CN116589571A - 乙型脑炎病毒抗体及其应用 - Google Patents
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Abstract
本发明涉及抗体技术领域,具体涉及一种乙型脑炎病毒抗体1B2及其应用。提供一种乙型脑炎病毒抗体1B2,其重链可变区的互补决定区CDR1具有如SEQ ID NO.1所示的氨基酸序列,CDR2具有如SEQ ID NO.2所示的氨基酸序列,CDR3具有如SEQ ID NO.3所示的氨基酸序列。轻链可变区的互补决定区CDR1具有如SEQ ID NO.4所示的氨基酸序列,CDR2具有如SEQ ID NO.5所示的氨基酸序列,CDR3具有如SEQ ID NO.6所示的氨基酸序列。
Description
技术领域
本发明涉及抗体技术领域,具体涉及一种乙型脑炎病毒抗体及其应用。
背景技术
流行性乙型脑炎简称乙脑,是日本脑炎病毒(Japanese Encephalitis Virus,JEV)引起的最常见的病毒性脑炎。为了与本世纪初曾流行于欧洲的昏睡性脑炎(又称甲型脑炎)相区别,我国卫生部定名为流行性乙型脑炎。乙型脑炎病毒属于虫媒病毒黄病毒科黄病毒属,基因组为单股正链RNA,两端为非编码区,整个基因组为一个开放阅读框,编码一个多蛋白质前体。流行性乙型脑炎是乙型脑炎病毒感染引起的人畜共患病,人通过蚊虫叮咬或密切接触感染。
目前已应用的乙型脑炎病毒疫苗为弱毒疫苗和灭活疫苗。其中灭活疫苗是世界卫生组织推荐的在全球应用的疫苗,虽然该疫苗具有较高的安全性,但是在免疫保护力上却不够理想,经常出现免疫失败的报道。更为重要的是,目前尚没有针对乙型脑炎病毒的治疗技术,导致了该病的死亡率高,即使康复造成的后遗症也高。由于乙型脑炎尚无特效疗法,早期准确的诊断和有效的预防是防治本病的主要手段。
因此,本领域需求有高特异性、结合能力和中和能力的乙型脑炎病毒抗体,以满足诊断、预防和治疗的需要。
发明内容
有鉴于此,第一方面,本发明提供了本发明的目的之一是提供一种乙型脑炎病毒抗体。本发明的另一目的是提供该抗体的应用及其相关产品。
为实现上述目的,本发明通过杂交瘤技术筛选到一株乙型脑炎病毒特异性的杂交瘤(克隆号记为1B2),制备小鼠腹水并纯化抗体,得到能够特异性结合乙型脑炎病毒的鼠源性单克隆抗体(mAb)1B2,该抗体对乙型脑炎病毒具有较高的亲和力,同时具有中和乙型脑炎病毒的能力。
具体地,本发明提供以下技术方案:
本发明提供乙型脑炎病毒抗体或其抗原结合片段,其重链可变区的互补决定区CDR1具有如SEQ ID NO.1所示的氨基酸序列,CDR2具有如SEQ ID NO.2所示的氨基酸序列,CDR3具有如SEQ ID NO.3所示的氨基酸序列。轻链可变区的互补决定区CDR1具有如SEQ IDNO.4所示的氨基酸序列,CDR2具有如SEQ ID NO.5所示的氨基酸序列,CDR3具有如SEQ IDNO.6所示的氨基酸序列。
优选地,所述乙型脑炎病毒抗体或其抗原结合片段的重链可变区具有如SEQ IDNO.7所示的氨基酸序列。轻链可变区具有如SEQ ID NO.8所示的氨基酸序列。
本发明中,所述抗原结合片段可选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体、双特异抗体或多特异抗体。
本发明提供含有所述乙型脑炎病毒抗体或其抗原结合片段的双特异抗体或多特异抗体。
基于上述抗体,本发明提供一种核酸分子,其编码所述乙型脑炎病毒抗体或其抗原结合片段。
根据上述提供的乙型脑炎病毒抗体或其抗原结合片段的氨基酸序列,本领域技术人员可以确定编码所述乙型脑炎病毒抗体或其抗原结合片段的核酸分子的核苷酸序列,并根据宿主细胞的密码子偏好性选择不同的核酸分子的核苷酸序列。
