CN101798347B - 抗高迁移率族蛋白b1的单克隆抗体 - Google Patents
抗高迁移率族蛋白b1的单克隆抗体 Download PDFInfo
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Abstract
本发明公开了抗高迁移率族蛋白B1的单克隆抗体及其应用。一种抗体,其重链可变区具有序列表中序列1所示的氨基酸残基序列,其轻链可变区具有序列表中序列2所示的氨基酸残基序列。实验证明杂交瘤细胞株3E8CGMCC No.2906分泌的单克隆抗体、衍生于该单克隆抗体的单链抗体3E8scFV、人-鼠嵌合抗体ch-3E8或Fab片段与人HMGB1的解离常数分别为1.3nM,70nM,1.3nM,10nM。上述单克隆抗体及其衍生物能够高亲和力特异性识别高迁移率族蛋白B1,并中和其生物学活性,可以用于治疗败血症,全身炎症反应综和症或急性肺损伤等炎症反应;可以用于治疗类风湿性关节炎,糖尿病或红斑狼疮等自身免疫性疾病。还可用于临床检验HMGB1的水平。
Description
技术领域
本发明涉及抗高迁移率族蛋白B1的单克隆抗体。
背景技术
HMGB-1(high mobility group box,I)是染色体蛋白中高迁移率家族的成员之一。HMGB-1是一条由219个氨基酸残基构成的、分子量为30kDa的单链多肽,其表达普遍而且量也较大。蛋白可被分为三大部分:两个同源的DNA结合区域(Boxes A和B)及酸性C末端。HMG boxes A和B是由相似的80个氨基酸片段形成的L型结构。
HMGB-1结合于双链DNA的小沟内,结合部位与DNA序列无关,但与其结构密切相关,通常位于节状DNA、弯曲DNA或十字交叉点处。HMGB-1可以迅速地弯曲DNA,促成核蛋白复合体的形成,进而促成DNA结合蛋白与其各自对应位点的相互作用。如p53转录因子不能直接与直线DNA结合,只有在HMGB-1先与DNA结合并使其弯曲后,p53才能有效地与其对应位点相结合,然后HMGB-1从复合体中脱离出来。HMGB1-/-鼠在GR应答基因的激活上有缺陷,因此会因血糖过低而在出生后不久就死去,此结果进一步支持了HMGB-1作为转录调控因子的结论。
HMGB-1存在于神经细胞的细胞间隙中,可以刺激神经突起的生长。HMGB1刺激平滑肌细胞(SMC)和原纤维细胞的迁移,还能够引起细胞骨架肌动蛋白的重组以及运动型细胞的形态改变。此外,HMGB-1与纤维蛋白酶原活化系统相关,因此在细胞外蛋白水解、细胞迁移、炎症、纤维蛋白溶解、伤口愈合和肿瘤入侵的调控中发挥作用。
HMGB-1不具有信号序列,它的分泌过程不通过经典的″粗面内质网-高尔基体″途径。在活化的单核细胞中,HMGB-1经过一个从核内到分泌小泡的再分配过程,然后经细胞外分泌得以释放。
研究表明,用LPS刺激RAW264.7细胞,应用SDS-PAGE分析细胞培养液中成分变化,18个小时后,发现有分子量为30-KD的蛋白出现。根据该因子的N端序列分析,将其归属于HMG-1,一种DNA结合的非组蛋白。HMG-1后改名为HMGB-1。
小鼠实验证实,注射LPS、IL-1或TNF-8小时后,单核巨噬细胞开始分泌HMGB1,并在随后的24h中血清HMGB1浓度维持较高水平,HMGB1抗体可以改善LPS引起的内毒素血症;反过来,HMGB1也可刺激单核巨噬细胞分泌某些促炎因子,如TNF-、IL-1、II-6、IL-8等。注射HMGB1小鼠出现内毒素休克样症状。人体检测也发现,脓毒症患者血清HMGB1水平升高,并且升高的程度与感染严重性相关。
当细胞坏死或受损时,核内的HMGB1可释放到胞外,引发单核巨噬细胞分泌促炎因子;而促炎因子又反过来促进HMGB1的分泌,这样正反馈环就形成了。在炎性反应的后期,这种正反馈效应对炎性反应的维持起到了相当重要的作用。在近期研究中,发现HMGB1的功能与全身炎症反应综合症,急性肺损伤,类风湿性关节炎,糖尿病以及脏器缺血造成的梗塞密切相关。
因此,需要一种有效抑制HMGB1行使促炎症因子功能的物质,以治疗炎症相关疾病。
发明内容
本发明的一个目的是提供分泌特异性结合人高迁移率族蛋白B1的单克隆抗体。
本发明所提供的抗体可以为如下1)、2)、3)或4)所述的抗体:
1)一种抗体,其重链可变区具有序列表中序列1所示的氨基酸残基序列,其轻链可变区具有序列表中序列2所示的氨基酸残基序列。
2)一种抗体,其轻链具有序列表中序列3所示的氨基酸残基序列,重链具有序列表中序列4所示的氨基酸残基序列。此抗体为人-鼠嵌合抗体.
