CN114989295B - 抗MERS-CoV单克隆抗体及其应用 - Google Patents
抗MERS-CoV单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明公开了抗MERS‑CoV的单克隆抗体及其制备方法于应用,将MERS‑CoV病毒的s蛋白的部分片段作为免疫原免疫小鼠,通过细胞融合和经过杂交瘤细胞的筛选,初步获得具有较高效价及敏感性的单克隆抗体细胞株。通过棋盘法筛选能够实现最佳配对的单克隆抗体,并对获得的最佳配对抗体进行测序列测定,进一步获得了抗体的重链以及轻链可变区序列。建立一种基于酶联免疫反应(ELISA)的双抗体夹心检测方法用于检测MERS‑CoV。采用所述配对抗体建立的双抗夹心法可以将MERS‑CoV感染者与SARS感染者以及正常阴性血清区分,并且对于临床样本的检测具有较高的符合率。采用本发明制备的单克隆抗体,可以应用于血清样本中MERS‑CoV的检测,以辅助MERS的诊断和预防,具有特异性强、灵敏度高、准确性好等优点。
Description
技术领域
本发明属于生物检测领域,具体涉及一种抗MERS-CoV单克隆抗体及其应用,以及基于所述单克隆抗体建立的ELISA检测方法。
背景技术
中东呼吸综合征(MERS)是由中东呼吸综合症冠状病毒(MERS corona virus,MERS-CoV)感染引起的一种急发传染性呼吸系统疾病,2012年最早在约旦和沙特发现。
MERS-CoV是一种人兽共患病病毒,单峰骆驼是主要的动物宿主和传播给人类的主要来源。该病毒在单峰骆驼中不会引起重大疾病,但通过无保护措施的鼻、眼分泌物、粪便以及牛奶和尿液与该病毒接触,已多次传播给人类。这种病毒也可能存在于受感染动物的器官和肉中。这种人兽共患病最常发生在阿拉伯半岛国家;目前在非洲、中东和南亚大部分地区的单峰骆驼体内也都发现了这种病毒。
患者主要表现为发热、咳嗽和气短,肺炎常见,偶见腹泻等胃肠炎症状,严重者可发展为肾衰竭后死亡。该病的潜伏期为2至14天,典型表现为急性呼吸道感染,起病急,高热(39℃~40℃),可伴有畏寒、寒战、咳嗽、胸痛、头痛、全身肌肉关节酸痛、乏力、食欲减退等症状。目前尚无可用的疫苗和特异性治疗方法,主要采用对症治疗和支持性疗法。虽然该病最初发生在中东地区,但随着贸易、旅游、宗教等的活动的开展,逐渐蔓延到欧洲、非洲、亚洲和北美洲等27个国家。
MERS-CoV的形态结构与分类学冠状病毒科下分α、β、γ和δ4个属,病原学调查确认病原体为一种新的冠状病毒,命名为MERS冠状病毒(MERS-CoV)。前2个属感染人及其他哺乳动物等,后2个属主要感染禽类等脊椎动物。冠状病毒形态结构在电镜下大同小异,多表现为日冕状或皇冠状,多为圆形或椭圆形,表面呈突起状,大的突起主要是刺突蛋白,小的有膜成分在内。
MERS-CoV是单股正链RNA病毒。全基因组一般超过30000个核苷酸,不同株间略有差别,基因组分析分为A、B两亚群,但A亚群只有少数株数,主要都在B亚群。
全基因组编码16种非结构蛋白和4种主要结构蛋白。其中以三聚体形式锚定在囊膜上的刺突糖蛋白(Spike,S)是免疫原性最强的结构蛋白,在病毒吸附,决定病毒毒力和组织嗜性,以及诱导保护性免疫等方面发挥重要作用。S蛋白全长共含有1353个氨基酸,由膜外N端的S1亚基和近膜端的S2亚基组成。研究表明,S1亚基能与宿主细胞表面的DPP4受体结合进而介导病毒进入宿主细胞。MERS-CoV的4个结构蛋白S、M、E、N中,以S蛋白最为重要。S蛋白以三聚体形式存在,以机体的二肽酶(DPP4;又称为CD26)为受体进入细胞,开始进行病毒的生命循环。DPP4主要存在于多种哺乳动物的肾、小肠、肝和前列腺等脏器的上皮细胞表面,在单峰骆驼中,DPP4存在于上呼吸道上皮细胞表面,而在人类中,DPP4则多在肺泡表面表达。S蛋白在进入DPP4前,裂解成S1和S2两个亚基,S1与DPP4的受体结合域(RBD)结合,此后S2细胞与MERS-CoV膜融合,促使病毒核酸进入细胞。
S蛋白既存在可使病毒遗传物质进入机体的RBD,又是进入宿主细胞的主要免疫原,因此是研制药物的靶点和候选疫苗的主要目标蛋白。MERS-CoV能感染多种动物,与DPP4能改变与S蛋白结合介面的静电荷有关。其他3种蛋白M、E、N及辅助蛋白,M主要在病毒的外层,起维护病毒形态的作用,与S蛋白相互作用,在E蛋白的辅助下,病毒RNA能进入细胞浆进行复制;E蛋白具有较多的嗜水域,为主在双脂膜的内层,并和N衣壳蛋白相连,在病毒出芽生殖中起作用,N衣壳蛋白最主要的作用在于裹胁、保护病毒的RNA;辅助蛋白的作用可能与病毒的致病性有关,现已明确,包括M蛋白在内,ORF4A,ORF4B和ORF5编码的蛋白是I型干扰素的拮抗剂。
目前认为传染源与带MERS病毒的骆驼有关,因此与骆驼有密切接触的人群(饲养人员、农场工人、屠宰场工人和兽医等)感染可能性较大,赴中东旅游接触了骆驼或其分泌物、饮用未消毒骆驼奶的游客也有可能感染。与病例有密切接触的医务人员、家属感染MERS-CoV的风险均较高。另外,对现有感染MERS病例研究显示,病例的平均年龄为50岁,76%的MERS病例至少存在一种基础性疾病,包括慢性肾衰、糖尿病、心脏病等,且死亡病例与其他MERS病例比较,其基础疾病患病率更高(86.8% VS42.4%,P<0.001)。因此,患有糖尿病、慢性肺部疾病、肾衰或者免疫力低下的人群也被认为是感染MERS-CoV的高风险人群。
