccgacag catcactttc 120
agttggaagt acaagaacaa cagtgacatt tctagtacaa gaggatttcc gagtgttctg 180
agaggcggga agtacgctgc tacatcccag gtgctgctgc cgtcaaaaga cgttatgcag 240
ggtaccgatg agcacgtagt gtgtaaggtg caacacccaa acggcaacaa agagaagaat 300
gtccccctgc cagtgattgc tgaactgccc cctaaagtct ccgtctttgt accccccaga 360
gacggattct tcggcaaccc acgaaagtcc aagctcatct gccaggccac gggattctct 420
cctcgccaga tccaggtgtc ttggttgagg gaggggaaac aggttggctc tggcgttacc 480
accgatcagg ttcaggccga ggccaaagag tccgggccaa ctacctataa ggttacaagt 540
accctgacta taaaagaaag tgactggctg tctcaatcca tgtttacatg cagagtcgat 600
catcgaggcc tgacgttcca acagaatgcc agttccatgt gcgtgccaga tcaagatacc 660
gctataagag tgttcgcaat ccctccttca ttcgcttcca tattccttac taagagcacc 720
aagctgacat gtcttgtgac tgatttgacc acttacgaca gcgttacaat cagctggacc 780
cgccagaatg gcgaagctgt gaaaacccac acaaacatct ccgagtctca ccccaacgct 840
acatttagcg ccgtcggcga ggcatccatc tgcgaggacg attggaacag tggggagcgg 900
ttcacgtgca ctgttacgca tacagacctg ccgtctcctc tcaaacaaac tatcagccga 960
cctaaaggag tggcactgca ccggccagac gtttacctgc tgccccctgc ccgggaacag 1020
ctgaacctgc gggaatccgc cacaattaca tgcctggtga ctggatttag cccagccgac 1080
gtgttcgtgc agtggatgca gagggggcag cccctctccc cggaaaaata cgtaacatcc 1140
gctcccatgc ctgagcctca ggcccctgga cggtatttcg cccatagtat tctgactgtg 1200
tctgaagagg aatggaatac aggcgaaaca tatacttgcg tggttgctca tgaggccctg 1260
cctaaccgag tcacagagag aacagtggat aaatctacag gcaagccgac cctctataat 1320
gtgtctctcg tgatgtcaga tacggctggc acttgctact ga 1362
<210> 6
<211> 323
<212> DNA
<213> 人工序列()
<400> 6
gtacggtggc ggcgccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60
gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 120
ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 180
gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga 240
aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 300
gcttcaacag gggagagtgt tag 323
<210> 7
<211> 8285
<212> DNA
<213> 人工序列()
<400> 7
cctgcagggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga 60
aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 120
ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 180
ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 240
ccactagtgg agccgagagt aattcataca aaaggaggga tcgccttcgc aaggggagag 300
cccagggacc gtccctaaat tctcacagac ccaaatccct gtagccgccc cacgacagcg 360
cgaggagcat gcgctcaggg ctgagcgcgg ggagagcaga gcacacaagc tcatagaccc 420
tggtcgtggg ggggaggacc ggggagctgg cgcggggcaa actgggaaag cggtgtcgtg 480
tgctggctcc gccctcttcc cgagggtggg ggagaacggt atataagtgc ggcagtcgcc 540
ttggacgttc tttttcgcaa cgggtttgcc gtcagaacgc aggtgagggg cgggtgtggc 600
ttccgcgggc cgccgagctg gaggtcctgc tccgagcggg ccgggccccg ctgtcgtcgg 660
cggggattag ctgcgagcat tcccgcttcg agttgcgggc ggcgcgggag gcagagtgcg 720
aggcctagcg gcaaccccgt agcctcgcct cgtgtccggc ttgaggccta gcgtggtgtc 780
cgcgccgccg ccgcgtgcta ctccggccgc actctggtct tttttttttt tgttgttgtt 840