本发明还提供一种生物材料,其含有编码所述乙型脑炎病毒抗体或其抗原结合片段的核酸分子,所述生物材料为表达盒、载体或宿主细胞。
以上所述的表达盒是指将所述核酸分子的上游或下游连接用于转录、翻译的调控元件得到的重组核酸分子。
以上所述的载体是指可将核酸分子插入其中的一种核酸运载工具,包括但不限于质粒、人工染色体、噬菌体、动物病毒等,可以为表达载体,也可以为克隆载体或非复制型载体。
以上所述的宿主细胞可以为微生物细胞(如:大肠杆菌、酵母细胞等)或动物细胞(如:昆虫细胞、CHO细胞、BHK细胞,HEK293细胞等用于表达、制备抗体的细胞),其中,动物细胞为不能够繁殖成为动物个体的细胞。
基于上述乙型脑炎病毒抗体或其抗原结合片段的功能,本发明提供所述乙型脑炎病毒抗体或其抗原结合片段、所述核酸分子或所述生物材料的如下任一种应用:
(1)在制备用于检测乙型脑炎病毒的试剂或试剂盒中的应用;
(2)在制备用于预防或治疗乙型脑炎病毒感染或乙型脑炎病毒感染引起疾病的药物中的应用;
(3)在制备用于中和乙型脑炎病毒毒力的产品中的应用。
以上(1)中所述的检测乙型脑炎病毒包括检测乙型脑炎病毒的存在或其水平。
以上(2)中所述的乙型脑炎病毒感染引起疾病包括流行性乙型脑炎及其相关疾病。
基于上述乙型脑炎病毒抗体或其抗原结合片段,本发明提供一种抗体偶联物,其为由所述乙型脑炎病毒抗体或其抗原结合片段与载体或药物偶联得到,或者由所述乙型脑炎病毒抗体或其抗原结合片段与经化学或生物标记得到。
以上所述的载体可为任意可与蛋白质偶联的载体或药物载体。
以上所述的化学标记包括同位素、胶体金、荧光素、生物素标记等。
以上所述的生物标记包括蛋白标记、酶标记等。
本发明提供一种检测试剂,其含有所述乙型脑炎病毒抗体或其抗原结合片段或含有所述抗体偶联物。
以上所述的试剂盒还可包含免疫学检测、流式细胞检测等所需的其它试剂。
本发明提供一种药物组合物,其含有所述乙型脑炎病毒抗体或其抗原结合片段或含有所述抗体偶联物。
以上所述的药物组合物还可包含药学上可接受的载体或赋形剂。所用载体或赋形剂的类型可根据药物组合物的剂型和给药方式进行选择。所述药物组合物还可包含具有其它功效的抗体或药物活性成分。
本发明的有益效果在于:本发明提供一种针对乙型脑炎病毒的单克隆抗体1B2,该抗体能够与乙型脑炎病毒特异性结合,具有高度特异性和高结合能力,可用于乙型脑炎病毒的免疫荧光染色和流式细胞染色等检测,即使在较高稀释度时,仍然具有较好的检测效果。同时,该抗体对乙型脑炎病毒具有高效的中和能力。该抗体为乙型脑炎病毒的检测和诊断提供了有效的工具。
附图说明
图1为本发明抗体1B2抑制乙脑病毒感染T98G细胞的结果图;
图2为阳性对照抑制乙脑病毒感染T98G细胞的结果图;
图3为阴性对照抑制乙脑病毒感染T98G细胞的结果图。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
实施例1、杂交瘤细胞和单克隆抗体的筛选和制备
(1)小鼠免疫
将免疫原为JEV(SA14)株选择雌性5周龄BALB/c小鼠4只,颈背部皮下两点。第15天时加强免疫1次;第30天时再加强免疫1次(同上)。在第45天时断尾采血,用间接ELISA的方法检测抗体水平,在第46天时对免疫效果最好的BALB/c小鼠使用不加佐剂的NS1蛋白100ug腹腔注射冲击免疫一次,三天后即可融合。
(2)SP2/0瘤细胞的制备
将冻存的SP2/0细胞复苏,培养至六孔板,然后收集细胞,注入BALB/c小鼠背部皮下。待10~14天后小鼠背部形成肿瘤时用颈椎脱臼法处死,75%酒精浸泡5min,消毒皮毛。将小鼠固定于解剖盘中,无菌状态下将肿瘤块剪下,置匀浆器中,加5mL1640基本培养基充分研磨后,补加1640基本培养基至10mL,静置5min,吸取上层的细胞悬液于另一离心管备用,再补加1640基本培养基至10mL,重复两次。1000rpm离心10min去上清,以20mL 1640基本培养基重悬细胞。在另一50ml离心管中加入15mL淋巴细胞分离液,将SP2/0细胞悬液轻轻地加在分离液之上,1000rpm离心10min,用吸管吸取位于界面致密的白色细胞层,用1640基本培养基洗2遍,计数后备用。
