CN112794901A - 一种抗smim15单克隆抗体及其应用 - Google Patents
一种抗smim15单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种抗SMIM15单克隆抗体及其应用,属于免疫学技术领域。该抗体是由杂交瘤细胞株1M5分泌产生的。其制备方法为:制备小鼠免疫原和检测用抗原;制备抗SMIM15杂交瘤细胞株1M5;SMIM15阳性单克隆抗体的筛选;单克隆抗体的亚型鉴定;SMIM15阳性单克隆抗体特异性检测;BALB/c小鼠体内生产单抗并纯化;SDS‑PAGE检测抗体纯度;并采用WB、IHC和ELISA对SMIM15单克隆抗体进行功能鉴定及应用。本申请由杂交瘤细胞株1M5直接分泌产生的抗体是一种免疫球蛋白,无毒,可应用于免疫印迹和免疫组化,也可作为活性成分制成与SMIM15相关疾病的诊断及治疗的试剂或药物。
Description
技术领域
本发明属于免疫学技术领域,涉及一种单克隆抗体,具体涉及一种抗SMIM15单克隆抗体及其应用。
背景技术
小整合膜蛋白15(Small Integral Membrane Protein 15,SMIM15,别名C5orf43)是一种蛋白质编码基因,在机体的结肠、甲状腺以及其他组织中普遍表达,已知SMIM15包括2个转录本,155个同源基因,SMIM15的主要功能是在各种生理病理情况下控制蛋白质的合成,进一步在细胞间的免疫识别以及物质运输等过程起到关键作用。
相比于正常组织,大多数癌组织中SMIM15的表达量较高,如胶质瘤、胃癌以及乳腺癌等,但是其相关机制以及研究未见有文献报道。近几年随着免疫治疗及免疫靶向治疗在多种疾病的综合治疗过程发挥重要作用,利用抗体将药物导向靶点细胞更是一种理想途径。当前对于蛋白质编码基因的研究逐步成为基础科研以及生物医学的重要靶标,使得用于医学科研和临床诊断的生物试剂市场需求量增加,就目前国内外文献的报道情况,尚未见具有人鼠交叉反应、高特异性抗SMIM15的单克隆抗体可用于免疫学方法的研究。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题是提供一种抗SMIM15单克隆抗体,本发明所要解决的另一技术问题是提供上述抗SMIM15单克隆抗体的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
本申请所提供的分泌抗SMIM15单克隆抗体A1M5的杂交瘤细胞株1M5,已于2020年11月18日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C2020247;所制备得到的抗SMIM15的单克隆抗体,命名为A1M5。
一种抗SMIM15的单克隆抗体A1M5,是由杂交瘤细胞株1M5分泌的。
采用杂交瘤细胞株1M5产生单克隆抗体A1M5的方法如下:
(1)为了增强免疫原性,从GenBank获取SMIM15基因的核苷酸片段序列(Accession:NM 001048249.4),人工合成多肽片段:CAWAEYVVEWAAKDPY,并将多肽片段与KLH偶联作为免疫原,命名为2019-348多肽-KLH,以常规方法免疫SPF BALB/c雌性小鼠;
(2)从免疫合格小鼠无菌取其脾细胞作为抗原致敏的B细胞,按常规方法,将B细胞与骨髓瘤细胞SP2/0株融合,然后利用常规的融合细胞HAT筛选方法进行筛选,进而获取融合细胞生长克隆;
(3)应用酶联免疫吸附试验(Enzyme-Linked ImmunoSorbent Assay,ELISA)方法筛选出抗SMIM15阳性而正常人血清阴性的单克隆抗体,最终选取9株稳定分泌抗SMIM15抗体的杂交瘤细胞,分别命名为1M4、1M5、1M6、1M7、1M8、1M9、1M10、1M11和1M15,它们分泌的单抗分别命名为A1M4、A1M5、A1M6、A1M7、A1M8、A1M9、A1M10、A1M11和A1M15;