MERS的诊断需要结合临床特点、流行病学因素和在呼吸道中检测到病毒。检测方法主要有病毒核酸检测和血清学检测。一种实时RT-PCR方法是通过目标区域上游的E基因(upE)或开放读取框来实现监测的。E基因用来筛选,而开放读取框1b用来确认;另一种实时RT-PCR方法是以核壳蛋白基因为目标来筛选和确认的。当两种实时RT-PCR方法的结果不一致时,合适的RT-PCR扩增子排序可以帮助确认结果。但是,血清学样本的结果需要谨慎解释,可能会由于交互效应而与其他冠状病毒感染混淆。
通常,血清学检测适用于在无法开展核酸扩增时在《国际卫生条例》下应用血清学来定义MERS-CoV感染病例、持续性暴发调查中的部分调查及血清学专项调查。应用血清学确认MERS-CoV感染的方法较多,其中2项免疫荧光试验和1项血清中和试验在德国用于确诊病例密切接触者的筛查,并在沙特阿拉伯用于人群血清流行率的调查;1项利用蛋白质芯片技术的提示具有较高的特异度;此外,还有2项ELISA试验也被报道用于MERS-CoV的检测。
MERS-CoV的准确检测是预防和后续治疗的基础,本研究旨在开发出一种对MERS-CoV具有高亲和性和高特异性的单克隆抗体,通过棋盘法筛选获得最佳配对的单克隆抗体,建立一种基于酶联免疫反应(ELISA)的双抗体夹心检测方法,以用于准确检测各种样品中MERS-CoV的含量,以辅助MERS的诊断和预防。
发明内容
本发明公开了抗MERS-CoV单克隆抗体,所述单克隆抗体的轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:6、7和8所示;和所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:17、18和19所示。
本发明公开了单克隆抗体,所述单克隆抗体的轻链可变区的氨基酸序列分别如SEQ ID NO:5所示;和所述单克隆抗体的重链可变区氨基酸序列分别如SEQ ID NO:16所示。
本发明公开了抗MERS-CoV单克隆抗体,所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:17、18和19所示;和所述单克隆抗体的轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:6、7和8所示;或,所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:22、23和24所示;和所述单克隆抗体的轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:11、12和13所示。
本发明公开了单克隆抗体,所述单克隆抗体的重链可变区氨基酸序列分别如SEQID NO:16所示;和所述单克隆抗体的轻链可变区的氨基酸序列分别如SEQ ID NO:5所示;或,所述单克隆抗体的重链可变区氨基酸序列分别如SEQ ID NO:21所示;和所述单克隆抗体的轻链可变区的氨基酸序列分别如SEQ ID NO:10所示。
本发明公开了所述的单克隆抗体,还包括恒定区,所述单克隆抗体的重链的恒定区为IgG1、IgG2、IgG3或IgG4的任意一种;所述单克隆抗体的轻链的恒定区为κ型或λ型。
本发明公开了编码所述的单克隆抗体的核苷酸分子。
本发明公开了所述的核苷酸分子,所述编码单克隆抗体重链的核苷酸分子序列如SEQ ID NO:15所示,所述编码单克隆抗体轻链的核苷酸分子序列如SEQ ID NO:4所示;或,所述编码单克隆抗体重链的核苷酸分子序列如SEQ ID NO:20所示,所述编码单克隆抗体轻链的核苷酸分子序列如SEQ ID NO:9所示。
本发明公开了表达载体,所述表达载体包含所述的核苷酸分子。
本发明公开了细胞,所述细胞包含所述的载体。
单克隆抗体,其特征在于,所述单克隆抗体由权利要求7所述的细胞表达获得。
本发明公开了所述的单克隆抗体在制备检测MERS-CoV的试剂盒中的用途。
一种检测MERS-CoV的试剂盒,所述试剂盒包括所述的单克隆抗体。
本发明公开了所述的试剂盒,所述试剂盒还包括终止液。
本发明公开了所述的试剂盒,所述试剂盒为ELISA检测试剂盒或胶体金检测试剂盒。
优选的,所述检测试剂盒还包括标准品。
优选的,所述试剂盒可以应用于MERS-CoV的定量检测。
优选的,所述试剂盒可以应用于MERS-CoV的快速检测。