3)一种抗体,其氨基酸残基序列如序列表中序列7所示。此抗体为单链抗体。
4)由杂交瘤细胞株抗人HMGB1杂交瘤3E8 CGMCC No.2906分泌得到的单克隆抗体。
由杂交瘤细胞株抗人HMGB1杂交瘤3E8 CGMCC No.2906分泌得到的单克隆抗体的Fab片段也属于本发明的保护范围。
上述1)中所述抗体的重链可变区的编码基因和轻链可变区的编码基因也属于本发明的保护范围。
上述2)中所述抗体的重链和轻链的编码基因也属于本发明的保护范围。所述抗体的轻链编码基因具体具有序列表中序列5的核苷酸序列,重链编码基因具体具有序列表中序列6的核苷酸序列。
上述3)中所述抗体的编码基因也属于本发明的保护范围。
上述任一所述抗体在制备以人高迁移率族蛋白B1为靶点的药物中的应用也属于本发明的保护范围。
所述以人高迁移率族蛋白B1为靶点的药物可以为用于治疗由人高迁移率族蛋白B1参与的炎症反应的药物、用于治疗由人高迁移率族蛋白B1参与的自身免疫性疾病的药物、或用于治疗由人高迁移率族蛋白B1参与的器官损伤性疾病的药物。
所述炎症反应为败血症、全身炎症反应综合症或急性肺损伤;所述自身免疫性疾病为类风湿性关节炎、糖尿病或红斑狼疮;所述器官损伤性疾病为心肌梗塞、脑梗、药物性或病毒性肝损伤。
上述任一所述抗体在检验人高迁移率族蛋白B1中的应用。
本发明的另一个目的是提供一种分泌特异性结合人高迁移率族蛋白B1的单克隆抗体的杂交瘤细胞系。
该杂交瘤细胞系为鼠杂交瘤抗人HMGB1杂交瘤3E8,已于2009年2月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区大屯路,中国科学院微生物研究所,邮编100101),保藏号为CGMCC No.2906。
实验证明杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体、衍生于该单克隆抗体的单链抗体3 E8scFV、人-鼠嵌合抗体ch-3E8或Fab片段与人HMGB1的解离常数分别为1.3nM,70nM,1.3nM,10nM。杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体、衍生于该单克隆抗体的单链抗体3E8scFV、人-鼠嵌合抗体ch-3E8或Fab片段可以阻断人HMGB1刺激Raw264.7细胞产生的IL-6上调,在LPS诱导的小鼠败血症模型中具有保护作用。
上述单克隆抗体及其衍生物能够高亲和力特异性识别高迁移率族蛋白B1,并中和其生物学活性,可以用于治疗败血症,全身炎症反应综和症或急性肺损伤等炎症反应;可以用于治疗类风湿性关节炎,糖尿病或红斑狼疮等自身免疫性疾病。还可用于临床检验HMGB1的水平。
附图说明
图1为ELISA检测杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体与人HMGB1的解离曲线(纵坐标是ELISA显色后的光吸收值)。
图2为杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体在HMGB1上的结合位点鉴定。
图3为杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体特异性鉴定。
图4为杂交瘤细胞株3E8 C6MCC No.2906分泌的单克隆抗体抑制人HMGB1刺激Raw264.7细胞产生的IL-6mRNA上调。
图5为注射LPS和注射LPS和杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的小鼠的存活情况。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、鼠杂交瘤CGMCC No.2906及其分泌的抗高迁移率族蛋白B1(HMGB1)的单克隆抗体的制备和鉴定
1、抗原的制备:
人HMGB1与结核杆菌(Mycobacterium tuberculosis,MT)Psts-1蛋白氨基酸326-344小肽(DQVHFQPLPPAVVKLSDAL)的融合蛋白(HMGB1-MT)的获得:将编码人HMGB1N端181氨基酸的cDNA与小肽通过PCR方法重组,得到融合基因HMGB1-MT;将融合基因克隆至删除了GST编码区的PET-41a载体(Novagen,USA),克隆的质粒在DH5α里扩增,再经由BL21中表达,得到融合蛋白HMGB1-MT。融合蛋白中带有6×hisTag,通过Ni-NTA亲和层析纯化。
2、免疫:
以步骤1得到的融合蛋白作为抗原免疫NZB/W F1小鼠,共免疫三次:10微克HMGB1-MT加完全弗氏佐剂皮下免疫(第一次免疫),14天后5微克HMGB1-MT加不完全弗氏佐剂皮下免疫(第二次免疫)。14天后5微克HMGB1-MT加不完全弗氏佐剂皮下第三次免疫;3天后的NZB/W F1小鼠取脾中的免疫细胞与鼠骨髓瘤SP2/0细胞以5∶1比例混合,用聚乙二醇进行融合,HAT筛选后得到杂交瘤。
3、杂交瘤筛选
GST-HMGB1重组蛋白的制备:将人HMGB1的编码区(序列如序列表中序列11所示)克隆至PET-41a载体(Novagen,USA)的EcoR I和HindIII酶切位点,转化至DH5α,抗性筛选得到阳性克隆,提取阳性克隆的质粒,测序,结果质粒中HMGB1的序列及插入方向正确;将阳性质粒转入大肠杆菌BL21,1mM IPTG诱导表达;纯化:融合蛋白中带有GST Tag,通过谷胱甘肽亲和层析纯化,具体步骤是,将诱导后的菌液5000rpm10min离心收集,弃上清,用PBS将菌体重悬,超声破碎,5000rpm离心10min,弃沉淀,将上清缓慢通过谷胱甘肽层析柱(Sigma),用谷胱甘肽洗脱液将挂在谷胱甘肽层析柱上的融合蛋白洗脱下来,得到GST-HMGB1重组蛋白。
分泌特异性结合HMGB1抗体的杂交瘤检测:以GST-HMGB1重组蛋白(10微克/毫升)作为包被原包板,杂交瘤上清(进行梯度稀释)为一抗,HRP偶联的羊抗小鼠IgG多克隆抗体(R&D Systems)为二抗进行ELISA检测。
亚克隆:用限制性稀释法多次克隆分泌特异性抗体的杂交瘤细胞,最后获得杂交瘤细胞株鼠杂交瘤抗人HMGB1杂交瘤3E8。该细胞株已于2009年2月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区大屯路,中国科学院微生物研究所,邮编100101),保藏号为CGMCC No.2906。
4、单克隆抗体的制备
体外培养方法:
将已经建立的杂交瘤细胞CGMCC No.2906置于细胞培养基中,置于37℃和5%CO2孵箱中培养,每隔2d换一次细胞培养液,待细胞浓度大于105个/ml时停止换液,持续培养到细胞全部死亡。1500rpm,离心10分钟,收集培养上清,上清含有高水平的单克隆抗体,-20℃保存备用。
所述细胞培养基为向DMEM培养基中添加胎牛血清,使胎牛血清在细胞培养基中的终浓度为20%(体积百分含量),所述细胞培养基的pH为7.4。
纯化:将体外培养得到的上清进行如下纯化,将上述上清缓慢通过蛋白A/G亲和层析柱(Pierce),再用0.1M Glycine/HCl(pH2.5)将挂在层析柱上的目的蛋白洗脱下来,得到纯化后的蛋白。
5、杂交瘤CGMCC No.2906分泌的抗体的性能检测
(1)ELISA检测杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体与HMGB1的解离曲线。