(4)将筛选出来的9株阳性细胞株进行亚类鉴定,最后得到9株IgG的阳性杂交瘤细胞株,亚型分别为G1、G2a、G2b、G2b、G2a、G2a、G2a、G2b和G2a;
(5)蛋白免疫印迹(Western blot,WB)检测SMIM15阳性单克隆抗体特异性,结果显示2019-348多肽-BSA与SMIM15阳性单克隆抗体(A1M4、A1M5、A1M6、A1M7、A1M8、A1M9、A1M10、A1M11和A1M15)均能发生特异性反应(图1);
(6)在动物腹腔内接种最终选择的杂交瘤细胞1M5,动物腹水中分离和纯化得到所需的抗SMIM15单克隆抗体A1M5;
(7)SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测A1M5抗体纯度,结果显示出现清晰两条带分别是重链和轻链(图2),说明抗体纯化的效果较高;
(8)WB对SMIM15抗体进行功能鉴定及应用,发现A1M5抗体与胃癌细胞、胶质瘤细胞和乳腺癌细胞中的天然抗原SMIM15均有较好的结合活性,表明纯化的A1M5抗体特异性较好,在WB中应用,能识别SMIM15天然抗原(图3)。
(9)免疫组织化学技术(Immunohistochemistry,IHC)对SMIM15抗体进行功能鉴定及应用,发现A1M5抗体有阳性染色结果(图4、乳腺癌;图5、胶质瘤;图6、胃癌),在乳腺癌、胶质瘤和胃癌的细胞质中表达,说明纯化的A1M5抗体效价、特异性较好,在IHC中应用,可与过表达SMIM15的组织结合。
(10)ELISA检测抗SMIM15单克隆抗体A1M5效价,发现A1M5抗体浓度低至93.75ng/mL与SMIM15的多肽抗原(浓度为0.0025μg/mL)有较好的结合活性,A1M5具有较好的ELISA反应滴度(图7)。
一种试剂盒,含有抗SMIM15的单克隆抗体A1M5。
所述的抗SMIM15的单克隆抗体A1M5在制备检测SMIM15抗原试剂中的应用。
所述的抗SMIM15的单克隆抗体A1M5在制备SMIM15相关疾病的治疗性药物、相关的诊断试剂及科研试剂中的应用。
有益效果:相比于现有技术,本发明的优点为:
本发明从GenBank获取SMIM15基因的核苷酸片段序列(Accession:NM_001048249.4),运用单克隆抗体制备技术制备的抗SMIM15单克隆抗体,制备方法简单,可由杂交瘤细胞株1M5直接分泌产生。该抗体是一种免疫球蛋白,不会通过身体表面接触和呼吸道而伤害健康,无毒;可以应用于免疫印迹和免疫组化,也可以潜在应用该抗体为活性成分制成与SMIM15相关疾病的诊断及治疗的试剂或药物。
附图说明
图1是WB检测2019-348多肽-BSA与SMIM15阳性单克隆抗体能否发生特异性反应,图中,抗体A1M4、A1M5、A1M6、A1M7、A1M8、A1M9、A1M10、A1M11和A1M15均能与SMIM15分子发生特异性结合;
图2是SDS-PAGE检测SMIM15单克隆抗体A1M5纯度,图中,出现清晰的两条带分别是重链(55KD)和轻链(25KD);
图3是WB检测抗SMIM15单克隆抗体A1M5与细胞来源的天然抗原反应结果,图中,出现清晰条带,A1M5与天然抗原SMIM15有较好的结合活性;
图4是IHC检测抗SMIM15单克隆抗体A1M5在乳腺癌组织中的表达情况图,图中,阳性定位在乳腺癌细胞的细胞质;
图5是IHC检测抗SMIM15单克隆抗体A1M5在胶质瘤组织中的表达情况图,图中,阳性定位在胶质瘤细胞的细胞质;
图6是IHC检测抗SMIM15单克隆抗体A1M5在胃癌组织中的表达情况图,图中,阳性定位在胃癌细胞的细胞质;
图7是ELISA检测抗SMIM15单克隆抗体A1M5与SMIM15偶联多肽的反应效价,显示A1M5抗体有较高的滴度反应。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。这些实施例仅用于说明本发明而不用于限制本发明的范围。以下实施例中如无特殊说明,所用实验方法均为常规方法。
