CN101880324B - 一种抗人SIRPα的单克隆抗体及其细胞株、制备方法和应用 - Google Patents
一种抗人SIRPα的单克隆抗体及其细胞株、制备方法和应用 Download PDFInfo
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Abstract
本发明属于医学生物工程技术领域,具体涉及一种抗人信号调节蛋白α(SIRPα)的单克隆抗体及其制备与应用。信号调节蛋白α(SIRPα)是属于免疫球蛋白超家族的成员(IgSF),组织分布主要在髓系(巨噬细胞,树突状细胞等),因此SIRPα在机体免疫调节中的作用已被日益重视。本发明提供了一种抗人SIRPα的单克隆抗体,是由保藏号为CGMCC No.3801的杂交瘤细胞株产生的。本发明还提供了该抗人SIRPα的单克隆抗体在制备检测SIRPα的试剂中的应用,以及制备刺激人巨噬细胞TNF-α表达药物中的应用。
Description
技术领域
本发明属于医学生物工程技术领域。具体地说,本发明涉及一种抗人信号调节蛋白α(SIRPα)的单克隆抗体、分泌该单克隆抗体的细胞株及该单克隆抗体的制备方法和应用。
背景技术
信号调节蛋白α(SIRPα)是属于免疫球蛋白超家族的成员(IgSF)。在1996年的MOLECULAR AND CELLULAR BIOLOGY杂志和1997年发表的NATURE杂志中,大鼠的蛋白酪氨酸磷酸酶SIRPα首先被鉴定。SIRPα也叫SHPS1、CD172α、P84、MYD1、BIT、PTPNS1等,是一种主要表达于髓系细胞的跨膜蛋白,GeneID:140885。SIRPα胞外区含有三个免疫球蛋白超家族(IgSF)结构域和多个糖基化位点。其胞浆区在大鼠,小鼠和人之间高度保守,带有两个免疫受体酪氨酸抑制基元(ITIMs,其特征氨基酸序列为“I/VxYxxL”),其中包含四个酪氨酸残基。由于其特殊的细胞定位和结构,决定了它在不同细胞中传递不同的信号,发挥不同的功能。
SIRPα在细胞内传递信号主要通过胞内酪氨酸残基的磷酸化。其胞外区的三个Ig样结构域SIRPα可被多种有丝分裂原活化而发生磷酸化,如血清、胰岛素、生长因子、EGF、PDGF以及神经营养因子等。SIRPα被磷酸化后能结合带有SH2结构域的蛋白磷酸酶SHP-1和SHP-2使之活化,并作为其底物被去磷酸化。SIRPα的胞外区与特异性配体相结合,也是其实现信号转导功能的重要途径。目前对SIRPα的特异性配体研究比较多的是另一种膜蛋白CD47。与SIRPα的严格组织细胞表达不同,CD47在大部分细胞类型都有表达,因此SIRPα与CD47复合体在SIRPα介导的信号转导中起着重要作用,包括介导细胞吞噬、迁移和细胞因子分泌等。
由于SIRPα的组织分布主要在髓系(巨噬细胞,树突状细胞等),因此SIRPα在机体免疫调节中的作用已被日益重视。总的来说,在免疫系统中SIRPα通过与CD47相互作用发挥负向调节作用。在天然免疫中,LPS可通过转录抑制和蛋白降解途径诱导巨噬细胞中SIRPα表达下调,SIRPα通过屏蔽SHP-2作用抑制MAPK、IKKs、NF-κB和IRF3激活,减少炎性因子IL-6、TNFα和IFNβ释放。但用SIRPα抗体与SIRPα结合,能促进巨噬细胞分泌一氧化氮,这从另一方面说明了SIRPα有可能在天然免疫中起到一定的正向调节作用。在SIRPα与获得性免疫关系的研究中,利用CD47-Fc段融合蛋白作为配体与DC表面SIRPα相互作用,能观察到DC表型和功能的抑制,包括DC成熟标志减少,细胞因子IL-12分泌减少。