实验方法:用HMGB1-MT(制备方法见步骤1)(2ug/ml)作为包被抗原包板,步骤4制备纯化得到的单抗作为一抗(以160ug/ml为起始浓度,二倍二倍进行稀释),羊抗小鼠IgG-HRP(R&D)(1∶2000)为二抗进行ELISA测定。
实验设3次重复,结果如图1所示,ELISA最大读数的50%相对应的抗体浓度为抗体的解离常数。表明杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体与HMGB1的解离常数为1.3nM。图1的横坐标是杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的浓度。
(2)抗体亚型鉴别实验:以GST-HMGB1重组蛋白(10微克/毫升)包板,步骤4制备纯化得到的单抗为一抗,HRP偶联的大鼠抗小鼠IgG1,IgG2a,或IgG2b单克隆抗体(BD Pharmingen)为二抗进行ELISA检测。实验证明杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体为IgG2b亚型。
(3)抗体结合位点鉴定:利用GST-A box、GST-B box和GST-A+B Box分别包板,实验方法用GST-A box、GST-B box和GST-A+B Box分别以2ug/ml作为包被抗原包板,步骤4制备的杂交瘤细胞CGMCC No.2906分泌的单抗作为一抗,羊抗小鼠IgG-HRP(R&D)(1∶2000)为二抗进行ELISA测定。
GST-A box的制备步骤:将人HMGB1 A box的编码区(序列如序列表中序列14所示)克隆至PET-41a载体(Novagen,USA)的BamHI和HindIII酶切位点,转化至DH5α,抗性筛选得到阳性克隆,提取阳性克隆的质粒,测序,结果质粒中HMGB1的序列及插入方向正确;将阳性质粒转入大肠杆菌BL21,1mM IPTG诱导表达;纯化:融合蛋白中带有GST Tag,通过谷胱甘肽亲和层析纯化,具体步骤是,将诱导后的菌液5000rpm 10min离心收集,弃上清,用PBS将菌体重悬,超声破碎,5000rpm离心10min,弃沉淀,将上清缓慢通过谷胱甘肽层析柱(Sigma),用谷胱甘肽洗脱液将挂在谷胱甘肽层析柱上的融合蛋白洗脱下来,得到GST-A box重组蛋白。
GST-B box的制备步骤:将人HMGB1 B box的编码区(序列如序列表中序列15所示)克隆至PET-41a载体(Novagen,USA)的BamHI和HindIII酶切位点,转化至DH5α,抗性筛选得到阳性克隆,提取阳性克隆的质粒,测序,结果质粒中HMGB1的序列及插入方向正确;将阳性质粒转入大肠杆菌BL21,1mM IPTG诱导表达;纯化:融合蛋白中带有GST Tag,通过谷胱甘肽亲和层析纯化,具体步骤是,将诱导后的菌液5000rpm 10min离心收集,弃上清,用PBS将菌体重悬,超声破碎,5000rpm离心10min,弃沉淀,将上清缓慢通过谷胱甘肽层析柱(Sigma),用谷胱甘肽洗脱液将挂在谷胱甘肽层析柱上的融合蛋白洗脱下来,得到GST-B box重组蛋白。
GST-A+B Box的制备步骤:将人HMGB1 A+Bbox的编码区(序列如序列表中序列11所示)克隆至PET-41a载体(Novagen,USA)的BamHI和HindIII酶切位点,转化至DH5α,抗性筛选得到阳性克隆,提取阳性克隆的质粒,测序,结果质粒中HMGB1的序列及插入方向正确;将阳性质粒转入大肠杆菌BL21,1mM IPTG诱导表达;纯化:融合蛋白中带有GST Tag,通过谷胱甘肽亲和层析纯化,具体步骤是,将诱导后的菌液5000rpm10min离心收集,弃上清,用PBS将菌体重悬,超声破碎,5000rpm离心10min,弃沉淀,将上清缓慢通过谷胱甘肽层析柱(Sigma),用谷胱甘肽洗脱液将挂在谷胱甘肽层析柱上的融合蛋白洗脱下来,得到GST-A+B box重组蛋白。
实验设3次重复,结果如图2所示,3E8只结合HMGB1中间的B Box。
(4)抗体特异性鉴定:用SDS-PAGE电泳分离人PBMC或Hela细胞的裂解液,以生物素标记的抗体进行western检测,Streptavidin-HRP为二抗,ECL显色。
人PBMC细胞,用Ficoll密度梯度离心法从人全血中分离得到(血液由北京市红十字血液中心提供);Hela细胞购自协和细胞中心,产品目录号为CCC0011。
Streptavidin-HRP购自中杉金桥,产品目录号为ZB-2404。
细胞裂解缓冲液:购自上海碧云天生物技术有限公司。
人PBMC或Hela细胞的裂解液的获得方法:人PBMC或Hela细胞用PBS洗2-3遍,离心收集细胞,按50万个细胞加100ul细胞裂解缓冲液液比例加入,同时加入PMSF,4度裂解30分钟后,12000rpm4度离心15分钟,取上清即可。
western检测方法:细胞裂解中获得的上清经10%SDS-PAGE电泳,转膜后用10%脱脂牛奶-TBS封闭(室温2小时),用TBS-T洗3遍,同3E8-biotin(1∶1000TBS稀释)室温反应1小时,再用用TBS-T洗3遍,与streptavidin-HRP(1∶8000)室温反应1小时,用TBS-T洗3遍后曝光。
实验设3次重复,结果如图3所示。(图3中CTL表示TBS)表明3E8抗体可以与细胞裂解液中内源性的HMGB1(大约30kd条带)特异性结合。
TBS配方:Tris-HCI缓冲液(0.5M pH7.6)100ml,NaCI 8.5~9g,双蒸水加至1000ml。
6、杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的生物活性鉴定
将Raw264.7细胞接种于细胞培养基中,向其中分别加入不同的刺激物,培养4小时;用real-time RT-PCR检测细胞中IL-6mRNA水平,所用引物序列为5’TGG GAAATC GTG GAA ATG AG 3’、5’CTC TGA AGG ACT CTG GCT TTG 3’。实验设3次重复。
细胞培养基组成:添加了10%胎牛血清的RPMI 1640培养基。
Raw264.7细胞购自协和细胞中心,产品目录号为CCC0146。
GST-HMGB1重组蛋白的制备方法:见步骤3。
各实验设如下处理:
处理1:不加任何刺激物;
处理2:只添加GST-HMGB1重组蛋白,其在培养体系中的终浓度为1ug/ml;
处理3:只添加3E8单抗(其在培养体系中的终浓度为50ug/ml);
处理4:添加GST-HMGB1重组蛋白(其在培养体系中的终浓度为1ug/ml)和步骤4制备纯化的单抗(其在培养体系中的终浓度为50ug/ml),具体方法是先将GST-HMGB1重组蛋白和步骤4制备纯化的单抗混合,再刺激Raw264.7细胞。
实验结果如图4所示(1表示处理1,2表示处理2,3表示处理3,4表示处理4),表明1ug/ml HMGB-1能显著上调IL-6mRNA的表达水平,50ug/ml的3E8抗体可以显著降低1ug/ml HMGB-1诱导的IL-6mRNA上调,中和HMGB-1活性。杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体可以阻断人HMGB1刺激Raw264.7细胞产生的IL-6mRNA上调。