实施例1
I、以下实施例中使用的主要试剂为:
(1)细胞融合用:
IMDM培养基;
IMDM完全培养基(含15%血清);
2.2%甲基纤维素(SIGMA;货号:M0262-100G);
新生牛血清;PEG1500(Roche;货号:78364);
HAT(Sigma;货号:H0262-10VL);
HT(Sigma;货号:H0137-10VL)。
(2)单克隆细胞筛选用:
包被液:碳酸钠-碳酸氢钠缓冲液(pH9.6);PBS缓冲液(pH7.4);
封闭液:含2%牛奶的PBS;
洗液:PBS-T(0.05%吐温,PBS);
显色液:1%A液+10%B液(A液:1%TMB in DMSO;B液:0.1%H2O2的柠檬酸缓冲液);
终止液:2M硫酸;
二抗:山羊抗小鼠IgG/HRP。
(3)单克隆细胞亚类鉴定用:
包被抗体(Southern Biotech);
封闭液:2%BSA+3%蔗糖in PBS;
显色液:0.2mL A液+10μL 30%H2O2in 10mL B液(A液:15mg/mL ABTS in H2O;B液:柠檬酸缓冲液,pH4.0);
各型亚类二抗(Southern Biotech)。
(4)SDS-PAGE凝胶电泳及WB用:
SDS-PAGE凝胶配制试剂盒(碧云天生物技术有限公司);
2×Loading Buffer(碧云天生物技术有限公司);
预染蛋白Marker(美国Thermo公司);
考马斯亮蓝R-250(碧云天生物技术有限公司);
PDVF膜(美国Millipore公司);
滤纸(碧云天生物技术有限公司);
IgG鼠二抗(美国abcam公司);
ECL显影液(苏州新赛美公司)。
(5)免疫组化用:
DAB染色液(增强聚合物法)试剂盒(福州迈新生物技术开发公司);
柠檬酸组织抗原修复液(100X)(福州迈新生物技术开发公司);
内源性过氧化物酶阻断剂(福州迈新生物技术开发公司);
反应增强液(福州迈新生物技术开发公司);
酶标抗小鼠/兔IgG聚合物(福州迈新生物技术开发公司)。
(6)ELISA用:
ELISA包被液(北京索莱宝科技有限公司);
ELISA终止液(北京索莱宝科技有限公司);
单组分TMB显色液(北京索莱宝科技有限公司);
羊抗小鼠IgG-HRP(北京索莱宝科技有限公司)。
II、以下实施例中使用的主要仪器为:
组织芯片系统(UT06,韩国,Unitma公司);倒置显微镜(DMIRB,德国,Leica公司);全自动酶标仪(SN209941,美国,Bio-Tek公司);二氧化碳水套式培养箱(HEPACLASS 100,美国,ThermoFisher公司);pH计(EL20,瑞士,MettlerToledo公司);垂直电泳转印系统(1658033,美国BIO-RAD公司);UVP凝胶成像系统(UVP Biosp,美国UVP公司)。
实施例1
1、制备小鼠免疫原和检测用抗原
多肽设计及合成2019-348多肽:CAWAEYVVEWAAKDPY-NH2(序列),多肽偶联:将偶联KLH后的多肽定量3.0mg/mL(命名:2019-348多肽-KLH)。
2、制备抗SMIM15杂交瘤细胞
用“2019-348多肽-KLH”,按60μg蛋白/只小鼠的量,皮下初次免疫4只SPF BALB/c雌性小鼠,编号为:1、2、3、4。皮下第一次加强免疫,免疫量为30μg蛋白/只。随后分别进行第二次、第三次、第四次、第五次加强免疫,免疫量均为30μg蛋白/只。眼眶取血,测血清效价。用免疫原50μg,腹腔冲击小鼠。
免疫效价检测步骤:
用“2019-348多肽-BSA”,2μg/mL,4℃包被过夜;2%脱脂奶粉,37℃封闭2h;血清从200倍开始2倍梯度稀释,空白对照(blank)为PBS,阴性对照(negative)为阴性血清200倍稀释;冲击小鼠做细胞融合实验;