gccctgctgc cttcgattgc cgttcagcaa taggggctaa caaagggagg gtgcggggct 900
tgctcgcccg gagcccggag aggtcatggt tggggaggaa tggagggaca ggagtggcgg 960
ctggggcccg cccgccttcg gagcacatgt ccgacgccac ctggatgggg cgaggcctgg 1020
ggtttttccc gaagcaacca ggctggggtt agcgtgccga ggccatgtgg ccccagcacc 1080
cggcacgatc tggcttggcg gcgccgcgtt gccctgcctc cctaactagg gtgaggccat 1140
cccgtccggc accagttgcg tgcgtggaaa gatggccgct cccgggccct gttgcaagga 1200
gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg 1260
aagagggcct ggtccctcac cggctgctgc ttcctgtgac cccgtggtcc tatcggccgc 1320
aatagtcacc tcgggctttt gagcacggct agtcgcggcg gggggagggg atgtaatggc 1380
gttggagttt gttcacattt ggtgggtgga gactagtcag gccagcctgg cgctggaagt 1440
catttttgga atttgtcccc ttgagttttg agcggagcta attctcgggc ttcttagcgg 1500
ttcaaaggta tcttttaaac ccttttttag gtgttgtgaa aaccaccgct aattcaaagc 1560
aaccggtatg gactggacct ggaggatcct cttcttggtg gcagcagcca caggagccca 1620
ctccctcgag tgcgcacgct ccaaccctgt tcccccttgt gagctgcgaa aattctccat 1680
ccgacacaag cagcgtcgcg gtcggctgcc tggctcaaga ctttctcccc gacagcatca 1740
ctttcagttg gaagtacaag aacaacagtg acatttctag tacaagagga tttccgagtg 1800
ttctgagagg cgggaagtac gctgctacat cccaggtgct gctgccgtca aaagacgtta 1860
tgcagggtac cgatgagcac gtagtgtgta aggtgcaaca cccaaacggc aacaaagaga 1920
agaatgtccc cctgccagtg attgctgaac tgccccctaa agtctccgtc tttgtacccc 1980
ccagagacgg attcttcggc aacccacgaa agtccaagct catctgccag gccacgggat 2040
tctctcctcg ccagatccag gtgtcttggt tgagggaggg gaaacaggtt ggctctggcg 2100
ttaccaccga tcaggttcag gccgaggcca aagagtccgg gccaactacc tataaggtta 2160
caagtaccct gactataaaa gaaagtgact ggctgtctca atccatgttt acatgcagag 2220
tcgatcatcg aggcctgacg ttccaacaga atgccagttc catgtgcgtg ccagatcaag 2280
ataccgctat aagagtgttc gcaatccctc cttcattcgc ttccatattc cttactaaga 2340
gcaccaagct gacatgtctt gtgactgatt tgaccactta cgacagcgtt acaatcagct 2400
ggacccgcca gaatggcgaa gctgtgaaaa cccacacaaa catctccgag tctcacccca 2460
acgctacatt tagcgccgtc ggcgaggcat ccatctgcga ggacgattgg aacagtgggg 2520
agcggttcac gtgcactgtt acgcatacag acctgccgtc tcctctcaaa caaactatca 2580
gccgacctaa aggagtggca ctgcaccggc cagacgttta cctgctgccc cctgcccggg 2640
aacagctgaa cctgcgggaa tccgccacaa ttacatgcct ggtgactgga tttagcccag 2700
ccgacgtgtt cgtgcagtgg atgcagaggg ggcagcccct ctccccggaa aaatacgtaa 2760
catccgctcc catgcctgag cctcaggccc ctggacggta tttcgcccat agtattctga 2820
ctgtgtctga agaggaatgg aatacaggcg aaacatatac ttgcgtggtt gctcatgagg 2880
ccctgcctaa ccgagtcaca gagagaacag tggataaatc tacaggcaag ccgaccctct 2940
ataatgtgtc tctcgtgatg tcagatacgg ctggcacttg ctactgatgt acagctagct 3000
ggccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 3060
aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 3120
gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 3180
tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggaa atgttaatta 3240
actagccatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 3300