本发明公开了抗MERS-CoV的单克隆抗体及其制备方法,将MERS-CoV病毒的s蛋白的部分片段作为免疫原免疫小鼠,通过细胞融合和经过杂交瘤细胞的筛选,初步获得具有较高效价及敏感性的单克隆抗体细胞株。通过棋盘法筛选能够实现最佳配对的单克隆抗体,并对获得的最佳配对抗体进行测序列测定,进一步获得了抗体的重链以及轻链可变区序列。建立一种基于酶联免疫反应(ELISA)的双抗体夹心检测方法用于检测MERS-CoV。采用所述配对抗体建立的双抗夹心法可以将MERS-CoV感染者与SARS感染者以及阴性血清区分,并且对于临床样本的检测具有较高的符合率。采用本发明制备的单克隆抗体,可以应用于血清样本中MERS-CoV的检测,以辅助MERS的诊断和预防,具有特异性强、灵敏度高、准确性好等优点。
附图说明
图1为重组质粒pET-28a-S酶切电泳图,其中1为重组质粒图,2为酶切后的质粒和S基因片段。
图2为表达的MERS-S蛋白,纯化后的电泳检测图。
图3为筛选的5个杂交瘤细胞分离纯化后的抗体的电泳图,其中,1为杂交瘤2C7,2为杂交瘤4D9,3为杂交瘤6E4,4为杂交瘤7G5,5为杂交瘤8F10。
图4为棋盘法筛选最佳配对抗体的柱状结果图。
图5为棋盘法筛选最佳配对抗体的曲线结果图。
图6为ELISA方法中不同包被浓度以及标记浓度的优化检测结果图。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1主要试剂及培养基的配制
1.LB培养基:胰蛋白胨1g、酵母粉0.5g、NaCl 1g、,加100mL去离子定容,高压灭菌,4℃保存备用。
2.10%SDS:SDS 5g,加去离子水50mL充分溶解,4℃保存。
3.完全1640培养液:含20%胎牛血清、1%双抗和1%L-G的基础1640培养液,4℃保存备用;
4.HAT培养液:含2% HAT的完全1640培养液。
5.HT培养液:含1%HT的完全1640培养液。
实施例2MERS-CoV的S蛋白的表达纯化
1.扩增引物设计以及表达载体的构建
首先采用抗原表位预测软件,对于NCBI上公开的MERS-CoV的S蛋白进行分析,选取含有相应的抗原优势表位的片段,并进一步设计引物扩增编码MERS-CoV的S蛋白的基因片段,由生物科技有限公司合成的S基因作为模板,采用PCR扩增、纯化、回收S基因片段,通过BamHⅠ和XhoI限制性内切酶双酶切后插入pET-28a表达载体,构建pET-28a-S基因重组质粒。
其中,上游引物:5’-ggattcgaaagttacgttgatgtag-3’(SEQ ID NO:1),(包含BamHⅠ酶切位点);
下游引物:5’-ctcgagtcatctgcgtatataacca-3’(SEQ ID NO:2),(包含XhoI酶切位点),扩增片段大小为963bp(未包含酶切位点)。重组质粒pET-28a-S为模板,利用合成的引物进行PCR。扩增产物经1%琼脂糖凝胶电泳鉴定正确后进行目的片段的回收。用BamHⅠ和XhoI双酶切重组质粒pET-28a-S,得到约为960bp(S片段基因)和约5300bp的质粒片段,重组质粒鉴定正确,质粒酶切的电泳图片参见图1。
2.MERS-CoV-S重组蛋白的表达与纯化
将构建的重组质粒转化大肠杆菌BL21(DE3),筛选获得阳性转化菌株后备用。
取20μl重组菌株BL21(DE3)/pET-28a-S接种于20ml LB液体培养基(含50μg/mLKan)中,37℃、180r/min培养。次日取少量菌液转接到200ml LB液体培养基(含50μg/mLKan)中,37℃、180r/min培养至OD600达到0.5-0.6时,加入终浓度为0.8mM的IPTG,25℃、200r/min诱导培养10小时。4℃、8000r/min离心6分钟收集菌体,加入10mL PBS缓冲液重悬菌体,经超声波破碎处理后,4℃、8500r/min离心6分钟收集菌体,收集上清液,经镍柱基质纯化后,取5微克重组蛋白进行SDS-PAGE检测。电泳检测结果参见图2。
实施例3抗MERS-CoV的杂交瘤细胞的筛选与制备
1.小鼠免疫与抗血清的检测
将纯化获得重组MERS-CoV-S蛋白稀释后,0.1%(V/V)甲醛灭活24小时后,作为免疫原免疫小鼠。采取皮下多点注射的方式对8周龄BALB/c小鼠(共5只)进行免疫(50μg/只),免疫3次。
初次免疫使用弗氏完全佐剂与目的蛋白等体积混合并充分乳化;第2次和第3次免疫用弗氏不完全佐剂等体积混合并乳化。其中,第3次免疫的过程中,有两只小鼠的生存状况不佳,出现厌食消瘦状况,因此,将这两只免疫小鼠舍弃,仅保留另外3只小鼠继续试验。第3次免疫后第10天取小鼠尾静脉血,通过间接ELISA法检测抗血清效价,检测结果参见表1,从ELISA的初步检测结果来看,编号为2的小鼠免疫效果最好。因此,后续将选择效价高的2号小鼠用于细胞融合。
表1:免疫小鼠多抗血清效价的测定
2.细胞融合与杂交瘤细胞的筛选
将SP2/0瘤细胞从液氮罐中复苏后,加入RPMI1640培养基于离心瓶中混合均匀,置于二氧化碳培养箱中培养备用。
取未经免疫的清洁级BALB/c小鼠一只,摘眼球采血后即把小鼠拉颈处死,剪开小鼠腹膜。吸取冷的HAT培养基,打入小鼠腹腔,轻轻挤压小鼠腹腔回抽出含有饲养细胞的培养液。
取初步测定血清效价较好的第2只免疫后的BALB/c小鼠,摘眼球拉颈处死小鼠,取其脾脏,并对脾脏进行研磨,之后取5mL预热的GNK洗液缓慢冲洗,制成单细胞悬液。