取小鼠脾细胞与SP2/0细胞(骨髓瘤细胞),采用PEG法进行融合,融合完细胞用半固体培养基(含HAT)进行筛选培养。
细胞融合步骤:
(1)将状态良好的SP2/0细胞轻柔地从培养瓶壁上吹打下来,吸入到50mL离心管中;
(2)小鼠摘眼球取血,然后拉颈处死,放入75%的酒精中浸泡5min;
(3)在平皿中倒入少量无血清的IMDM,将细胞筛及注射器内芯放入平皿中;用剪刀和镊子取下小鼠的脾脏,放到细胞筛上;用注射器的内芯轻轻地将脾充分碾碎,将碾好的细胞吸入到装有SP2/0的离心管中,离心1500rad/min,5min;
(4)用剪刀和镊子取下小鼠的胸腺,碾碎;将碾好的胸腺细胞吸到15mL离心管中,再加入1mL的HAT,放在孵箱中备用;
(5)将离心好的细胞,倒掉上清,用无血清的IMDM将细胞小心轻柔地吹匀,离心(1500rad/min,5min);
(6)将离心好的细胞上清倒掉,拍打离心管底悬浮细胞,将离心管放入37℃温水中,1min内缓慢加入1mLPEG(Roche),温水静置1min,随后缓慢加入2mL的无血清IMDM,接着2min内缓慢加入8mL无血清IMDM,1000rad/min,离心5min;
(7)倒掉上清,加入10mL的血清,小心地将细胞吹匀,倒入前面准备好的胸腺细胞;再加入25mL灭过菌的半固体培养基,充分混匀;然后均匀倒入30个细胞培养皿中;将细胞培养皿放入湿盒中,然后放入孵箱中培养。
3、抗SMIM15阳性单克隆抗体的筛选
两周后,挑10板×93个细胞单克隆,培养于96孔细胞培养板(事先用胸腺细胞铺板,100μL/孔);
3天后,用“2019-348多肽-BSA”包板,对挑选的克隆采用ELISA方法,做第一次筛选,得到15株阳性杂交瘤细胞株,编号:1M1、1M2、1M3、1M4、1M5、1M6、1M7、1M8、1M9、1M10、1M11、1M12、1M13、1M14和1M15。
实验步骤:
(1)用包被液(碳酸钠-碳酸氢钠缓冲液,pH9.6)稀释“2019-348多肽-BSA”,终浓度为2μg/mL,100μL/孔,4℃过夜;后用PBST(含0.05%吐温的PBS)洗涤3次;
(2)封闭液(含2%牛奶的PBS)封闭,200μL/孔,37℃孵箱,2h,PBST洗涤3次;
(3)加入一抗(细胞培养上清)、阴性对照(SP2/0培养上清)、空白对照(PBS)、阳性对照(阳性血清,PBS稀释1000倍),均为100μL/孔,37℃孵箱,1h;PBST洗涤3次;
(4)加入PBS稀释20000倍的二抗,100μL/孔,37℃孵育1h;取出后用PBST洗涤3次;
(5)显色,每孔加100μL显色液,显色时间为5min;
(6)每孔加入50μL终止液终止;
(7)双波长(450,630)测吸光值,对免疫蛋白筛选呈阳性的杂交瘤细胞株,共15株。
将15株阳性的细胞株,用“2019-348多肽-BSA”再次包板,采用ELISA方法,做第二次筛选,最终选取9株稳定分泌抗SMIM15抗体的杂交瘤细胞,分别命名为1M4、1M5、1M6、1M7、1M8、1M9、1M10、1M11和1M15,它们分泌的单抗分别命名为A1M4、A1M5、A1M6、A1M7、A1M8、A1M9、A1M10、A1M11和A1M15。
4、单克隆细胞的亚型鉴定
将筛选出来的9株阳性细胞株进行亚类鉴定,最后得到9株IgG的阳性杂交瘤细胞株,亚型分别为G1、G2a、G2b、G2b、G2a、G2a、G2a、G2b和G2a。
实验步骤:
(1)用100mM PBS(pH 7.4)稀释包被抗体(Southern Biotech)至0.5μg/mL,每孔加0.1mL,4℃,过夜;
(2)PBST洗2次,每孔加入200μL封闭液,37℃孵育2h;
(3)PBST洗3次,每孔加入100μL杂交瘤上清,37℃孵育1h;