7、抗HMGB1抗体在败血症小鼠模型中的应用
C57BL/6雌性小鼠购自北京维通利华实验动物技术有限公司。LPS购自Sigma,产品目录号为L2880。
8周龄C57BL/6雌性小鼠40只,重19.5g--20.5g,按体重匹配均分为5组:PBS组(LPS+PBS),对照抗体组(LPS+mIg)和三个抗体剂量组(LPS+3E8)。实验前在SPF级动物房中同笼48小时,自由进食及水。注射前2小时禁食。PBS组和抗体组小鼠依体重给予LPS(Sigma 0111:B4)22.5mg/kg。抗体组在给LPS后,给予杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体2.5mg/kg、1.25mg/kg、0.62mg/kg。两种药物均用无菌PBS稀释到终体积200ul,腹腔内注射。将各组分笼喂养,观察给药后情况。实验设3次重复。5组小鼠在注射后12小时后,观察无明显异常。但在20hr后,均出现相似的颤抖,体毛直立,对外界刺激不敏感表现。24小时后,LPS组开始有小鼠死亡;各组动物存活情况变化见图5。说明杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体在LPS诱导的小鼠败血症模型中具有保护作用。
实施例2、抗HMGB1人-鼠嵌合抗体ch-3E8的制备
鼠抗体在人体内会产生免疫排斥反应,因此只能用于急症的治疗,一旦人体内产生针对鼠抗体的抗抗体,作为药物的鼠抗体就会失效。为了克服这一缺点,本发明对鼠抗体进行了人源化,制备了人-鼠嵌合抗体ch-3E8。该抗体的可变区序列来自鼠抗体-杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体,不变区序列来自人IgG1。这种抗体保持了鼠单抗的抗原结合特异性,同时降低了在人体内诱导的免疫排斥反应。
制备嵌合抗体的步骤如下:
从杂交瘤细胞株3E8 CGMCC No.2906中分离纯化mRNA,用oligo-dT合成第一链cDNA。用PCR方法分别扩增抗体轻链和重链可变区,轻链引物为5’GAY ATT GTG MTSACM CAR WCT MCA 3’和5’CTC CAG ATG TTA ACT GCT CAC 3’;重链引物为:5’ATGSAR GTN MAG CTG SAG SAG TC 3’和5’GGT CAA GGT CAC TGG CTC AGG 3’。其中,R=G或A,Y=T或C,M=A或C,S=G或C,W=T或A,N=G或A或C或T。将PCR产物分别克隆到T载体中,测序。结果表明杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的重链可变区的编码基因具有序列表中序列8的核苷酸序列,编码的重链可变区的序列如序列表中序列1所示;杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的轻链可变区的编码基因具有序列表中序列9的核苷酸序列,编码的轻链可变区的序列如序列表中序列2所示。
人的轻链和重链不变区基因的获得方法:从人PBMC中分离纯化mRNA,用oligo-dT合成第一链cDNA,用PCR方法分别扩增人轻链和重链不变区基因。轻链引物为5’ttccat act cca gcg ctg cac cat ctg tct tca tct tcc cg3’和,5’cct cac tct agagtc gcg gcc gcc taa cac tct ccc ctg ttg aag ctc ttt g 3’,重链引物为5’gcgtcg acc aag ggc cca tcg gtc ttc c 3’和5’acc ctc act cta gag tcg cgg ccgctc att tac ccg gag aca ggg aga ggc t 3’。
单克隆抗体的轻链可变区基因与人的轻链不变区基因的连接:用PCR方法先将轻链可变区基因的3’端加上人轻链不变区基因5’端的一段序列,引物为5’gct gctgct gtg gtt ccc cgg ctc gcg atg cga tat tgt gat gac tca gg 3’和gac aga tggtgc agc cac agt ccg ttt tat ttc caa ctt tg,再以带有一段人轻链不变区基因的轻链可变区基因和人轻链不变区基因为模板进行PCR反应,引物为5’gct gct gct gtggtt ccc cgg ctc gcg atg cga tat tgt gat gac tca gg 3’和5’cct cac tct agagtc gcg gcc gcc taa cac tct ccc ctg ttg aag ctc ttt g 3’。因轻链可变区基因的3’端已带有人轻链不变区基因5’端的一段序列,因此在PCR的退火阶段,两段基因可自行连接。
单克隆抗体的重链可变区基因与人的重链不变区基因的连接:用PCR方法先将重链可变区基因的3’端加上人重链不变区基因5’端的一段序列,引物为5’gat ttcgcg att tta aaa ggt gtc cag tgc cag gtt cag ctg cag 3’和5’gat ggg ccc ttggtc gac gca cac cca ggg gcc agt gga t3’,再以带有一段人重链不变区基因的重链可变区基因和人重链不变区基因为模板进行PCR反应,引物为5’gat ttc gcg att ttaaaa ggt gtc cag tgc cag gtt cag ctg cag 3’和5’acc ctc act cta gag tcg cggccg ctc att tac ccg gag aca ggg aga ggc t 3’。因重链可变区基因的3’端已带有人重链不变区基因5’端的一段序列,因此在PCR的退火阶段,两段基因可自行连接。
将杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的轻链和重链可变区基因分别与人的轻链和重链不变区基因连接,构建可编码嵌合抗体ch-3E8的融合基因。该嵌合抗体的轻链的编码基因具有序列表中序列6的核苷酸序列,其编码序列为自序列5的5′端第1位-657位核苷酸,编码具有序列表中序列8的氨基酸残基序列的ch-3E8轻链;该嵌合抗体重链的编码基因具有序列表中序列6的核苷酸序列,其编码序列为自序列6的5′端第1位-1398位核苷酸,编码具有序列表中序列4的氨基酸残基序列的ch-3E8重链。
嵌合抗体轻链表达载体pCI-gpt-3E8L的构建:将该嵌合抗体轻链的编码基因(序列9所示)插入到有选择性标记(鸟嘌呤磷酸核糖转移酶,gpt)和基因表达调控区(CMV启动子,终止子)的表达载体pCI-gpt的Nru I和Afe I位点中,得到该嵌合抗体轻链表达载体pCI-gpt-3E8L。
表达载体pCI-gpt的构建方法(以promega载体pCI为模版构建):从Hela细胞(购自协和细胞中心,产品目录号为CCC0011)中分离纯化mRNA,用oligo-dT合成第一链cDNA。用PCR方法扩增出gpt(gpt基因序列见序列表中序列12),引物为5’ccgtcg cga agc gct atg agc gaa aaa tac atc gtc acc tgg gac3’和5’tta gcg accgga gat tgg cgg gac gaa tac 3’。将gpt插入pCI的HindIII和BamH I位点。