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3360
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3420
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 3480
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3540
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3600
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3660
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3720
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3780
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3840
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3900
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3960
tgctcacatg ttcttaatta acctgcaggc gttacataac ttacggtaaa tggcccgcct 4020
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 4080
acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 4140
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 4200
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 4260
tacatctacg tattagtcat cgctattacc atgatgatgc ggttttggca gtacatcaat 4320
gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 4380
gggagtttgt tttgactagt ggagccgaga gtaattcata caaaaggagg gatcgccttc 4440
gcaaggggag agcccaggga ccgtccctaa attctcacag acccaaatcc ctgtagccgc 4500
cccacgacag cgcgaggagc atgcgcccag ggctgagcgc gggtagatca gagcacacaa 4560
gctcacagtc cccggcggtg gggggagggg cgcgctgagc gggggccagg gagctggcgc 4620
ggggcaaact gggaaagtgg tgtcgtgtgc tggctccgcc ctcttcccga gggtggggga 4680
gaacggtata taagtgcggt agtcgccttg gacgttcttt ttcgcaacgg gtttgccgtc 4740
agaacgcagg tgagtggcgg gtgtggcttc cgcgggcccc ggagctggag ccctgctctg 4800
agcgggccgg gctgatatgc gagtgtcgtc cgcagggttt agctgtgagc attcccactt 4860
cgagtggcgg gcggtgcggg ggtgagagtg cgaggcctag cggcaacccc gtagcctcgc 4920
ctcgtgtccg gcttgaggcc tagcgtggtg tccgccgccg cgtgccactc cggccgcact 4980
atgcgttttt tgtccttgct gccctcgatt gccttccagc agcatgggct aacaaaggga 5040
gggtgtgggg ctcactctta aggagcccat gaagcttacg ttggatagga atggaagggc 5100
aggaggggcg actggggccc gcccgccttc ggagcacatg tccgacgcca cctggatggg 5160
gcgaggcctg tggctttccg aagcaatcgg gcgtgagttt agcctacctg ggccatgtgg 5220
ccctagcact gggcacggtc tggcctggcg gtgccgcgtt cccttgcctc ccaacaaggg 5280
tgaggccgtc ccgcccggca ccagttgctt gcgcggaaag atggccgctc ccggggccct 5340
gttgcaagga gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc 5400
cacacaaagg aagagggcct tgcccctcgc cggccgctgc ttcctgtgac cccgtggtct 5460
atcggccgca tagtcacctc gggcttctct tgagcaccgc tcgtcgcggc ggggggaggg 5520
gatctaatgg cgttggagtt tgttcacatt tggtgggtgg agactagtca ggccagcctg 5580
gcgctggaag tcattcttgg aatttgcccc tttgagtttg gagcgaggct aattctcaag 5640
cctcttagcg gttcaaaggt attttctaaa cccgtttcca ggtgttgtga aagccaccgc 5700
taattcaaag caatccggaa tgttgccatc acaactcatt gggtttctgc tgctctgggt 5760
tccagctagc cgcggtgaat tcgtttaaac gtacggtggc ggcgccatct gtcttcatct 5820
tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata 5880
acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc caatcgggta 5940
actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc ctcagcagca 6000
ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc gaagtcaccc 6060
atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt tagggatccc 6120
gtacgcctag gagcaggttt ccccaatgac acaaaacgtg caacttgaaa ctccgcctgg 6180
tctttccagg tctagagggg taacactttg tactgcgttt ggctccacgc tcgatccact 6240
ggcgagtgtt agtaacagca ctgttgcttc gtagcggagc atgacggccg tgggaactcc 6300
tccttggtaa caaggaccca cggggccaaa agccacgccc acacgggccc gtcatgtgtg 6360
caaccccagc acggcgactt tactgcgaaa cccactttaa agtgacattg aaactggtac 6420
ccacacactg gtgacaggct aaggatgccc ttcaggtacc ccgaggtaac acgcgacact 6480
cgggatctga gaaggggact ggggcttcta taaaagcgct cggtttaaaa agcttctatg 6540
cctgaatagg tgaccggagg tcggcacctt tcctttgcaa ttactgaccc tatgaataca 6600
ctgactgttt gacaattaat catcggcata gtatatcggc atagtataat acgactcact 6660
ataggagggc caccatgaag aaacctgaac tgacagcaac ttctgttgag aagtttctca 6720
ttgaaaaatt tgattctgtt tctgatctca tgcagctgtc tgaaggtgaa gaaagcagag 6780
ccttttcttt tgatgttgga ggaagaggtt atgttctgag ggtcaattct tgtgctgatg 6840
gtttttacaa agacagatat gtttacagac actttgcctc tgctgctctg ccaattccag 6900
aagttctgga cattggagaa ttttctgaat ctctcaccta ctgcatcagc agaagagcac 6960
aaggagtcac tctccaggat ctccctgaaa ctgagctgcc agctgttctg caacctgttg 7020
ctgaagcaat ggatgccatt gcagcagctg atctgagcca aacctctgga tttggtcctt 7080
ttggtcccca aggcattggt cagtacacca cttggaggga tttcatttgt gccattgctg 7140
atcctcatgt ctatcactgg cagactgtga tggatgacac agtttctgct tctgttgctc 7200
aggcactgga tgaactcatg ctgtgggcag aagattgtcc tgaagtcaga cacctggtcc 7260
atgctgattt tggaagcaac aatgttctga cagacaatgg cagaatcact gcagtcattg 7320
actggtctga agccatgttt ggagattctc aatatgaggt tgccaacatt tttttttgga 7380
gaccttggct ggcttgcatg gaacaacaaa caagatattt tgaaagaaga cacccagaac 7440
tggctggttc ccccagactg agagcctaca tgctcagaat tggcctggac caactgtatc 7500
aatctctggt tgatggaaac tttgatgatg ctgcttgggc acaaggaaga tgtgatgcca 7560
ttgtgaggtc tggtgctgga actgttggaa gaactcaaat tgcaagaagg tctgctgctg 7620
tttggactga tggatgtgtt gaagttctgg ctgactctgg aaacaggaga ccctccacaa 7680
gacccagagc caaggaatga atattagcta gattatccct aatacctgcc accccactct 7740
taatcagtgg tggaagaacg gtctcagaac tgtttgtttc aattggccat ttaagtttag 7800
tagtaaaaga ctggttaatg ataacaatgc atcgtaaaac cttcagaagg aaaggagaat 7860
gttttgtgga ccactttggt tttctttttt gcgtgtggca gttttaagtt attagttttt 7920
aaaatcagta ctttttaatg gaaacaactt gaccaaaaat ttgtcacaga attttgagac 7980
ccattaaaaa agttaaatga gaaacctgtg tgttcctttg gtcaacaccg agacatttag 8040
gtgaaagaca tctaattctg gttttacgaa tctggaaact tcttgaaaat gtaattcttg 8100
agttaacact tctgggtgga gaatagggtt gttttccccc cacataattg gaaggggaag 8160
gaatatcatt taaagctatg ggagggttgc tttgattaca acactggaga gaaatgcagc 8220
atgttgctga ttgcctgtca ctaaaacagg ccaaaaactg agtccttggg ttgcatagaa 8280