DC表面的SIRPα与T细胞表面的CD47相互结合,双向负调节DC和T细胞功能:一方面,抑制DC活化,下调其抗原递呈能力;另一方面,抑制T细胞增殖和杀伤功能。但是也有研究用抗-SIRPα抗体刺激SIRPα的胞外区可以有效降低静息性记忆T细胞的增殖。研究人员还证实了SIRPα突变体小鼠对实验诱导的自身免疫性脑脊髓炎、胶原性关节炎和2,4二硝基-1-氟苯诱导的接触过敏有很强的抵抗能力;同样,CD47缺失小鼠也能抑制包括胶原性关节炎和接触性过敏在内的自身免疫性疾病,这说明了SIRPα在一定程度上促进了自身免疫性疾病的产生。SIRPα的这一功能主要与SIRPα抑制Th17细胞分泌IL-17相关,Th17是机体一类重要的调节性T细胞,是免疫系统反应过激的抑制因素,在SIRPα突变体小鼠诱导自身免疫性疾病模型中,可以检测到IL-17表达上调。除了对免疫细胞功能的调节外,SIRPα对免疫细胞的发育也起了至关重要的作用。在小鼠脾细胞中,SIRPα在CD8a+CD11c+DCs细胞膜上表达大大强于CD8a-CD11c+DCs,当转基因鼠表达突变SIRPα时,脾细胞中CD8a+CD11c+DCs比例明显减少。
检测SIRPα在临床实践中也有现实意义。SIRPα在某些白血病细胞中下调,检测SIRPα的表达可以帮助确定其亚型;检测SIRPα的表达可以帮助鉴定DC亚群;检测SIRPα的表达也可以确定巨噬细胞的活化状态。而现有的SIRPα抗体没有很好的能同时进行免疫印迹、流式检测和免疫组化检测的。并且现有的功能性SIRPα抗体多为封闭型或抑制型,而没有激活型的。
发明内容
本发明所要解决的技术问题之一是:提供抗人信号调节蛋白α(SIRPα)的单克隆抗体。
本发明所要解决的技术问题之二是:提供产生抗人信号调节蛋白α(SIRPα)的单克隆抗体的杂交瘤细胞株。
本发明所要解决的技术问题之三是:提供抗人信号调节蛋白α(SIRPα)的单克隆抗体的制备方法。
本发明所要的解决的技术问题之四是:提供抗人信号调节蛋白α(SIRPα)的单克隆抗体在检测SIRPα中的应用。
本发明还要的解决的技术问题之五是:提供抗人信号调节蛋白α(SIRPα)的单克隆抗体在制备刺激人巨噬细胞TNF-α等表达药物中的应用。
本发明的具体技术方案如下:
a)合成作为半抗原的多肽,其序列为:RVTTVSESTKRENMDFSISISC:
b)将上述多肽与匙孔嘁血蓝蛋白(KLH)偶联,作为免疫原免疫小鼠;
c)取免疫鼠的脾细胞,与小鼠骨髓瘤细胞融合;
d)经多轮筛选出与合成多肽反应阳性的细胞克隆,作为抗人SIRPα蛋白单克隆抗体的杂交瘤细胞株,该杂交瘤细胞株已经于2010年4月12日提交位于中国北京的中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)保藏,保藏号为:CGMCC No.3801;
e)通过在小鼠体内接种杂交瘤细胞生产腹水抗体,将腹水进行纯化,得到抗人SIRPα的单克隆抗体;或者通过体外细胞培养,分离纯化,得到抗人SIRPα的单克隆抗体。
本发明提供了一种抗人SIRPα的单克隆抗体,该单克隆抗体由保藏号为保藏号为:CGMCC No.3801的杂交瘤细胞株产生,其识别位于SIRPα氨基酸序列RVTTVSESTKRENMDFSISIS的抗原表位。
本发明还提供了一种杂交瘤细胞株,其保藏号为:CGMCC No.3801。
本发明还提供了一种抗人SIRPα的单克隆抗体的制备方法,包括体外培养保藏号为CGMCC No.3801的杂交瘤细胞株,然后从培养液中回收抗人SIRPα的单克隆抗体。
在本发明中,多肽可用多肽合成仪合成。多肽片段可用戊二醛连接法等与KLH等载体蛋白偶联,作为免疫原免疫小鼠,并用于单克隆抗体筛选。