嵌合抗体重链表达载体pCI-DHFR-3E8H的构建:将该嵌合抗体重链的编码基因(序列8所示)插入到有选择性标记(二氢叶酸还原酶DHFR)和基因表达调控区(CMV启动子,终止子)的表达载体pCI-DHFR的Nru I和Not I位点,得到该嵌合抗体重链表达载体pCI-DHFR-3E8H。
表达载体pCI-DHFR的构建方法(以promega载体pCI为模版构建):从Hela细胞(购自协和细胞中心,产品目录号为CCC0011)中分离纯化mRNA,用oligo-dT合成第一链cDNA。用PCR方法扩增出DHFR(DHFR基因序列见序列表中序列12),引物为5’ccg gtc gcg agc ggc cgc atg gtt cga cca ttg aac tgc atc gtc gcc3’和5’aagttt gaa gtc tac gag aag aaa gac taa 3’。将DHFR插入pCI的HindIII和BamH I位点。
用电转染的方法将含有嵌合抗体基因的表达载体pCI-gpt-3E8L和pCI-DHFR-3E8H一同导入到哺乳动物细胞NS/0(ECACC购买)中。用霉酚酸酯(Mycophenolate)在含黄嘌呤(Xanthine)的培养基中筛选转化细胞,获得稳定转染的细胞株,记作NS/3E8。
细胞NS/3E8的培养方法:用含10%胎牛血清的RPMI 1640培养基培养。
按照实施例1的方法用ELISA鉴定抗体的分泌。
实验设3次重复。
结果显示得到的ch-3E8抗体保留了杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的特异性和亲和力,ch-3E8抗体与HMGB1的解离常数为1.3nM。
实施例3、抗HMGB1单链抗体3E8 scFV的制备
PCR方法分别扩增杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的轻链和重链可变区基因,然后用PCR方法将重链和轻链可变区用富含甘氨酸和丝氨酸的15氨基酸片段连接起来,获得抗HMGB1单链抗体3E8 scFV的编码基因序列(序列10)。
将3E8 scFV的编码基因序列克隆到表达载体pET-26b(Novagen,USA)的BamHI和Xho I位点,克隆的质粒在DH5α里扩增,再经由BL21中表达。表达条件为用1mMIPTG(购自北京好友新创科技发展有限公司)诱导2小时。融合蛋白中带有6×his Tag,通过Ni-NTA亲和层析纯化。表达的3E8 scFV具有序列表中序列7的氨基酸残基序列。
按照实施例1的方法用ELISA鉴定抗HMGB1单链抗体3E8scFV,实验设3次重复。结果显示抗HMGB1单链抗体3E8scFV保留了杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的特异性和亲和力,抗HMGB1单链抗体3B1scFV与HMGB1的解离常数为70nM。
实施例4、抗HMGB1的Fab片段3E8Fab的制备
利用ImmunoPureFab制备试剂盒(Pierce)中的固定化木瓜蛋白酶消化杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体,将全长抗体降解成为Fab和Fc片段。酶解后的产物用试剂盒中提供的固定化蛋白A柱纯化得到Fab的抗体片段。按照实施例1的方法用ELISA鉴定杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的Fab片段3E8Fab。实验设3次重复。结果显示杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的Fab片段3E8Fab保留了杂交瘤细胞株3E8 CGMCC No.2906分泌的单克隆抗体的特异性和亲和力,抗HMGB1单链抗体3E8Fab与HMGB1的解离常数为10nM。
序列表
<110>中国科学院生物物理研究所
<120>抗高迁移率族蛋白B1的单克隆抗体
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Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
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Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly
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Asn Gly Asn Ile Phe Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser
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Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile
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Arg
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Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly
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Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile
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Ser Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His
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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
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Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
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Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
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Gly Arg Ile Tyr Pro Gly Asp Gly Asp Met Ala Tyr Asn Gly Lys Phe
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Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