agctg 8285
<210> 8
<211> 7930
<212> DNA
<213> 人工序列()
<400> 8
cctgcagggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga 60
aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 120
ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 180
ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 240
ccactagtgg agccgagagt aattcataca aaaggaggga tcgccttcgc aaggggagag 300
cccagggacc gtccctaaat tctcacagac ccaaatccct gtagccgccc cacgacagcg 360
cgaggagcat gcgctcaggg ctgagcgcgg ggagagcaga gcacacaagc tcatagaccc 420
tggtcgtggg ggggaggacc ggggagctgg cgcggggcaa actgggaaag cggtgtcgtg 480
tgctggctcc gccctcttcc cgagggtggg ggagaacggt atataagtgc ggcagtcgcc 540
ttggacgttc tttttcgcaa cgggtttgcc gtcagaacgc aggtgagggg cgggtgtggc 600
ttccgcgggc cgccgagctg gaggtcctgc tccgagcggg ccgggccccg ctgtcgtcgg 660
cggggattag ctgcgagcat tcccgcttcg agttgcgggc ggcgcgggag gcagagtgcg 720
aggcctagcg gcaaccccgt agcctcgcct cgtgtccggc ttgaggccta gcgtggtgtc 780
cgcgccgccg ccgcgtgcta ctccggccgc actctggtct tttttttttt tgttgttgtt 840
gccctgctgc cttcgattgc cgttcagcaa taggggctaa caaagggagg gtgcggggct 900
tgctcgcccg gagcccggag aggtcatggt tggggaggaa tggagggaca ggagtggcgg 960
ctggggcccg cccgccttcg gagcacatgt ccgacgccac ctggatgggg cgaggcctgg 1020
ggtttttccc gaagcaacca ggctggggtt agcgtgccga ggccatgtgg ccccagcacc 1080
cggcacgatc tggcttggcg gcgccgcgtt gccctgcctc cctaactagg gtgaggccat 1140
cccgtccggc accagttgcg tgcgtggaaa gatggccgct cccgggccct gttgcaagga 1200
gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg 1260
aagagggcct ggtccctcac cggctgctgc ttcctgtgac cccgtggtcc tatcggccgc 1320
aatagtcacc tcgggctttt gagcacggct agtcgcggcg gggggagggg atgtaatggc 1380
gttggagttt gttcacattt ggtgggtgga gactagtcag gccagcctgg cgctggaagt 1440
catttttgga atttgtcccc ttgagttttg agcggagcta attctcgggc ttcttagcgg 1500
ttcaaaggta tcttttaaac ccttttttag gtgttgtgaa aaccaccgct aattcaaagc 1560
aaccggtatg gactggacct ggaggatcct cttcttggtg gcagcagcca caggagccca 1620
ctccctcgag tgcgcacccg ctagcaccaa gggcccatcg gtcttccccc tggcaccctc 1680
ctccaagagc acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc 1740
cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc 1800
ggctgtccta cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag 1860
cagcttgggc acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt 1920
ggacaagaaa gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc 1980
acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct 2040
catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc 2100
tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc 2160
gcgggaggag cagtacaaca gcacgtaccg ggtggtcagc gtcctcaccg tcctgcacca 2220
ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc 2280
catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct 2340
gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg 2400
cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta 2460
caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac 2520
cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc 2580
tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat gatgtacagc 2640
tagctggcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 2700
gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 2760
aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 2820
ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggaaatgtt 2880
aattaactag ccatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 2940
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 3000
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 3060
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 3120
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 3180
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 3240
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 3300
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 3360
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 3420
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 3480
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 3540
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 3600
ccttttgctc acatgttctt aattaacctg caggcgttac ataacttacg gtaaatggcc 3660
cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 3720
tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 3780
cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 3840
acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 3900
ggcagtacat ctacgtatta gtcatcgcta ttaccatgat gatgcggttt tggcagtaca 3960
tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 4020
tcaatgggag tttgttttga ctagtggagc cgagagtaat tcatacaaaa ggagggatcg 4080
ccttcgcaag gggagagccc agggaccgtc cctaaattct cacagaccca aatccctgta 4140
gccgccccac gacagcgcga ggagcatgcg cccagggctg agcgcgggta gatcagagca 4200
cacaagctca cagtccccgg cggtgggggg aggggcgcgc tgagcggggg ccagggagct 4260
ggcgcggggc aaactgggaa agtggtgtcg tgtgctggct ccgccctctt cccgagggtg 4320
ggggagaacg gtatataagt gcggtagtcg ccttggacgt tctttttcgc aacgggtttg 4380
ccgtcagaac gcaggtgagt ggcgggtgtg gcttccgcgg gccccggagc tggagccctg 4440
ctctgagcgg gccgggctga tatgcgagtg tcgtccgcag ggtttagctg tgagcattcc 4500
cacttcgagt ggcgggcggt gcgggggtga gagtgcgagg cctagcggca accccgtagc 4560
ctcgcctcgt gtccggcttg aggcctagcg tggtgtccgc cgccgcgtgc cactccggcc 4620
gcactatgcg ttttttgtcc ttgctgccct cgattgcctt ccagcagcat gggctaacaa 4680
agggagggtg tggggctcac tcttaaggag cccatgaagc ttacgttgga taggaatgga 4740
agggcaggag gggcgactgg ggcccgcccg ccttcggagc acatgtccga cgccacctgg 4800
atggggcgag gcctgtggct ttccgaagca atcgggcgtg agtttagcct acctgggcca 4860
tgtggcccta gcactgggca cggtctggcc tggcggtgcc gcgttccctt gcctcccaac 4920
aagggtgagg ccgtcccgcc cggcaccagt tgcttgcgcg gaaagatggc cgctcccggg 4980
gccctgttgc aaggagctca aaatggagga cgcggcagcc cggtggagcg ggcgggtgag 5040
tcacccacac aaaggaagag ggccttgccc ctcgccggcc gctgcttcct gtgaccccgt 5100