用于分泌抗人SIRPα的单克隆抗体的杂交瘤细胞株可采用本技术领域常规的方法制备。比如,取经免疫的小鼠或大鼠的脾细胞,与小鼠或大鼠的骨髓瘤细胞如SP2/0细胞进行融合,经筛选后即可得到杂交瘤细胞。
单克隆抗体的获得可采用本技术领域常规的方法。一种方法是,通过在哺乳动物比如小鼠体内接种杂交瘤细胞产生腹水抗体,取出腹水进行纯化而得到。另一种方法是,通过体外培养杂交瘤细胞而获得。
本发明的杂交瘤细胞株,也称杂交瘤细胞系92CT57.39.3,所制备的单克隆抗体是一种免疫球蛋白,其可特异性结合于人SIRPα蛋白。经鉴定,所制备的免疫球蛋白是IgG型的单克隆抗体。
本发明还提供了上述抗人SIRPα的单克隆抗体在制备检测SIRPα的试剂中的应用。
上述抗人SIRPα的单克隆抗体在在制备检测SIRPα的试剂中的应用,其中的检测是指流式细胞术、Western Blot和免疫组化检测。
本发明还提供了上述抗人SIRPα的单克隆抗体在制备刺激人巨噬细胞TNF-α表达药物中的应用。
本发明为SIRPα的临床治疗应用提供了新的思路。
应当明确,在本发明中单克隆抗体和单抗系同义语,可以互换使用。本发明的抗人SIRPα的单克隆抗体有时也以单克隆抗体92CT57.39.3表示。
附图说明
图1是单克隆抗体92CT57.39.3流式细胞术检测人SIRPα蛋白的结果。
A为同型对照抗体0.4%;B为SIRPα单克隆抗体92.8%。
图2是单克隆抗体92CT57.39.3 Western Blot检测人SIRPα蛋白的结果。
A为细胞系Huh-7/hSIRPα-4Y裂解液;B为细胞系Jurkat裂解液。
图3是单克隆抗体92CT57.39.3免疫组化检测人SIRPα蛋白的结果。
A为肝癌组织(100X);B为肝癌组织(200X);
C为癌旁肝组织(100X);C为癌旁肝组织(200X)。
具体实施方式
下面结合附图及实施例对本发明进行详细描述,但本发明的实施不仅限于此。
实施例1:抗人SIRPα的单克隆抗体的制备和纯化
1.抗原的合成和偶联
抗原多肽设计参考GenBank,GeneID:140885,具体序列为:RVTTVSESTKRENMDFSISISC(SEQ ID NO:1),由百奇生物科技(上海)有限公司通过多肽自动合成仪用固相多肽合成法合成。
多肽与匙孔嘁血蓝蛋白(KLH)的偶联采用《分子克隆实验指导》第2版,萨姆布鲁克著,金冬雁译,科学出版社,北京,1999,第856页描述的戊二醛连接法。
2.单克隆抗体92CT57.39.3的制备和纯化
将上述1中纯化的多肽KLH偶联物100μg以PBS稀释后与等体积完全弗氏佐剂(CFA)混匀乳化,免疫5-6周龄的雌性BALB/c小鼠5只,双肩周围的皮肤下进行皮下注射和双后腿进行肌肉注射,每个区域大约用1/8的免疫原,将剩余1/2的免疫原进行腹腔注射。1周后加强免疫50μg,完全弗氏佐剂,腹腔注射。2周后加强免疫50μg,不完全弗氏佐剂(IFA),腹腔注射。3周及以后,每周直接腹腔注射50μg加强免疫。第5周起,每周尾静脉采血,ELISA测定效价,应用多肽KLH偶联物抗原1.25μg/ml包被,10%FCS封闭,将血清稀释后加入,应用羊抗小鼠IgG标记HRP作为二抗,OPD显色,使用酶标仪(Bio-Rad 550型)在492nm读数。
第3次采血的ELISA结果OD值均大于0.75。取转染了人SIRPα蛋白的稳定细胞系Huh-7/hSIRPα-4Y 1×106,重悬于100μl含1%BSA的PBS中,加入1μl血清4℃作用30分钟,PBS洗涤2次,重悬于100μl含1%BSA的PBS中,加入1μg PE标记的二抗4℃作用20分钟,PBS洗涤2次,重悬于500μlPBS中,用Beckman Coulter MoFloTM XDP流式细胞仪检测。