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Ala Arg Ser Leu Asp Gly Tyr Val Gly Tyr Val Met Asp Tyr Trp Gly
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Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
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Val Tyr Pro Leu Ala Pro Gly Cys Ala Ser Thr Lys Gly Pro Ser Val
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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
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Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
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Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
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gatattgtga tgactcaggc tgcaccctct gtacctgtca ctcctggaga gtcagtatcc 60
atctcctgca ggtctagtaa gagtctcctg catcgtaatg gcaacatttt tttgtattgg 120
ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 180
tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc 240
agtagagtag agactgagga tgtgggtgtt tattactgta tgcaacatct agaatatcct 300
ttcacgttcg gctcggggac aaagttggaa ataaaacgga ctgtggctgc accatctgtc 360
ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420
ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480
tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540
agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600
gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag 660
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caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcctc agtgaagatt 60
tcctgcaaag cttctggcta cgcattcagt aactactgga tgaactgggt gaagcagagg 120
cctggaaagg gtcttgagtg gatcggacgg atttatcctg gagatggaga tatggcctac 180
aatgggaagt tcaggggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct acttctgtgc aagatctttg 300
gatggttacg tcgggtatgt tatggactat tggggtcaag gaacctcagt caccgtctcc 360
tcagccaaaa caacaccccc atcagtctat ccactggccc ctgggtgtgc gtcgaccaag 420
ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 480
ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 540
gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 600
ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660
gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 720
aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 780
ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840
gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900
gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960
gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1020
aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1080
cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 1140
caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200
gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260
ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320
gtcttctcat gctccgtgat gcatgagggt ctgcacaacc actacacgca gaagagcctc 1380
tccctgtctc cgggtaaa 1398
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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr
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35 40 45
Gly Arg Ile Tyr Pro Gly Asp Gly Asp Met Ala Tyr Asn Gly Lys Phe
50 55 60
Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
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Ala Arg Ser Leu Asp Gly Tyr Val Gly Tyr Val Met Asp Tyr Trp Gly
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Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
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Val Tyr Pro Leu Ala Pro Gly Cys Gly Ser Gly Ser Ser Gly Ser Gly
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Ser Ser Gly Ser Gly Ser Ser Asp Ile Val Met Thr Gln Ala Ala Pro
145 150 155 160
Ser Val Pro Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser
165 170 175
Ser Lys Ser Leu Leu His Arg Asn Gly Asn Ile Phe Leu Tyr Trp Phe
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Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser
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Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
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Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu Thr Glu Asp Val Gly
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Gly Thr Lys Leu Glu Ile Lys Arg
260
<210>8
<211>408
<212>DNA
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<220>
<223>
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caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcctc agtgaagatt 60
tcctgcaaag cttctggcta cgcattcagt aactactgga tgaactgggt gaagcagagg 120
cctggaaagg gtcttgagtg gatcggacgg atttatcctg gagatggaga tatggcctac 180
aatgggaagt tcaggggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct acttctgtgc aagatctttg 300
gatggttacg tcgggtatgt tatggactat tggggtcaag gaacctcagt caccgtctcc 360
tcagccaaaa caacaccccc atcagtctat ccactggccc ctgggtgt 408
<210>9
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<212>DNA
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<223>
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gatattgtga tgactcaggc tgcaccctct gtacctgtca ctcctggaga gtcagtatcc 60
atctcctgca ggtctagtaa gagtctcctg catcgtaatg gcaacatttt tttgtattgg 120
ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 180
tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc 240
agtagagtag agactgagga tgtgggtgtt tattactgta tgcaacatct agaatatcct 300
ttcacgttcg gctcggggac aaagttggaa ataaaacgg 339
<210>10
<211>792
<212>DNA
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<223>
<400>10
caggttcagc tgcagcagtc tggacctgag ctggtgaagc ctggggcctc agtgaagatt 60
tcctgcaaag cttctggcta cgcattcagt aactactgga tgaactgggt gaagcagagg 120
cctggaaagg gtcttgagtg gatcggacgg atttatcctg gagatggaga tatggcctac 180
aatgggaagt tcaggggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct acttctgtgc aagatctttg 300
gatggttacg tcgggtatgt tatggactat tggggtcaag gaacctcagt caccgtctcc 360
tcagccaaaa caacaccccc atcagtctat ccactggccc ctgggtgtgg ctctggctct 420
tctggctctg gctcttctgg ctctggctct tctgatattg tgatgactca ggctgcaccc 480
tctgtacctg tcactcctgg agagtcagta tccatctcct gcaggtctag taagagtctc 540
ctgcatcgta atggcaacat ttttttgtat tggttcctgc agaggccagg ccagtctcct 600
cagctcctga tatatcggat gtccaacctt gcctcaggag tcccagacag gttcagtggc 660
agtgggtcag gaactgcttt cacactgaga atcagtagag tagagactga ggatgtgggt 720
gtttattact gtatgcaaca tctagaatat cctttcacgt tcggctcggg gacaaagttg 780
