CN116284418A - 抗基质金属蛋白酶3的单克隆抗体及其制备和应用 - Google Patents
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Abstract
本发明公开了抗基质金属蛋白酶3的单克隆抗体及其制备和应用,所述单克隆抗体为兔单克隆抗体A或兔单克隆抗体B,兔单克隆抗体A的重链CDR区氨基酸序列如SEQ ID NO.1~3所示且其轻链CDR区氨基酸序列如SEQ ID NO.4~6所示,兔单克隆抗体B的重链CDR区氨基酸序列如SEQ ID NO.7~9所示且其轻链CDR区氨基酸序列如SEQ ID NO.10~12所示;兔单克隆抗体A和兔单克隆抗体B可以用于开发双抗夹心法酶联免疫检测试剂盒,具有检测灵敏度高、稳定性好等优点;而且本发明通过单B细胞克隆技术生产兔单克隆抗体A和兔单克隆抗体B,成本更为低廉,生产更易放大,具有更高的推广价值。
Description
技术领域
本发明属于单克隆抗体技术领域,具体涉及一种抗基质金属蛋白酶3的单克隆抗体及其制备方法和应用。
背景技术
基质金属蛋白酶(matrix metalloproteinases,MMPs)是一个大家族,因其需要Ca2+、Zn2+等金属离子作为辅助因子而得名。MMPs几乎能降解细胞外基质中的各种蛋白成分,破坏肿瘤细胞侵袭的组织学屏障,在肿瘤侵袭转移中起关键性作用,被认为是肿瘤浸润转移中主要的蛋白水解酶。MMPs家族目前已分离鉴别出26个成员,编号分别为MMP-1~26。
基质金属蛋白酶3(MMP-3)是MMPs家族一员,其能够降解聚集的软骨连结蛋白纤维结合素、核心蛋白及Ⅳ、Ⅶ、Ⅸ型的胶原的显著活性,能导致软骨的降解。强直性脊柱炎患者的血清MMP-3水平显著高于正常人群,能有效反映出患者的疾病活动及骨关节破坏程度,能为临床判断、病情进展及疗效提供科学依据。MMP-3还是类风湿关节炎新的诊断指标,冠心病新的预测因子。因此,开发MMP-3单克隆抗体用于诊断试剂中,对上述疾病的临床诊断具有重要意义。
单克隆抗体具有靶点性强、成药性强、副作用小等优点。目前诊断试剂盒所用单克隆抗体大多数通过杂交瘤细胞分泌产生,是将B淋巴细胞与骨髓瘤细胞进行融合,再采用有限稀释法经过多轮筛选从而获得能产生抗原特异性抗体的杂交瘤细胞。但从脾细胞分离的B细胞并非都能有效的用于融合,有效融合比例仅为1:105~106,而融合后的杂交瘤细胞也仅有极少部分可分泌抗原特异性抗体。而且小鼠杂交瘤技术免疫原性高、半衰期短,临床效果有限;人杂交瘤技术则具有融合效率低、亚克隆易丢失等缺点。另外,还可以采用噬菌体文库技术、EBV转化B淋巴细胞技术等方法获得单克隆抗体,但是噬菌体文库展示技术在建库过程中易出现轻重链随机配对的情况,且研发周期长,EBV转化B细胞技术的抗体产量较低。而单B细胞抗体克隆表达技术可有效解决上述问题,可实现抗原特异性小鼠单克隆抗体的快速克隆和筛选鉴定分析。
发明内容
有鉴于此,本发明通过利用单B细胞抗体克隆技术开发出了高亲和力的临床诊断可用的抗MMP-3兔单克隆抗体,而且生产放大方便,批间差可控,成本更为低廉,更好满足诊断试剂对抗体稳定性和重复性的要求。
为了实现上述目的,本发明的技术方案具体如下:
本发明第一方面提供了一种抗基质金属蛋白酶3的单克隆抗体,具体为兔单克隆抗体A或兔单克隆抗体B;
其中,兔单克隆抗体A重链CDR区氨基酸序列如下所示:
CDR-H1:SYDMS;
CDR-H2:SIDTVGSAYYASWAKG;
CDR-H3:SFYMPAFDP;
兔单克隆抗体A轻链CDR区氨基酸序列如下所示:
CDR-L1:QASESVYSNNRLA;
CDR-L2:LASTLAS;
CDR-L3:AGYKIITDGIS;
兔单克隆抗体B重链CDR区氨基酸序列如下所示:
CDR-H1:NYCCGC;
CDR-H2:CIYGSSDNTYYATWAKG;