(4)PBST洗3次,用封闭液1:10000(κ,λ)或1:20000(其它的)稀释的HRP标记的抗体每孔0.1mL,分别加入适当的孔中,37℃孵育1h;
(5)PBST洗3次;每孔加50μL底物溶液,10-20min内于双波长(450,630)测吸光值,最终得到9株阳性单克隆细胞株的亚型,分别为G1、G2a、G2b、G2b、G2a、G2a、G2a、G2b和G2a。
5、单克隆抗体特异性检测
(1)按说明装好蛋白垂直电泳槽;
(2)配制12%分离胶:室温下静置50min至凝胶聚合;
(3)配制浓缩胶:待分离胶完全聚合后配制5%浓缩胶;
(4)上样:待浓缩胶完全聚合后,拔取梳子,加入SDS甘氨酸电泳缓冲液并清理上样孔,将准备好的样品(2019-348多肽-BSA)加入上样孔中;
(5)电泳:接通电源,以80V恒压电流50min,待蛋白预染Marker分开之后转换为120V恒压电流继续电泳约40min。
(6)转膜:300mA电流下转膜90min。
(7)一抗:转膜结束后,封闭液封闭2h,随后给与稀释好的一抗(9株杂交瘤细胞分泌的单抗)。
(8)二抗:采用HRP标记的山羊抗鼠IgG的二抗,按照1∶5000稀释比例配制后室温孵育2h;
(9)显影:UVP凝胶成像系统显影并拍照保存。
结果发现SMIM15单克隆抗体能与SMIM15的偶联多肽(2019-348多肽-BSA)特异性结合,形成阳性条带。表明9株杂交瘤细胞株分泌的阳性上清均具有识别SMIM15分子的良好特异性(图1)。
6、BALB/c小鼠体内生产单抗并纯化
6周龄小鼠每只腹腔接种0.5mL降植烷,10天后腹腔接种RPMI-1640基础培养基稀释的1M5和1M15细胞悬液,同时用骨髓瘤细胞SP2/0做为对照组,每只小鼠接种5×106个细胞;10天后收集腹水,离心,取上清,用二氧化硅粉末去除杂质,接着用Protein G亲和层析柱(Amersham)对抗体进行纯化,纯化完毕后分装冻存于-80℃。
7、SDS-PAGE检测SMIM15单克隆抗体纯度
(1)按说明装好蛋白垂直电泳槽;
(2)配制12%分离胶:室温下静置50min至凝胶聚合;
(3)配制浓缩胶:待分离胶完全聚合后配制5%浓缩胶;
(4)上样:待浓缩胶完全聚合后,拔取梳子,加入SDS甘氨酸电泳缓冲液并清理上样孔,将准备好的样品(纯化抗体)按预定顺序加入上样孔中;
(5)电泳:接通电源,以80V恒压电流50min,待蛋白预染Marker分开之后转换为120V恒压电流继续电泳约40min。
(6)电泳结束后关闭电源取出凝胶,室温下以考马斯亮蓝R-250染液平缓染色4h,然后以脱色液充分脱色后观察蛋白条带,拍照。
结果表明,SMIM15单克隆抗体A1M5经SDS-PAGE电泳,出现清晰的两条带,分别为重链和轻链,表明纯化抗体具有较高的纯化效果(图2)。
8、WB对SMIM15抗体进行功能鉴定及应用
(1)将数量为1×107的胃癌细胞(MKN-1)、胶质瘤细胞(U87-mg)和乳腺癌细胞(MCF-7)分别在RAPI裂解液中裂解,取得细胞总蛋白。
(2)如先前所述进行凝胶电泳及转膜,一抗为稀释后的A1M5(1∶500),二抗为HRP标记的山羊抗鼠IgG(1∶5000),并进行显影保存。
结果表明,A1M5与多种肿瘤细胞中天然抗原SMIM15有较好的结合活性,在WB应用中能识别SMIM15天然抗原(图3)。
9、IHC对SMIM15抗体进行功能鉴定及应用
(1)70℃的烘箱中烤片(乳腺癌、胶质瘤和胃癌切片)90min,60℃烘片60min,随后进行2次二甲苯脱蜡,每次5min,脱蜡完成后依次过100%、95%、70%酒精,每次5min;
(2)在微波炉中进行抗原修复,先100%功率修复2.5min,再20%功率修复15min,待冷却后用PBS冲洗;
(3)用3%过氧化氢孵育30min,PBST冲洗2min×3次;
(4)用含5%BSA的PBS溶液封闭30min;
(5)孵育一抗(纯化抗体),4℃过夜;
(6)从4℃取出组织芯片后常温放置半小时,PBST冲洗2min×3次;