用PEG1500将分离得到的脾细胞与提前复苏的SP2/0骨髓瘤细胞按3:1至5:1的比例融合,融合后使用HAT及HT培养基进行换液培养。培养约4d后吸取细胞培养上清液,将表达的MERS-S蛋白包被酶联免疫96孔板,通过间接ELISA筛选阳性杂交瘤细胞,并进一步采用有限稀释法进行亚克隆,转移至24孔细胞培养板进行扩大培养。待细胞长至1/2孔底时,取上清测其效价值和敏感性,筛选获得5株具有较高效价(达到1:2.56X105)及敏感性杂交瘤细胞株,其中6E4的敏感性更好(表2)。
表2:筛选的杂交瘤的效价及敏感性
杂交瘤编号 | 效价 | 敏感性 |
2C7 | 1:2.56X105 | 1.056ng/mL |
4D9 | 1:2.56x105 | 0.723ng/mL |
6E4 | 1:2.56x105 | 2.314ng/mL |
7G5 | 1:2.56x105 | 0.561ng/mL |
8F10 | 1:2.56x105 | 0.432ng/mL |
实施例4抗MERS单克隆抗体的大量制备与纯化
1.腹水的制备
用经产的BALB/C母鼠腹腔大量的制备单克隆抗体,用经产母鼠是因为其腹腔内的空间较大,便于产生更多的抗体。首先在母鼠腹腔内注射1ml灭菌的液体石蜡。约7天后,用纯1640培养基清洗并悬浮杂交瘤细胞(尽量去除杂蛋白和FBS),计数,并用500ul纯1640培养基重悬约5×106个细胞,注射到母鼠的腹腔中。
大约7-10天后母鼠腹腔中已出现大量的腹水,要立即抽取收集。用较粗的针头(12号针头)从腹股沟处插入腹腔内,后使腹水自然流出并用离心管收集。3-5天后,母鼠的腹腔会继续变大,可继续收集腹水,收集的腹水5000r/min离心15min,上清上层的油状不溶物质弃掉,取上清,保存于-20℃中。
2.单克隆抗体的纯化
取出保存的腹水,加入3倍体积的醋酸盐缓冲液,充分混合均匀后用NaOH溶液调pH至4.3。测量上述溶液总体积,按0.025mL/mL的量缓慢加入合适体积的辛酸,4℃缓慢搅拌45min。4℃,5000rpm离心1h,收集中间液体并使用滤纸过滤。加入1/10体积的0.2M的PBS,充分混合均匀后调pH至7.4。按照0.2778g/mL的量在上述溶液中加入固体硫酸铵,4℃缓慢搅拌30min。4℃,8000rpm离心1h,弃上清,使用0.02M的PBS充分溶解沉淀。4℃条件下透析过夜并换液2-3次。收取透析液,-20℃保存备用。将纯化后的抗体采用电泳检测,其中5株杂交瘤在进行抗体大量制备纯化后的电泳结果参见图3。纯化后的抗体含有两条特异性的条带,与IgG的重链和轻链的大小也一致。
实施例5棋盘法筛选最佳配对抗体
用碳酸盐缓冲液(CBS0.05mol/L,pH9.6)作为稀释单抗介质,稀释纯化获得2C7、4D9、6E4、7G5、8F10这5株单抗分别稀释到约2μg/mL,按照100μL/孔的规格加入ELISA板,4℃过夜包被。弃去并在纸上拍尽包被液,ELISA板用PBST(PBS pH 7.4,0.05% Tween-20)清洗2-3次,弃去孔中液体,晾干得到抗体包被板。
采用改良的过碘酸钠法将单克隆抗体标记HRP,按照1:5000的稀释倍数之后,作为酶标二抗使用
棋盘滴定法实验,每种包被抗体制备的包被板分别和其它4种抗体的HRP标记物配对,检测不同浓度的MERS-S抗原。具体操作:包被板孔中加入不同浓度的MERS-S抗原,温育后洗涤2-3次。每孔加入HRP标记的抗MERS单克隆抗体,温育后洗涤2-3次。加入底物TMB进行显色后,加硫酸中止反应,置酶标仪(OD450nm)中测定A值。筛选结果参见柱状图4以及曲线图图5。
根据筛选的图4以及图5的结果所表现出来的线性关系以及检测最低限及最高限之间的数值关系可知,抗体6E4和7G5之间进行配对的检测效果较好,而抗体2C7和4D9之间进行配对的检测效果较好,并且抗体6E4和7G5之间的配对检测效果优于抗体2C7和4D9之间的配对。
实施例6单克隆抗体6E4和7G5可变区基因的获取
分别提取杂交瘤细胞株6E4和7G5的总RNA,并根据BIO TEC公司逆转录试剂盒的操作方法,分别反转录制备获得对应的cDNA序列。
根据已知的小鼠抗体轻链恒定区序列,设计相应的扩增引物:5’
-TCACTGCCATCAATCTTCCAC-3’,SEQ ID NO:3)和试剂盒中的接头引物进行PCR扩增,获得相应的杂交瘤分泌的抗MERS-CoV鼠单克隆抗体轻链片段,并于pGEM-T载体构建后测序。
测序后获得抗MERS-CoV鼠单克隆抗体6E4轻链可变区基因序列为:gatattcaggaaacccagacccgcagcgtgctgagcgcggcgctgggcagccgcgtgaccattagctgcagcatggatattgcgaactatgtgaacgaatatcagtggtatcagagcccgaaaccggattttgatgtgaaactgagcatttatgaagtgctgatttatcgcagcgtgcgcctgcagagcggcgtgccgagccgctttagcaaaaaaggcagcctggatgattatagcctgaccattagctgctttgaaccggaagatgtggaaatttattgcaacgattgccagattctgggcagcgaactgccggaatttggcgattttaccaaaattgaaattctgcgc(SEQ ID NO:4)。