在免疫小鼠的同时准备小鼠骨髓瘤细胞SP2/0细胞(购自ATCC)。最后一次加强免疫3天后,取流式检测效果最好的小鼠的脾细胞,与SP2/0细胞比例5∶1,在PEG1500的作用下进行融合反应,植入96孔板,37℃、5%CO2条件下培养。培养14天后,镜下检测生长出克隆细孔为融合阳性孔,计算总融合率>95%。尽量选择单克隆细胞孔的上清进行ELISA检测。ELISA阳性的克隆用流式细胞术检测Huh-7/hSIRPα-4Y细胞,筛选出来的克隆亚克隆两次,每次流式检测效果最好的克隆。筛选得到1个克隆92CT57.39.3。
6-8周龄的雌性BALB/c小鼠用石蜡油腹腔注射10天后,取杂交瘤细胞以2×106个细胞/只进行腹腔注射。7-14天后从小鼠腹腔抽出富含抗体的腹水进行检测和纯化。纯化采用Protein G亲和层析法。Protein G亲和层析柱用PBS平衡柱子后取腹水过柱,然后用PBS洗柱至OD值接近零,以50nmol/L的甘氨酸盐酸溶液洗脱,收集洗脱液,测定各收集管的OD值,保留峰值区的洗脱液,透析纯化。
实施例2:抗人SIRPα的单克隆抗体的鉴定和检测应用
1.单克隆抗体的类型鉴定
利用小鼠单克隆抗体的Ig类与亚类特异性抗体进行ELISA检测,鉴定结果见表1。结果表明单克隆抗体的重链为IgG1型,轻链为κ链。
表1:单克隆抗体92CT57.39.3的Ig类与亚类鉴定结果
92CT57.39.3 | 阴性对照 | 阳性对照 | |
IgG1 | 3.4102 | 0.0809 | 3.1634 |
IgG2a | 0.0716 | 0.0581 | 3.186 |
IgG2b | 0.0865 | 0.0611 | 3.1889 |
IgG3 | 0.1084 | 0.1021 | 3.3212 |
IgM | 0.0659 | 0.0621 | 3.3335 |
IgA | 0.0631 | 0.0568 | 3.4426 |
Igκ | 4 | 0.0611 | 3.4046 |
Igλ | 0.0625 | 0.0536 | 2.9594 |
2.单克隆抗体的流式检测应用
1×106Huh-7/hSIRPα-4Y细胞,重悬于100μl含1%BSA的PBS中,加入1μg抗体4℃作用30分钟,PBS洗涤2次,重悬于100μl含1%BSA的PBS中,加入1μg PE标记的二抗4℃作用20分钟,PBS洗涤2次,重悬于500μlPBS中,用Beckman Coulter MoFloTM XDP流式细胞仪检测,检测结果见图1。结果表明,相较于对照同型抗体,该单克隆抗体能特异性地结合于Huh-7/hSIRPα-4Y细胞。
3.单克隆抗体的Western Blot检测应用
变性的蛋白样品进行SDS-PAGE蛋白电泳;通过电转仪将蛋白转移至硝酸纤维素膜(Schleicher&Schcell公司);含5%BSA的TBST封闭1h,TBST洗一次,5min;含4%BSA的TBST稀释抗SIRPα单克隆抗体92CT57.39.3一抗(1μg/ml),孵育2h,TBST洗三次,每次5min;含4%BSA的TBST稀释IR-800标记的抗小鼠二抗,孵育1h,TBST洗四次,每次5min;用红外线激光扫描仪Odyssey扫描。结果见图2。结果表明,该单克隆抗体能较为特异地识别人SIRPα分子。
4.单克隆抗体的免疫组化检测应用