(3)免疫脾细胞的制备
取免疫的BALB/c鼠的脾脏,加3mL 1640基本培养基于匀浆器中研磨,制备脾细胞,再补加10mL 1640基本培养基于匀浆器中,同上重复2次。1000rpm离心10min去上清,细胞重悬后计数。
(4)饲养细胞的制备
取未免疫的BALB/c鼠,用吸管吸3mL 1640液注入小鼠腹腔,再将液体吸出放置于50mL离心管中,再重复一次,此为腹腔巨噬细胞。同上操作制备脾细胞悬液,并放入腹腔巨噬细胞管中。1000rpm离心10min去上清,细胞重悬后计数,用HAT培养基悬浮后,置于37℃,5%CO2培养箱中待用。
(5)细胞融合
将SP2/0骨髓瘤细胞与免疫脾细胞以体积比为1:10比例在50mL离心管中混匀,1500rpm离心10min。倒空上清,将装有细胞混合物的离心管放于37℃水浴中。然后在1min内慢慢滴入预温至37℃的融合用PEG0.8mL,边加边轻轻用吸管尖搅拌。继续搅拌30秒后静置1min。然后慢慢加入37℃预温的1640基本培养基10mL,并不断轻轻地搅拌。最后缓慢加入30mL 1640基本培养基。1000rpm离心5min,去上清后37℃放置5-8min。用HAT培养基悬浮,同时也用HAT培养基悬浮饲养脾细胞与融合后的细胞混合,根据需要补加适量的HAT培养基,均匀滴加入96孔细胞培养板中,约250μL/孔。置于37℃,5%CO2培养箱中培养。融合后于第5天补加1滴HAT培养基,第8-10天时换HT培养基100uL。待融合细胞集落长至培养孔1/4时,进行抗体检测。
(6)分泌单抗的杂交瘤细胞的筛选
采用间接ELISA方法检测。用NS1蛋白包被96孔酶联板,并用0.5%的BSA37℃封闭1h,用洗涤液洗三次,然后加入杂交瘤细胞的培养上清100uL,同时设阴、阳性血清对照,于37℃温育30min,取出后洗涤三次,加入1:5000倍稀释的HRP标记的羊抗鼠IgG,37℃反应30min,取出后洗涤三次,加入底物显色,用0.25%HF终止反应之后,在630nm的波长下检测OD值。同时设SP2/0细胞的培养上清作为阴性对照,OD630大于阴性对照3倍的样品为阳性。
克隆化后,经ELISA初筛,筛选到3株对乙脑具有结合能力的单克隆抗体,编号分别为a-c,其中,b和c对乙脑具有较好的结合能力,b的杂交瘤克隆号记为1B2。
将1B2杂交瘤注射入经降植烷致敏的小鼠腹腔内,约10天后收获小鼠腹水,采用Protein G进行纯化,获得纯化单克隆抗体,浓度为2mg/ml。
培养1B2杂交瘤细胞,提取细胞总RNA,RT-PCR扩增抗体重链(1.4kb)及轻链基因(0.8kb),并将正确DNA条带经内切酶消化后与载体pcomb3连接,进而转化x-blue感受态细胞,最后选择阳性克隆进行测序,得到抗体重链及轻链全长序列。
经测序,单克隆抗体1B2的重链可变区的互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO.1-3所示,轻链可变区的互补决定区CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO.4-6所示。重链可变区的氨基酸序列如SEQ ID NO.7所示,轻链可变区的氨基酸序列如SEQ ID NO.8所示。
实施例2、本发明抗体抑制乙型脑炎病毒感染细胞