抗MERS-CoV鼠单克隆抗体6E4轻链可变区的序列为:
DIQETQTRSVLSAALGSRVTISCSMDIANYVNEYQWYQSPKPDFDVKLSIYEVLIYRSVRLQSGVPSRFSKKGSLDDYSLTISCFEPEDVEIYCNDCQILGSELPEFGDFTKIEILR(SEQ ID NO:5所示)。
按照抗体CDR结构分析网站中对于抗体的CDR分析定义方法,获得抗MERS-CoV鼠单克隆抗体6E4的轻链可变区的CDR序列,其中:CDR1序列为:SMDIANYVNEYQ(SEQ ID NO:6);CDR2序列为:EVLIYRSVRLQS(SEQ ID NO:7);CDR3序列为:NDCQILGSELPE(SEQ ID NO:8)。
获得抗MERS-CoV鼠单克隆抗体7G5轻链可变区基因序列为:gatattagcctgacccgcagcccgagcaccctgagcgcgaccccgcaggaaagcgtgagcctgagctgcaaacagctgattagcagctatctgatgtggacccagtggtatcagagcaaaagccatgaaagcctggaa ctgaccgaaaaagtggaactgatttatagcaacagccatctgcataccggcattccgtttagctttagcggcgatgatagcggcaccgatttttttctgaaagtgaacgtgaacagcaccgaagattttggcgaatatttttgcagctgccaggatcagaacaccagcgaacgcggcgattttggccaggcgaccaaactgctggaaaaa(SEQ ID NO:9);
抗MERS-CoV鼠单克隆抗体7G5对应的轻链可变区的序列为:DISLTRSPSTLSATPQESVSLSCKQLISSYLMWTQWYQSKSHESLELTEKVELIYSNSHLHTGIPFSFS GDDSGTDFFLKVNVNSTEDFGEYFCSCQDQNTSERGDFGQATKLLEK(SEQ ID NO:10)。
按照抗体CDR结构分析网站中对于抗体的CDR分析定义方法,获得抗MERS-CoV鼠单克隆抗体7G5的轻链可变区的CDR序列,其中:CDR1序列为:KQLISSYLMWTQ(SEQ ID NO:11);CDR2序列为:VELIYSNSHLHT(SEQ ID NO:12);CDR3序列为:SCQDQNTSERGD(SEQ ID NO:13)。
根据已知的小鼠抗体重链恒定区序列,设计相应的扩增引物:5’
-CTCAGGGAARTARCCYTTGAC-3’,SEQ ID NO:14)和试剂盒中的接头引物进行PCR扩增,获得相应杂交瘤分泌的抗MERS-CoV鼠单克隆抗体重链片段,并于pGEM-T载体构建后测序。
获得抗MERS-CoV鼠单克隆抗体6E4重链可变区基因序列为:gaagtggatctgagcgaaagcctgggcggccaggtgaaagtgaccggcagcctgaaactggatgaagcggcgagcggcagcatgcatggcggcgatgtgaaagaaggcgcgagcctgtggctgtgggtgcgcgtgaccccggaaattgaactggaatggcgcgatgcgcagggcgtggcgtttattagcgcgggcgaaagcagcacctatcgctttaccaccagccgcgataacgcgcgcagcgatcgctatctgcagatgagcgaactgcgcaaactggataccgcgtttatttattgcggccgctatcgcgtgaccctgagcaccctgcaggaatatagctggggccagggcgattgggtgaccaccgatgcg(SEQ ID NO:15)。
抗MERS-CoV鼠单克隆抗体6E4的重链可变区的序列为:
EVDLSESLGGQVKVTGSLKLDEAASGSMHGGDVKEGASLWLWVRVTPEIELEWRDAQGVAFISAGESSTYRFTTSRDNARSDRYLQMSELRKLDTAFIYCGRYRVTLSTLQEYSWGQGDWVTTDA(SEQ ID NO:16);
按照抗体CDR结构分析网站中对于抗体的CDR分析定义方法,获得抗MERS-CoV鼠单克隆抗体6E4的重链可变区的CDR序列,其中:CDR1序列为:SMHGGDVKEGASLWL(SEQ ID NO:17);CDR2序列为:QGVAFISAGESST(SEQ ID NO:18);CDR3序列为:YRVTLSTLQEYS(SEQ ID NO:19)。