ggtctatcgg ccgcatagtc acctcgggct tctcttgagc accgctcgtc gcggcggggg 5160
gaggggatct aatggcgttg gagtttgttc acatttggtg ggtggagact agtcaggcca 5220
gcctggcgct ggaagtcatt cttggaattt gcccctttga gtttggagcg aggctaattc 5280
tcaagcctct tagcggttca aaggtatttt ctaaacccgt ttccaggtgt tgtgaaagcc 5340
accgctaatt caaagcaatc cggaatgttg ccatcacaac tcattgggtt tctgctgctc 5400
tgggttccag ctagccgcgg tgaattcgtt taaacgtacg gtggcggcgc catctgtctt 5460
catcttcccg ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct 5520
gaataacttc tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc 5580
gggtaactcc caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag 5640
cagcaccctg acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt 5700
cacccatcag ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttaggg 5760
atcccgtacg cctaggagca ggtttcccca atgacacaaa acgtgcaact tgaaactccg 5820
cctggtcttt ccaggtctag aggggtaaca ctttgtactg cgtttggctc cacgctcgat 5880
ccactggcga gtgttagtaa cagcactgtt gcttcgtagc ggagcatgac ggccgtggga 5940
actcctcctt ggtaacaagg acccacgggg ccaaaagcca cgcccacacg ggcccgtcat 6000
gtgtgcaacc ccagcacggc gactttactg cgaaacccac tttaaagtga cattgaaact 6060
ggtacccaca cactggtgac aggctaagga tgcccttcag gtaccccgag gtaacacgcg 6120
acactcggga tctgagaagg ggactggggc ttctataaaa gcgctcggtt taaaaagctt 6180
ctatgcctga ataggtgacc ggaggtcggc acctttcctt tgcaattact gaccctatga 6240
atacactgac tgtttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 6300
tcactatagg agggccacca tgaagaaacc tgaactgaca gcaacttctg ttgagaagtt 6360
tctcattgaa aaatttgatt ctgtttctga tctcatgcag ctgtctgaag gtgaagaaag 6420
cagagccttt tcttttgatg ttggaggaag aggttatgtt ctgagggtca attcttgtgc 6480
tgatggtttt tacaaagaca gatatgttta cagacacttt gcctctgctg ctctgccaat 6540
tccagaagtt ctggacattg gagaattttc tgaatctctc acctactgca tcagcagaag 6600
agcacaagga gtcactctcc aggatctccc tgaaactgag ctgccagctg ttctgcaacc 6660
tgttgctgaa gcaatggatg ccattgcagc agctgatctg agccaaacct ctggatttgg 6720
tccttttggt ccccaaggca ttggtcagta caccacttgg agggatttca tttgtgccat 6780
tgctgatcct catgtctatc actggcagac tgtgatggat gacacagttt ctgcttctgt 6840
tgctcaggca ctggatgaac tcatgctgtg ggcagaagat tgtcctgaag tcagacacct 6900
ggtccatgct gattttggaa gcaacaatgt tctgacagac aatggcagaa tcactgcagt 6960
cattgactgg tctgaagcca tgtttggaga ttctcaatat gaggttgcca acattttttt 7020
ttggagacct tggctggctt gcatggaaca acaaacaaga tattttgaaa gaagacaccc 7080
agaactggct ggttccccca gactgagagc ctacatgctc agaattggcc tggaccaact 7140
gtatcaatct ctggttgatg gaaactttga tgatgctgct tgggcacaag gaagatgtga 7200
tgccattgtg aggtctggtg ctggaactgt tggaagaact caaattgcaa gaaggtctgc 7260
tgctgtttgg actgatggat gtgttgaagt tctggctgac tctggaaaca ggagaccctc 7320
cacaagaccc agagccaagg aatgaatatt agctagatta tccctaatac ctgccacccc 7380
actcttaatc agtggtggaa gaacggtctc agaactgttt gtttcaattg gccatttaag 7440
tttagtagta aaagactggt taatgataac aatgcatcgt aaaaccttca gaaggaaagg 7500