切片置于60℃恒温箱中烘烤20分钟并脱蜡至水;3%H2O2(80%甲醇)室温10分钟灭活内源性过氧化物酶,PBS洗3次各5分钟;0.01M枸橼酸钠缓冲溶液(pH6.0)高压修复抗原,PBS洗3次各5分钟;山羊血清封闭,室温20分钟,甩去多余液体;滴加以抗体稀释液稀释的抗SIRPα单克隆抗体92CT57.39.3一抗(10μg/ml),室温孵育2h,PBS洗3次各5分钟;滴加HRP标记的二抗50μl,室温孵育1h,PBS洗3次各5分钟;DAB显色5-10分钟,在显微镜下掌握染色程度,PBS洗3次各5分钟;苏木精复染2分钟,盐酸酒精分化,自来水冲洗10-15分钟;脱水、透明、封片、拍照。结果见图3。结果表明,该单克隆抗体能较为特异地识别人SIRPα分子。
实施例3:抗人SIRPα的单克隆抗体的应用
人单核细胞系THP-1细胞4×105,培养基中加入100ng/ml的豆蔻佛波醇乙酯(PMA),24小时后换成普通培养基,继续培养48小时,重新换成普通培养基,加入相应抗体至20μg/ml,6小时后收集上清,测定其中TNF-α的浓度(使用达科为,DKW12-1720试剂盒)。结果见表2。结果表明,该抗体能刺激THP-1细胞分泌TNF-α达对照的3倍左右。
表2:单克隆抗体92CT57.39.3体刺激THP-1细胞后上清中TNFα的浓度测定结果
TNF-α(pg/ml) | |
THP-1上清 | 1.25 |
THP-1上清+IgG1,κ同型对照抗体(20μg/ml,BioLegend,400123) | 1.19 |
THP-1上清+SIRPα单克隆抗体(20μg/ml) | 3.67 |
SEQUENCE LISTING
<110>中国人民解放军第二军医大学
<120>一种抗人SIRPα的单克隆抗体及其细胞株、制备方法和应用
<130>说明书,权利要求书
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<170>PatentIn version 3.1
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Arg Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe
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Claims (6)
1.一种抗人SIRPα的单克隆抗体,其特征在于该单克隆抗体由保藏号为:CGMCC No.3801的杂交瘤细胞株产生,其识别位于SIRPα氨基酸序列如SEQID NO:1所示的抗原表位。
2.一种杂交瘤细胞株,其保藏号为:CGMCC No.3801。
3.根据权利要求1所述的抗人SIRPα的单克隆抗体的制备方法,其特征在于该方法包括体外培养保藏号为CGMCC No.3801的杂交瘤细胞株,然后从培养液中回收抗人SIRPα的单克隆抗体。
4.根据权利要求1所述的抗人SIRPα的单克隆抗体在制备检测SIRPα的试剂中的应用。
5.根据权利要求4所述的抗人SIRPα的单克隆抗体在制备检测SIRPα的试剂中的应用,其中的检测是指流式细胞术、Western Blot或免疫组化检测。
6.根据权利要求1所述的抗人SIRPα的单克隆抗体在制备刺激人巨噬细胞TNF-α表达药物中的应用。
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CN114040925B (zh) * | 2019-12-24 | 2023-04-28 | 礼新医药科技(上海)有限公司 | 抗SIRPα单克隆抗体及其用途 |
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