获得抗MERS-CoV鼠单克隆抗体7G5重链可变区基因序列为:gaagtgcagggccaggtgagcggcctgaaactggtggtggaaggcgcgagcgtgctgaaaagctgcaccagcaccggctttctgagcggcatgggcctggtgatgccgggcggcagcaaccatagctgggtgcaggaagtgccggaaattgtgctggaatggagcggcgattggagcgcgaactatagccaggatggcctgattaccagcaaagcgaccgcgagcgcggataccaccgatagcaccgcgtatctgcagctgagcgaatttgatagcgaagataccgcggtgagctttagctgcgcgagcaactggctgatgctgtatatggcgagcctgaactggggccagggcgaagatgataccgtgcagagc(SEQ ID NO:20)。
抗MERS-CoV鼠单克隆抗体7G5的重链可变区的序列为:
EVQGQVSGLKLVVEGASVLKSCTSTGFLSGMGLVMPGGSNHSWVQEVPEIVLEWSGDWSANYSQDGLITSKATASADTTDSTAYLQLSEFDSEDTAVSFSCASNWLMLYMASLNWGQGEDDTVQS(SEQ ID NO:21)。
按照抗体CDR结构分析网站中对于抗体的CDR分析定义方法,获得抗MERS-CoV鼠单克隆抗体7G5的重链可变区的CDR序列,其中:CDR1序列为:LSGMGLVMPGGSNHS(SEQ ID NO:22);CDR2序列为:DWSANYSQDGLIT(SEQ ID NO:23);CDR3序列为:SNWLMLYMASLN(SEQ ID NO:24)。
实施例7配对抗体的使用的浓度优化
使用不同浓度(0.5、1、2、3、4μg/mL)的抗MERS-CoV单抗6E4包被96孔微孔板(包被液为CB),封闭过夜。弃去孔中液体,晾干备用。
包被板孔中加入不同浓度(50pg/mL、100pg/mL、200pg/mL、500pg/mL、1000pg/mL)的表达的MERS-CoV-S抗原,37℃温育1小时,洗涤3次。每孔加入分别稀释1:1000、1:2000、1:5000、1:10000的HRP标记的抗MERS-CoV单抗7G5,反应1小时后,洗涤2-3次。加入底物DAB进行显色后,加硫酸中止反应,置酶标仪中(于490nm处)测定A值。检测结果见图6。优化后的单抗6E4的优化的包被浓度是2μg/mL,HRP标记单抗7G5的优化稀释浓度是1:5000。
实施例8抗体的特异性检测
采用实施例7中优化后的抗体的浓度,制备获得相应的包被浓度以及标记浓度的单抗ELISA检测试剂盒,并对临床样本进行检测,临床样本包括:10份阴性血清、10份MERS-CoV患者感染血清以及10份SARS感染者血清,上述血清分别采用对应的MERS-CoV以及SARS的RT-PCR方法进行确认。采用上述ELISA试剂盒检测,检测结果的OD值表明,10份MERS-CoV患者感染血清明显高于另外20份血清,采用所述试剂盒能够特异性的区分出MERS-CoV患者,检测的具体结果参见表3。
表3:ELISA试剂盒检测30份血清样本的检测结果
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
阴性血清 | 0.036 | 0.027 | 0.031 | 0.028 | 0.157 | 0.239 | 0.205 | 0.233 | 0.286 | 0.217 |
MERS-CoV患者 | 2.816 | 3.014 | 1.957 | 2.068 | 2.374 | 2.854 | 2.883 | 2.749 | 1.764 | 2.551 |
SARS患者 | 0.087 | 0.139 | 0.065 | 0.113 | 0.027 | 0.210 | 0.187 | 0.097 | 0.432 | 0.312 |
实施例9抗体的准确性检测
采用实施例7中优化后的抗体的浓度,制备获得相应的包被浓度以及标记浓度的单抗ELISA检测试剂盒,并对临床样本进行检测,临床样本共计60份。阳性判断标准为,检测的OD值大于实施例8的表3中10份阴性血清测定的平均OD值的2.5倍以上。检测得到26份阳性样本,以及35份阴性样本,将检测结果采用RT-PCR检测方法进行比较,发现本申请的夹心法的阳性符合率为96.29%,具有较高的阳性符合率。准确性检测结果参见表4。
表4:临床样本的准确性检测
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表信息:
DTD版本: V1_3
文件名: MERS单抗.xml
软件名称: WIPO Sequence
软件版本: 2.2.0
生成日期: 2023-04-19
基本信息:
当前申请 / 知识产权局: CN
当前申请 / 申请号: 202210694642 .0
当前申请 / 申请日: 2022-06-17
当前申请 / 申请人档案名: 2023
申请人姓名或名称: 上海优晶生物科技有限公司
申请人姓名或名称 / 语言: zh
申请人姓名或名称 / 拉丁名称: Shanghai Eugene Biotech Co., Ltd.