(7)滴加二抗增强液,室温孵育20min,PBST冲洗2min×3次;
(8)滴加二抗,室温30min后,PBST冲洗2min×3次;
(9)用DAB进行显色,显色完全后立即终止显色;
(10)放入苏木素中30s,过盐酸酒精分化,而后流水冲洗10min;
(11)依次放入70%、95%、100%酒精,每次5min,过两次二甲苯,每次5min,最后自然晾干,中性树胶封片。
染色结果见A1M5抗体有阳性染色结果(图4、乳腺癌;图5、胶质瘤;图6、胃癌),在乳腺癌、胶质瘤和胃癌的细胞质中表达,说明纯化的A1M5抗体效价、特异性较好,在IHC中应用,可与过表达SMIM15的组织结合。
10、ELISA检测抗SMIM15单克隆抗体A1M5效价
(1)用包被液(碳酸钠-碳酸氢钠缓冲液,pH9.6)稀释SMIM15的偶联多肽(2019-348-BSA),终浓度分别为0.01μg/mL、0.005μg/mL、0.0025μg/mL,100μL/孔,4℃过夜;后用PBST(含0.05%吐温的PBS)洗涤3次;
(2)封闭液(含2%牛奶的PBS)封闭,200μL/孔,37℃孵箱,2h,PBST洗涤3次;
(3)加入不同浓度的纯化抗体A1M5(初始浓度1.2mg/mL,分别按照1∶200、1∶400、1∶800、1∶1600、1∶3200、1∶6400、1∶12800浓度梯度稀释,阴性对照为PBS),100μL/孔,37℃孵箱,1h;PBST洗涤3次;
(4)加入PBS稀释3000倍的二抗(山羊抗小鼠IgG/HRP,SouthernBiotech),100μL/孔,37℃孵箱,1h;取出后用PBST洗涤3次;
(5)显色,显色液100μL/孔,显色时间为10min左右;
(6)每孔加入50μL终止液(2M硫酸)终止;
(7)双波长(450,630)测吸光值。
经间接ELISA实验结果表明,A1M5抗体浓度低至93.75ng/mL与SMIM15的多肽抗原(浓度为0.0025μg/mL)有较好的结合活性,A1M5具有较好的ELISA反应滴度(图7)。
综上所述,获得的单克隆抗体A1M5是目的抗SMIM15单抗,具有高特异性识别SMIM15蛋白抗原,可用于IHC的免疫学研究,也可潜在用于SMIM15相关疾病的诊断和治疗。
序列表
<110> 南通大学附属医院
<120> 一种抗SMIM15单克隆抗体及其应用
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> 人工合成多肽(artificial)
<400> 1
Cys Ala Trp Ala Glu Tyr Val Val Glu Trp Ala Ala Lys Asp Pro Tyr
1 5 10 15
Claims (7)
1.一种抗SMIM15的单克隆抗体A1M5,是由保藏号为CCTCC NO:C2020247的杂交瘤细胞株1M5所分泌的。
2.根据权利要求1所述的抗SMIM15的单克隆抗体A1M5,其特征在于,其属于G2a亚型。
3.根据权利要求1所述的抗SMIM15的单克隆抗体A1M5,其特征在于,其免疫小鼠用的抗原是将N-端氨基酸序列如SEQ ID NO:1所示的氨基酸残基序列与KLH偶联得到。
4.一种试剂盒,其特征在于,含有如权利要求1或权利要求2或权利要求3所述的任意一种单克隆抗体。
5.权利要求1所述的抗SMIM15的单克隆抗体A1M5在制备检测SMIM15抗原试剂中的应用。
6.权利要求1所述的抗SMIM15的单克隆抗体A1M5在制备SMIM15相关疾病的治疗性药物、相关的诊断试剂及科研试剂中的应用。
7.分泌权利要求1所述的抗SMIM15的单克隆抗体A1M5的杂交瘤细胞,是保藏号为CCTCCNO:C2020247的杂交瘤细胞株1M5。
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