CDR-H3:GFGDDYVFNL;
兔单克隆抗体B轻链CDR区氨基酸序列如下所示:
CDR-L1:QASQSISSYLA;
CDR-L2:RASTLAS;
CDR-L3:QQHNMISNIDNF。
本发明第二方面提供了如上述的单克隆抗体在建立非诊断目的的MMP-3的酶联免疫检测方法中的应用。
进一步地,所述酶联免疫检测方法为利用双抗夹心原理的酶联免疫检测方法。更进一步地,在该方法中,所述兔单克隆抗体A为包被抗体,所述兔单克隆抗体B为标记抗体。
本发明第三方面提供了如上述的单克隆抗体在制备用于检测MMP-3的试剂或试剂盒中的应用。
进一步地,所述MMP-3包括重组表达的MMP-3,或血清中的MMP-3。
本发明第三方面提供了如上述的单克隆抗体的制备方法,具体采用单B细胞抗体克隆技术制备,包括以下步骤:
S1、使用MMP-3重组蛋白免疫兔,免疫结束后收集脾脏并分离脾细胞;
S2、筛选特异性B淋巴细胞,并提取其总RNA,合成cDNA,再利用巢式PCR获得抗体可变区及恒定区序列;
S3、将扩增的抗体基因插入载体中,转染293F细胞;
S4、收集培养上清并纯化,即得单克隆抗体。
进一步地,步骤S1所述MMP-3重组蛋白的氨基酸序列如SEQ ID NO.13所示。
进一步地,步骤S2所述巢式PCR所用的引物序列如SEQ ID NO.14~28所示。
进一步地,步骤S4所述纯化具体为:将上清与PB缓冲液混合后上样protein G亲和层析柱,将含有所述抗体的洗脱液超滤浓缩后置于PBS中透析。
本发明的有益效果为:
(1)本发明开发了能够用于免疫诊断试剂中的抗MMP-3兔单克隆抗体对,其制备成的双抗夹心法酶联免疫检测试剂能实现临床血清中MMP-3的精确检测,打破了市面上仅有鼠单抗及羊多抗用于试剂盒的现状。
(2)本发明利用单B细胞技术的重组抗体,不仅生产放大方便,批间差可控,而且成本更低廉;经验证,相对于外购试剂盒或外购抗体对,本发明提供的抗体对能更好的满足诊断试剂对抗体亲和力,特异性,均一性等方面的要求,具有很高的操作性和推广价值。
附图说明
图1为实施例1中通过单B细胞技术制备抗MMP-3兔单克隆抗体的实验流程图;
图2为实施例2中抗体对检测临床血清样本的结果图。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
下述实施例中,若无特殊说明,均为常规方法;所述试剂和材料,若无特殊说明,均可从商业途径获得。
实施例1抗MMP-3兔单克隆抗体对的制备
采用哺乳动物细胞表达MMP-3蛋白并纯化,对所制备的MMP-3重组蛋白进行表征,表征结果如下表所示:
名称 | 蛋白浓度mg/mL | 活性浓度mg/mL | 纯度SDS-PAGE |
MMP-3重组蛋白 | 2 | 1.5 | 90% |
上表中的活性浓度通过胶乳比浊试剂盒进行检测。由上表可知,本例提供的MMP-3重组蛋白具有较高的纯度和活性。而且,经检测本例提供的MMP-3重组蛋白的氨基酸序列如SEQ ID NO.13所示。
采用上述的MMP-3重组蛋白作为免疫原免疫新西兰兔子,并通过单B细胞抗体克隆技术制备兔单克隆抗体,该实验的流程图如图1所示,具体包括如下步骤:
(1)取3月龄的新西兰兔子,按照1mg/只剂量免疫,每次免疫相隔2周,免疫3-4次后,取兔子耳朵静脉血2mL,检测血清中抗体效价滴度,效价达到50万以上停止免疫。
(2)脾细胞分离:停止免疫一周后处死兔子,收集脾脏,研磨脾脏成细胞悬液;另外取淋巴细胞分离液5mL于15mL灭菌离心管中,吸取脾细胞悬液,在离分离液上方1cm处,沿试管壁缓慢加入,使脾细胞悬液重叠于分离液上,并与分离液形成明显界面;室温,离心2000rpm,20min,此时离心管中出现明显分层,小心吸取中间呈白膜状的单个核细胞层,加5倍以上体积的PBS洗2次,每次离心1500rpm,10min,去掉上层清液,加入1640培养基1mL混匀,取少量进行细胞计数。用台盼蓝染液检查所分离的细胞活性,此时的活细胞比例应达到95%。