发明名称: 抗MERS-CoV单克隆抗体及其应用 ( zh )
序列总量: 24
序列:
序列号 (ID): 1
长度: 25
分子类型: DNA
特征 位置/限定符:
- source, 1..25
> mol_type, other DNA
> organism, synthetic construct
残基:
ggattcgaaa gttacgttga tgtag 25
序列号 (ID): 2
长度: 25
分子类型: DNA
特征 位置/限定符:
- source, 1..25
> mol_type, other DNA
> organism, synthetic construct
残基:
ctcgagtcat ctgcgtatat aacca 25
序列号 (ID): 3
长度: 21
分子类型: DNA
特征 位置/限定符:
- source, 1..21
> mol_type, other DNA
> organism, synthetic construct
残基:
tcactgccat caatcttcca c 21
序列号 (ID): 4
长度: 351
分子类型: DNA
特征 位置/限定符:
- source, 1..351
> mol_type, other DNA
> organism, synthetic construct
残基:
gatattcagg aaacccagac ccgcagcgtg ctgagcgcgg cgctgggcagccgcgtgacc 60
attagctgca gcatggatat tgcgaactat gtgaacgaat atcagtggtatcagagcccg 120
aaaccggatt ttgatgtgaa actgagcatt tatgaagtgc tgatttatcgcagcgtgcgc 180
ctgcagagcg gcgtgccgag ccgctttagc aaaaaaggca gcctggatgattatagcctg 240
accattagct gctttgaacc ggaagatgtg gaaatttatt gcaacgattgccagattctg 300
ggcagcgaac tgccggaatt tggcgatttt accaaaattg aaattctgcg c 351
序列号 (ID): 5
长度: 117
分子类型: AA
特征 位置/限定符:
- source, 1..117
> mol_type, protein
> organism, synthetic construct
残基:
DIQETQTRSV LSAALGSRVT ISCSMDIANY VNEYQWYQSP KPDFDVKLSIYEVLIYRSVR 60
LQSGVPSRFS KKGSLDDYSL TISCFEPEDV EIYCNDCQIL GSELPEFGDF TKIEILR117
序列号 (ID): 6
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
SMDIANYVNE YQ 12
序列号 (ID): 7
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
EVLIYRSVRL QS 12
序列号 (ID): 8
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
NDCQILGSEL PE 12
序列号 (ID): 9
长度: 348
分子类型: DNA
特征 位置/限定符:
- source, 1..348
> mol_type, other DNA
> organism, synthetic construct
残基:
gatattagcc tgacccgcag cccgagcacc ctgagcgcga ccccgcaggaaagcgtgagc 60
ctgagctgca aacagctgat tagcagctat ctgatgtgga cccagtggtatcagagcaaa 120
agccatgaaa gcctggaact gaccgaaaaa gtggaactga tttatagcaacagccatctg 180
cataccggca ttccgtttag ctttagcggc gatgatagcg gcaccgatttttttctgaaa 240
gtgaacgtga acagcaccga agattttggc gaatattttt gcagctgccaggatcagaac 300
accagcgaac gcggcgattt tggccaggcg accaaactgc tggaaaaa 348
序列号 (ID): 10
长度: 116
分子类型: AA
特征 位置/限定符:
- source, 1..116
> mol_type, protein
> organism, synthetic construct
残基:
DISLTRSPST LSATPQESVS LSCKQLISSY LMWTQWYQSK SHESLELTEKVELIYSNSHL 60
HTGIPFSFSG DDSGTDFFLK VNVNSTEDFG EYFCSCQDQN TSERGDFGQA TKLLEK116
序列号 (ID): 11
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
KQLISSYLMW TQ 12
序列号 (ID): 12
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
VELIYSNSHL HT 12
序列号 (ID): 13
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
SCQDQNTSER GD 12
序列号 (ID): 14
长度: 21
分子类型: DNA
特征 位置/限定符:
- source, 1..21
> mol_type, other DNA
> organism, synthetic construct
残基:
ctcagggaar tarccyttga c 21
序列号 (ID): 15
长度: 375
分子类型: DNA
特征 位置/限定符:
- source, 1..375
> mol_type, other DNA
> organism, synthetic construct
残基:
gaagtggatc tgagcgaaag cctgggcggc caggtgaaag tgaccggcagcctgaaactg 60
gatgaagcgg cgagcggcag catgcatggc ggcgatgtga aagaaggcgcgagcctgtgg 120