agaatgtttt gtggaccact ttggttttct tttttgcgtg tggcagtttt aagttattag 7560
tttttaaaat cagtactttt taatggaaac aacttgacca aaaatttgtc acagaatttt 7620
gagacccatt aaaaaagtta aatgagaaac ctgtgtgttc ctttggtcaa caccgagaca 7680
tttaggtgaa agacatctaa ttctggtttt acgaatctgg aaacttcttg aaaatgtaat 7740
tcttgagtta acacttctgg gtggagaata gggttgtttt ccccccacat aattggaagg 7800
ggaaggaata tcatttaaag ctatgggagg gttgctttga ttacaacact ggagagaaat 7860
gcagcatgtt gctgattgcc tgtcactaaa acaggccaaa aactgagtcc ttgggttgca 7920
tagaaagctg 7930
Claims (10)
1.抗EV71、CA16、CA10和CA6广谱中和抗体杂交瘤细胞株的制备方法,其特征在于,包括如下步骤:
步骤(1),免疫原的制备:以纯化的EV71和CA16病毒为免疫原;
步骤(2),动物免疫:取6~8周龄Balb/c小鼠,以2周间隔进行四次免疫,程序为:首次免疫,弗氏完全佐剂与等量免疫原充分混合乳化后,背部皮下多点注射;第二次免疫和第三次免疫,佐剂换为弗氏不完全佐剂,方法和计量同首次免疫;第四次采用腹腔注射未加佐剂的免疫原,注射量同首次免疫;3天后,取免疫小鼠脾脏细胞用于制备杂交瘤细胞;
步骤(3),杂交瘤细胞株的制备和筛选:将处于对数生长期的SP2/0细胞与步骤(2)免疫小鼠脾脏细胞通过PEG4000的作用下进行细胞融合,利用1×HAT选择性培养基培养融合后的杂交瘤细胞,细胞融合后10-14天,通过酶联免疫吸附试验检测细胞上清以筛选能特异性分泌抗EV71、CA16、CA10和CA6广谱中和抗体的杂交瘤细胞株。
2.根据权利要求1所述的抗EV71、CA16、CA10和CA6广谱中和抗体杂交瘤细胞株的制备方法,其特征在于,步骤(2)中,首次免疫注射量为50ul 107TCID50/ml EV71和 50ul107TCID50/ml CA16;
步骤(3)中,将处于对数生长期的SP2/0细胞与步骤(2)免疫小鼠脾脏细胞通过PEG4000的作用下进行细胞融合,利用1×HAT选择性培养基培养融合后的杂交瘤细胞,具体方法为:
选择6~8周龄Balb/c小鼠,取其腹腔巨噬细胞制备饲养层细胞;取处于对数生长期的SP2/0细胞与步骤(2)免疫小鼠脾脏细胞按照1∶10的细胞数量比例进行混和,采用聚乙二醇4000进行细胞融合,细胞融合后获得的杂交瘤细胞加入已含有饲养层细胞的培养板中,使用含有1×HAT选择性培养基,在37℃、5%CO2条件下培养;
还包括杂交瘤细胞的克隆化,具体是:通过有限稀释法对杂交瘤细胞株进行亚克隆培养,连续培养多次,直至克隆孔内抗体检测100%阳性为止,扩大培养并冻存。
3.广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体的制备方法,其特征在于,采用权利要求1~2任意一项所述的抗EV71、CA16、CA10和CA6广谱中和抗体杂交瘤细胞株的制备方法制得的抗EV71、CA16、CA10和CA6广谱中和抗体杂交瘤细胞株;包括如下步骤:
提取抗EV71、CA16、CA10和CA6广谱中和抗体杂交瘤细胞株的RNA,逆转录获取cDNA;然后巢式PCR扩增抗体重链可变区,同时巢式PCR扩增抗体轻链可变区;之后采用PVITRO1-hygro-mcs-IgM/κ连接载体对抗体20-IgM可变区进行连接,获取IgM型抗体;同时,采用PVITRO1-hygro-mcsIgG/κ连接载体对抗体20-IgM可变区进行连接,获取IgG型抗体;接着将连接得到的阳性单克隆抗体转染至CHO-S细胞,后纯化,即得广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体和IgG型单克隆抗体;所述的PVITRO1-hygro-mcs-IgM/κ连接载体的DNA序列如SEQ ID NO.7所示;所述的PVITRO1-hygro-mcsIgG/κ连接载体的DNA序列如SEQ ID NO.8所示。
4.采用权利要求3所述的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体的制备方法制备得到的抗EV71、CA16、CA10和CA6人-鼠嵌合广谱中和IgM型单克隆抗体以及其IgG型单克隆抗体。
5.一种广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体,其特征在于,包括重链可变区和轻链可变区,其所述重链可变区的氨基酸序列如序列表中SEQ ID NO.1所示,所述轻链可变区的氨基酸序列如序列表中SEQ ID NO.2所示。
6.一种如权利要求5所述的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体的编码DNA,其特征在于,包括重链可变区和轻链可变区;
所述重链可变区的编码DNA序列如SEQ ID NO.3所示;
所述轻链可变区的编码DNA序列如SEQ ID NO.4所示。
7.一种表达载体,其特征在于,包含重链恒定区DNA序列和轻链恒定区DNA序列,用于表达如权利要求5所述的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体;
所述重链恒定区的编码DNA序列如SEQ ID NO.5所示;
所述轻链恒定区的编码DNA序列如SEQ ID NO.6所示。
8.一种宿主细胞,其特征在于,包含如权利要求7所述的表达载体。
9.药物组合物,其特征在于,包括如权利要求4或5所述的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体和药学上可接受的载体。
10.权利要求4或5所述的广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体在制备治疗或预防EV71、CA16、CA10和CA6感染引起的手足口病的药物中的应用。
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