gaaataaaac gg 792
<210>11
<211>510
<212>DNA
<213>人工序列
<220>
<223>
<400>11
atgggcaaag gagatcctaa gatgggcaaa ggagatccta agaagccgag aggcaaaatg 60
tcatcatatg cattttttgt gcaaacttgt cgggaggagc ataagaagaa gcacccagat 120
gcttcagtca acttctcaga gttttctaag aagtgctcag agaggtggaa gaccatgtct 180
gctaaagaga aaggaaaatt tgaagatatg gcaaaagcgg acaaggcccg ttatgaaaga 240
gaaatgaaaa cctatatccc tcccaaaggg gagacaaaaa agaagttcaa ggatcccaat 300
gcacccaaga ggcctccttc ggccttcttc ctcttctgct ctgagtatcg cccaaaaatc 360
aaaggagaac atcctggcct gtccattggt gatgttgcga agaaactggg agagatgtgg 420
aataacactg ctgcagatga caagcagcct tatgaaaaga aggctgcgaa gctgaaggaa 480
aaatacgaaa aggatattgc tgcatatcga 510
<210>12
<211>459
<212>DNA
<213>人工序列
<220>
<223>
<400>12
atgagcgaaa aatacatcgt cacctgggac atgttgcaga tccatgcacg taaactcgca 60
agccgactga tgccttctga acaatggaaa ggcattattg ccgtaagccg tggcggtctg 120
gtaccgggtg cgttactggc gcgtgaactg ggtattcgtc atgtcgatac cgtttgtatt 180
tccagctacg atcacgacaa ccagcgcgag cttaaagtgc tgaaacgcgc agaaggcgat 240
ggcgaaggct tcatcgttat tgatgacctg gtggataccg gtggtactgc ggttgcgatt 300
cgtgaaatgt atccaaaagc gcactttgtc accatcttcg caaaaccggc tggtcgtccg 360
ctggttgatg actatgttgt tgatatcccg caagatacct ggattgaaca gccgtgggat 420
atgggcgtcg tattcgtccc gccaatctcc ggtcgctaa 459
<210>13
<211>564
<212>DNA
<213>人工序列
<220>
<223>
<400>13
atggttcgac cattgaactg catcgtcgcc gtgtcccaaa atatggggat tggcaagaac 60
ggagaccgac cctggcctcc gctcaggaac gagttcaagt acttccaaag aatgaccaca 120
acctcttcag tggaaggtaa acagaatctg gtgattatgg gtaggaaaac ctggttctcc 180
attcctgaga agaatcgacc tttaaaggac agaattaata tagttctcag tagagaactc 240
aaagaaccac cacgaggagc tcattttctt gccaaaagtt tggatgatgc cttaagactt 300
attgaacaac cggaattggc aagtaaagta gacatggttt ggatagtcgg aggcagttct 360
gtttaccagg aagccatgaa tcaaccaggc cacctcagac tctttgtgac aaggatcatg 420
caggaatttg aaagtgacac gtttttccca gaaattgatt tggggaaata taaacttctc 480
ccagaatacc caggcgtcct ctctgaggtc caggaggaaa aaggcatcaa gtataagttt 540
gaagtctacg agaagaaaga ctaa 564
<210>14
<211>237
<212>DNA
<213>人工序列
<220>
<223>
<400>14
atgggcaaag gagatcctaa gaagccgaga ggcaaaatgt catcatatgc attttttgtg 60
caaacttgtc gggaggagca taagaagaag cacccagatg cttcagtcaa cttctcagag 120
ttttctaaga agtgctcaga gaggtggaag accatgtctg ctaaagagaa aggaaaattt 180
gaagatatgg caaaagcgga caaggcccgt tatgaaagag aaatgaaaac ctatatc 237
<210>15
<211>207
<212>DNA
<213>人工序列
<220>
<223>
<400>15
cccaagaggc ctccttcggc cttcttcctc ttctgctctg agtatcgccc aaaaatcaaa 60
ggagaacatc ctggcctgtc cattggtgat gttgcgaaga aactgggaga gatgtggaat 120
aacactgctg cagatgacaa gcagccttat gaaaagaagg ctgcgaagct gaaggaaaaa 180
tacgaaaagg atattgctgc atatcga 207
Claims (11)
1.由杂交瘤细胞株鼠杂交瘤CGMCC No.2906分泌产生的单克隆抗体。
2.杂交瘤细胞株鼠杂交瘤,其保藏编号为CGMCC No.2906。
3.一种抗体,其重链可变区的氨基酸序列为序列表中序列1所示的氨基酸残基序列,其轻链可变区的氨基酸序列为序列表中序列2所示的氨基酸残基序列。
4.一种抗体,其轻链的氨基酸序列为序列表中序列3所示的氨基酸残基序列,重链的氨基酸序列为序列表中序列4所示的氨基酸残基序列。
5.一种抗体,其氨基酸残基序列如序列表中序列7所示。
6.由杂交瘤细胞株鼠杂交瘤CGMCC No.2906分泌产生的单克隆抗体的Fab片段。
7.权利要求3中所述重链可变区的编码基因和所述轻链可变区的编码基因。
8.权利要求4中所述重链和轻链的编码基因。
9.权利要求5所述抗体的编码基因。
10.权利要求1、3或4所述的抗体、或权利要求5所述的抗体在制备以人高迁移率族蛋白B1为靶点的药物中的应用。
11.权利要求1、3或4所述的抗体、或权利要求5所述的抗体在制备预防和/或治疗LPS诱导的小鼠败血症药物中的应用。
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