ctgtgggtgc gcgtgacccc ggaaattgaa ctggaatggc gcgatgcgcagggcgtggcg 180
tttattagcg cgggcgaaag cagcacctat cgctttacca ccagccgcgataacgcgcgc 240
agcgatcgct atctgcagat gagcgaactg cgcaaactgg ataccgcgtttatttattgc 300
ggccgctatc gcgtgaccct gagcaccctg caggaatata gctggggccagggcgattgg 360
gtgaccaccg atgcg 375
序列号 (ID): 16
长度: 125
分子类型: AA
特征 位置/限定符:
- source, 1..125
> mol_type, protein
> organism, synthetic construct
残基:
EVDLSESLGG QVKVTGSLKL DEAASGSMHG GDVKEGASLW LWVRVTPEIELEWRDAQGVA 60
FISAGESSTY RFTTSRDNAR SDRYLQMSEL RKLDTAFIYC GRYRVTLSTLQEYSWGQGDW 120
VTTDA 125
序列号 (ID): 17
长度: 15
分子类型: AA
特征 位置/限定符:
- source, 1..15
> mol_type, protein
> organism, synthetic construct
残基:
SMHGGDVKEG ASLWL 15
序列号 (ID): 18
长度: 13
分子类型: AA
特征 位置/限定符:
- source, 1..13
> mol_type, protein
> organism, synthetic construct
残基:
QGVAFISAGE SST 13
序列号 (ID): 19
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
YRVTLSTLQE YS 12
序列号 (ID): 20
长度: 375
分子类型: DNA
特征 位置/限定符:
- source, 1..375
> mol_type, other DNA
> organism, synthetic construct
残基:
gaagtgcagg gccaggtgag cggcctgaaa ctggtggtgg aaggcgcgagcgtgctgaaa 60
agctgcacca gcaccggctt tctgagcggc atgggcctgg tgatgccgggcggcagcaac 120
catagctggg tgcaggaagt gccggaaatt gtgctggaat ggagcggcgattggagcgcg 180
aactatagcc aggatggcct gattaccagc aaagcgaccg cgagcgcggataccaccgat 240
agcaccgcgt atctgcagct gagcgaattt gatagcgaag ataccgcggtgagctttagc 300
tgcgcgagca actggctgat gctgtatatg gcgagcctga actggggccagggcgaagat 360
gataccgtgc agagc 375
序列号 (ID): 21
长度: 125
分子类型: AA
特征 位置/限定符:
- source, 1..125
> mol_type, protein
> organism, synthetic construct
残基:
EVQGQVSGLK LVVEGASVLK SCTSTGFLSG MGLVMPGGSN HSWVQEVPEIVLEWSGDWSA 60
NYSQDGLITS KATASADTTD STAYLQLSEF DSEDTAVSFS CASNWLMLYMASLNWGQGED 120
DTVQS 125
序列号 (ID): 22
长度: 15
分子类型: AA
特征 位置/限定符:
- source, 1..15
> mol_type, protein
> organism, synthetic construct
残基:
LSGMGLVMPG GSNHS 15
序列号 (ID): 23
长度: 13
分子类型: AA
特征 位置/限定符:
- source, 1..13
> mol_type, protein
> organism, synthetic construct
残基:
DWSANYSQDG LIT 13
序列号 (ID): 24
长度: 12
分子类型: AA
特征 位置/限定符:
- source, 1..12
> mol_type, protein
> organism, synthetic construct
残基:
SNWLMLYMAS LN 12
END
Claims (10)
1.抗MERS-CoV单克隆抗体,其特征在于,所述单克隆抗体的轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:6、7和8所示;和所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:17、18和19所示。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述单克隆抗体的轻链可变区的氨基酸序列分别如SEQ ID NO:5所示;和所述单克隆抗体的重链可变区氨基酸序列分别如SEQID NO:16所示。
3.根据权利要求1-2任一所述的单克隆抗体,还包括恒定区,所述单克隆抗体的重链的恒定区为IgG1、IgG2、IgG3或IgG4的任意一种;所述单克隆抗体的轻链的恒定区为κ型或λ型。
4.编码权利要求1-3任一权利要求所述的单克隆抗体的核苷酸分子。
5.根据权利要求4所述的核苷酸分子,其特征在于,所述编码单克隆抗体轻链的核苷酸分子序列如SEQ ID NO:4所示,所述编码单克隆抗体重链的核苷酸分子序列如SEQ ID NO:15所示。
6.表达载体,其特征在于,所述表达载体包含权利要求4或5所述的核苷酸分子。
7.细胞,其特征在于,所述细胞包含权利要求6所述的载体。
8.单克隆抗体,其特征在于,所述单克隆抗体由权利要求7所述的细胞表达获得。
9.权利要求1-3任一权利要求所述的单克隆抗体在制备检测MERS-CoV的试剂盒中的用途。
10.一种检测MERS-CoV的试剂盒,其特征在于,所述试剂盒包括权利要求1-3任一权利要求所述的单克隆抗体。
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