(3)特异B淋巴细胞筛选:分离后的细胞用1640培养基稀释后计数,并将细胞稀释后加入载有饲养层细胞的96孔细胞培养板中培养富集,再用可视化筛选方法筛选特异性B淋巴细胞。具体为:将富集的B淋巴细胞经有限稀释至载有饲养细胞的96孔板中培养,然后将培养上清与结合有MMP-3重组蛋白的磁珠孵育,洗涤后与已制备好的sfGFP-SPA融合蛋白共孵育(sfGFP为绿色荧光蛋白,直接可观察到绿色荧光,SPA蛋白可与抗体的Fc端特异性结合),经洗涤后观察磁珠的颜色即可快速判定所检测的B淋巴细胞是否为阳性细胞。
(4)抗体基因克隆:从检测到的阳性B淋巴细胞中,提取特异性B淋巴细胞中的总RNA,利用cDNA5'末端快速扩增技术(5'RACE)合成cDNA,再利用巢式PCR获得抗体可变区及恒定区序列。具体为:以cDNA为模板,进行第一轮巢式PCR扩增,再以第一轮扩增的PCR产物为模板,进行第二轮巢式PCR扩增。
其中第一轮巢式PCR扩增所用引物包括:
第二轮巢式PCR扩增所用引物包括:
PCR扩增程序为:94℃变性,2分钟;94℃变性20秒,58℃退火20秒,72℃延伸60秒,进行40个PCR循环延伸;72℃终延伸5分钟。
(5)抗体转染表达。将扩增所获得的抗体基因插入pcDNA3.4载体中,重轻链质粒共转染293F细胞,具体步骤如下:
①选择密度1.5*10^6~2.0*10^6/mL,活率95%以上的293F悬浮细胞进行转染。
②以转10mL为一个小单位作例进行转染,取15μg质粒(浓度5μg/uL,约3μL体积)用无血清培养基稀释到约30μL。
③每10mL,取45μL的Buffer加入45μL的无血清培养混合均匀后取90μL与上一步的质粒稀释液混匀,静置5分钟后加入到待转染细胞培养瓶中。
④放于8%CO2,37℃恒温培养摇床中培养。
⑤转染第三天收样,用50mL的离心管收集上清液,3000rpm,5min通过离心除去死细胞以及细胞碎片。转移上清到另一个洁净的50mL离心管中,用0.22μm的滤器过滤,过滤后的样品即为待纯化的样品。
(6)抗体纯化,具体步骤如下:
①取protein G亲和层析柱,纯水洗至少10倍柱床体积。
②结合缓冲液冲洗至少10倍柱床体积。
③将样品与PB缓冲以1:4的比例混合,然后用0.22μm滤膜过滤。
④以5-6秒/滴的速度上样,收集穿透,穿透继续上样2-3次。
⑤上样结束后,继续用PB缓冲冲洗柱子至G250检测无色。
⑥用洗脱缓冲液洗脱所结合的IgG,收集洗脱峰,直至G250检测无色。
⑦收集过程中,迅速用pH9.0tris-HCl溶液中和洗脱所得的IgG液。
⑧洗脱结束后水洗至少10倍柱床体积,用20%乙醇封闭层析柱,置于4℃备用。
⑨洗脱峰超滤浓缩,装入透析袋,置于PBS中4℃透析过夜。
对纯化后所获得的多个抗体进行滴度测试:包被ProteinG,加入如下表所示梯度稀释的抗体,再加入生物素化的MMP-3重组蛋白,最后加入HRP标记的链霉亲和素,根据OD值排序抗体亲和力强弱,结果如下表:
选取2号和3号抗体进行测序,测序结果如下:
2号抗体重链CDR区氨基酸序列为:
CDR-H1:SYDMS(SEQ ID NO.1),
CDR-H2:SIDTVGSAYYASWAKG(SEQ ID NO.2),
CDR-H3:SFYMPAFDP(SEQ ID NO.3);
2号抗体轻链CDR区氨基酸序列为:
CDR-L1:QASESVYSNNRLA(SEQ ID NO.4),
CDR-L2:LASTLAS(SEQ ID NO.5),
CDR-L3:AGYKIITDGIS(SEQ ID NO.6)。
3号抗体重链CDR区氨基酸序列为:
CDR-H1:NYCCGC(SEQ ID NO.7),
CDR-H2:CIYGSSDNTYYATWAKG(SEQ ID NO.8),