将本发明筛选的抗体1B2、1B3,以及阴性对照和阳性对照分为三组与JEV(SA14)株进行孵育,孵育2h小时后,将孵育物放入事先准备好的T98G细胞中进行感染,感染24h后,放入荧光显微镜下进行观察,结果如图1~3所示。从图中可以看出,加入本发明抗体1B2的孵育物,与阳性对照一样,均能做到对JEV的感染抑制,在图中并不能观察到JEV对细胞的感染,而反观阴性对照,在显微镜下观察到大量的JEV对细胞感染,而另一株抗体1B1也能够抑制JEV的感染,但并不十分完全,仍然能观察到JEV病毒的感染。实验结果表明本发明筛选的抗体1B2能够很好的抑制JEV的感染。
实施例3、本发明抗体对乙型脑炎病毒的中和能力
为了进一步定量本申请筛选的抗体的中和能力,进行了噬斑实验。
提前一天准备90-100%密度的单层细胞。先制备2.0×105个BHK-21细胞/mL细胞悬液,每孔1mL接种至12孔板中。
稀释抗病毒药物:根据抗病毒药物测试浓度用MEM依次稀释8个梯度(原始浓度1mg/mL)。
稀释病毒:将病毒取出在冰上融化,按照测试需要用MEM将病毒稀释为180PFU/孔。
中和:分别取60μL稀释好的抗体与540μL病毒加至48孔板中,混匀后在37℃培养箱孵育1h。
感染:提前将细胞用D-Hanks洗一遍,每孔加500μL病毒和抗体孵育液,放在37℃培养箱孵育1h。
换液:病毒孵育液弃掉在消毒剂里,用D-Hanks洗一遍细胞,每孔加入1mL半固体培养基,放在37℃培养4-5天。
染色:小心吸去半固体培养基,用PBS洗三遍细胞。每孔加入500μL组织固定液固定细胞15min,再向每个孔加入适量0.5%的结晶紫,刚好覆盖即可。染色>5min。吸去结晶紫,加入PBS清洗,直到噬斑清楚为止,计数噬斑,绘制曲线并计算半数中和浓度,如表1所示。结果显示,抗体1B2的半数中和浓度为0.1046μg/ml,表明该抗体对JEV具有较强的中和活性。
表1
半数中和浓度 | |
1B2 | 0.1046 |
1B1 | 0.7535 |
阴性对照(不加抗体) | / |
阳性对照(利巴韦林) | 0.5022 |
Claims (8)
1.乙型脑炎病毒抗体,其特征在于,其重链可变区的互补决定区CDR1的氨基酸序列如SEQ ID NO.1所示,CDR2的氨基酸序列如SEQ ID NO.2所示,CDR3的氨基酸序列如SEQ IDNO.3所示;轻链可变区的互补决定区CDR1的氨基酸序列如SEQ ID NO.4所示,CDR2的氨基酸序列如SEQ ID NO.5所示,CDR3的氨基酸序列如SEQ ID NO.6所示。
2.根据权利要求1所述的乙型脑炎病毒抗体,其特征在于,其重链可变区的氨基酸序列如SEQ ID NO.7所示;轻链可变区的氨基酸序列如SEQ ID NO.8所示。
3.核酸分子,其特征在于,其编码权利要求1或2所述的乙型脑炎病毒抗体。
4.生物材料,其特征在于,其含有权利要求3所述的核酸分子,所述生物材料为表达盒、载体或宿主细胞。
5.权利要求1或2所述的乙型脑炎病毒抗体或权利要求3所述的核酸分子或权利要求4所述的生物材料的如下任一种应用:
(1)在制备用于检测乙型脑炎病毒的试剂或试剂盒中的应用;
(2)在制备用于预防或治疗乙型脑炎病毒感染或乙型脑炎病毒感染引起疾病的药物中的应用;
(3)在制备用于中和乙型脑炎病毒毒力的产品中的应用。
6.一种抗体偶联物,其特征在于,其为由权利要求1或2所述的乙型脑炎病毒抗体与载体或药物偶联得到,或者由权利要求1或2所述的乙型脑炎病毒抗体与经化学或生物标记得到。
7.一种检测试剂,其特征在于,其含有权利要求1或2所述的乙型脑炎病毒抗体或权利要求6所述的抗体偶联物。
8.一种药物组合物,其特征在于,其含有权利要求1或2所述的乙型脑炎病毒抗体或权利要求6所述的抗体偶联物。
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