CDR-H3:GFGDDYVFNL(SEQ ID NO.9);
3号抗体轻链CDR区氨基酸序列为:
CDR-L1:QASQSISSYLA(SEQ ID NO.10),
CDR-L2:RASTLAS(SEQ ID NO.11),
CDR-L3:QQHNMISNIDNF(SEQ ID NO.12)。
实施例2兔单克隆抗体对在制备酶联免疫检测试剂中的应用
以实施例1制备的2号抗体为捕获抗体,3号抗体为标记抗体,采用双抗夹心酶联免疫检测法测试多例外购试剂盒赋值的临床血清样本。
在微孔板上固相化的捕获抗体(即2号抗体),其与血清样本中待检测的MMP-3结合,再与HRP标记的MMP-3抗体(即3号抗体)结合,形成抗原抗体复合物,催化相应的底物发光,测定其相对发光强度(RLU),样本RLU值随MMP3浓度的增加而升高。
其检测结果具体如图2所示,从图2可知:通过测试多例临床血清样本,样本背景值用高质量商品试剂盒赋值,自研2号配对3号抗体测试相关性高达0.99,与商品试剂盒高度一致。
综上所述,本发明通过单B细胞克隆技术获得的兔单克隆抗体对用于免疫诊断试剂时具有优异的定量检测效果,且得益于单B细胞克隆技术具有成本低廉、批间差小、稳定性高等优点,对于强直性脊柱炎、类风湿关节炎、冠心病等临床诊断具有重要意义。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种抗基质金属蛋白酶3的单克隆抗体,其特征在于,所述单克隆抗体为兔单克隆抗体A或兔单克隆抗体B;
所述兔单克隆抗体A的重链CDR-H1、CDR-H2和CDR-H3的氨基酸序列分别如SEQ IDNO.1、SEQ ID NO.2和SEQ ID NO.3所示,且其轻链CDR-L1、CDR-L2和CDR-L3的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6所示;
所述兔单克隆抗体B的重链CDR-H1、CDR-H2和CDR-H3的氨基酸序列分别如SEQ IDNO.7、SEQ ID NO.8和SEQ ID NO.9所示,且其轻链CDR-L1、CDR-L2和CDR-L3的氨基酸序列分别如SEQ ID NO.10、SEQ ID NO.11和SEQ ID NO.12所示。
2.如权利要求1所述的单克隆抗体在建立非诊断目的的MMP-3的酶联免疫检测方法中的应用。
3.根据权利要求2所述的应用,其特征在于,所述酶联免疫检测方法为利用双抗夹心原理的酶联免疫检测方法。
4.根据权利要求3所述的应用,其特征在于,所述兔单克隆抗体A为包被抗体,所述兔单克隆抗体B为标记抗体。
5.如权利要求1所述的单克隆抗体在制备用于检测MMP-3的试剂或试剂盒中的应用。
6.根据权利要求5所述的应用,其特征在于,所述MMP-3包括重组表达的MMP-3,或血清中的MMP-3。
7.一种制备权利要求1所述单克隆抗体的方法,其特征在于,包括以下步骤:
S1、使用MMP-3重组蛋白免疫兔,免疫结束后收集脾脏并分离脾细胞;
S2、筛选特异性B淋巴细胞,并提取其总RNA,合成cDNA,再利用巢式PCR获得抗体可变区及恒定区序列;
S3、将扩增的抗体基因插入载体中,转染293F细胞;
S4、收集培养上清并纯化,即得单克隆抗体。
8.根据权利要求7所述的方法,其特征在于,所述MMP-3重组蛋白的氨基酸序列如SEQID NO.13所示。
9.根据权利要求7所述的方法,其特征在于,步骤S2所述巢式PCR所用的引物序列如SEQID NO.14~28所示。
10.根据权利要求7所述的方法,其特征在于,步骤S4所述纯化具体为:将上清与PB缓冲液混合后上样protein G亲和层析柱,将含有所述单克隆抗体的洗脱液超滤浓缩后置于PBS中透析。
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