CN109694856B - 抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株LGMN-2及其用途 - Google Patents
抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株LGMN-2及其用途 Download PDFInfo
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Abstract
本发明公开了抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株LGMN‑2及其用途。本发明所提供的杂交瘤细胞株,名称为LGMN‑2,保藏号为CGMCC NO.12995。本发明所提供的抗人Legumain蛋白的单克隆抗体,由名称为LGMN‑2,保藏号为CGMCC NO.12995的杂交瘤细胞株分泌产生。本发明的单克隆抗体具有较高的效价,间接法效价最高达1:2.56×108。本发明的单克隆抗体与Legumain同家族蛋白Cathepsin B,Cathepsin H和Cathepsin L无交叉反应,具有较好的特异性。
Description
技术领域
本发明涉及生物医药工程技术领域,尤其涉及抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株LGMN-2及其用途。
背景技术
Legumain(C13)(EC 3.4.22.34)又称天冬酰胺内肽酶(asparaginyl endopep-tidase,AEP),是一种溶酶半胱氨酸蛋白酶,是半胱氨酸蛋白酶C13家族一个新成员。人的Legumain基因定位于14号染色体,编码由433个氨基酸残基组成的多肽链,去糖基化后成为具有生物活性的成熟酶,与小鼠具有83%的同源性。人的legumain蛋白具有3种形式,分别为56kD的酶原前体,46kD和36kD成熟酶。Legumain的活性催化中心是由组氨酸(His)和半胱氨酸(Cys)组成的催化二联体,是Legumain发挥催化活性所必需的,也是每一个C13家族成员必有的结构。
与正常组织相比,Legumain在小鼠和人类多种实体瘤组织中高表达,尤其是在肿瘤细胞、新生血管内皮细胞和肿瘤相关巨噬细胞(Tumor-Associated Macrophages,TAMs)中,而在体外培养的相应肿瘤细胞株中却不表达。Liu等最先报道Legumain与肿瘤发生发展有关。他们通过Western blot和免疫组化分析,发现在多种鼠肿瘤细胞中,Legumain表达增高,而在体外培养的各肿瘤细胞株中则不表达。Liu等还检测了人正常组织及多种实体瘤组织,结果显示在正常组织中,Legumain表达极低,而在多种实体瘤组织中(如乳腺、结肠、肺、前列腺、卵巢肿瘤和中枢神经系统恶性肿瘤)Legumain高表达。
侵袭和转移是恶性肿瘤的播散方式,也是影响肿瘤患者的治疗效果和预后的重要因素。Legumain与整合素形成复合物,在实体肿瘤的低氧和酸性微环境中,具有激活肿瘤细胞Pro-MMP2和Pro-Cathepsin L的功能,促进肿瘤生长、侵袭和转移。为了证实Legumain的表达是否与肿瘤的分化程度、坏死、凋亡及预后相关。Murthy等通过Western blot和免疫组织化学方法,对164例原发性结肠癌、34例远端正常粘膜组织、89例癌旁组织及33例淋巴结转移癌组织样本进行临床病理检测,结果表明,在原发性结肠癌中,Legumain表达明显高于远端正常组织和癌旁组织(P<0.05),但与淋巴结转移癌组织样本无显著差异(P>0.05)。同时,Legumain在肿瘤患者瘤体(P=0.01)或基质中的表达(P=0.04)水平较低时,其预后较好。利用这种相关性,可将Legumain作为疾病诊断和预后判断的指标之一。
Legumain作为一种新的生物学指标,目前临床上暂时没有针对其的检测方法,在监测肿瘤的发生发展中的应用还属空白。
发明内容
基于上述领域的空白,本发明提供了一种用于检测Legumain蛋白的试剂盒。
本发明所提供的用于检测Legumain蛋白的试剂盒,包括由如下单克隆抗体A和单克隆抗体B的组成的单克隆抗体对:
单克隆抗体A:由名称为LGMN-1,保藏号为CGMCC NO.12994的杂交瘤细胞株分泌产生;
单克隆抗体B:由名称为LGMN-2,保藏号为CGMCC NO.12995的杂交瘤细胞株分泌产生。
本发明还提供了一种杂交瘤细胞株,名称为LGMN-1,保藏号为CGMCC NO.12994。
本发明所提供的杂交瘤细胞株LGMN-1CGMCC NO.12994于2016年10月24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:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101)。
一种抗人Legumain蛋白的单克隆抗体,由杂交瘤细胞株LGMN-1分泌产生。
本发明还提供了一种杂交瘤细胞株,名称为LGMN-2,保藏号为CGMCC NO.12995。
本发明所提供的杂交瘤细胞株LGMN-2CGMCC NO.12995于2016年10月24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:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101)。
一种抗人Legumain蛋白的单克隆抗体,由杂交瘤细胞株LGMN-2分泌产生。
所述单克隆抗体在作为抗原抗体反应中识别人Legumain蛋白的抗体用途也属于本发明的保护范围。
本发明首先利用分子克隆的方法构建了人Legumain真核表达质粒pCDNA3.1-tpa-hLGMN,利用瞬时转染的方法转染HEK293T细胞,采用亲和层析和离子交换层析对蛋白进行纯化,从细胞培养上清中纯化得到目的蛋白。
本发明利用体外表达的人Legumain蛋白作为抗原,制备了两种人Legumain单克隆抗体,这两种单克隆抗体分别由编号为CGMCC NO.12994和CGMCC NO.12995的杂交瘤细胞株所分泌。
具体而言,本发明采用小鼠骨髓瘤细胞杂交瘤技术制备人Legumain的单克隆抗体,通过以下技术实现:通过将免疫人Legumain抗原的小鼠脾细胞与骨髓瘤细胞融合,分离出能够分泌抗人Legumain单克隆抗体的杂交瘤细胞,接种小鼠腹腔,制备含有抗人Legumain抗体的腹水,经过后期处理并验证,得到特异性好、抗体水平高且能长期保存的抗人Legumain单克隆抗体。
对本发明的单克隆抗体,经生物标记或化学标记后获得的标记单克隆抗体也在本专利的保护范围。
进一步地,经酶标记的上述单克隆抗体属于本专利的保护范围。
所述的酶为辣根过氧化物酶或碱性磷酸酶。
本发明提供的抗人Legumain单克隆抗体有如下特点和优点:
(1)本发明的单克隆抗体具有较高的效价,间接法效价最高达1∶2.56×108。
(2)本发明的单克隆抗体与Legumain同家族蛋白Cathepsin B,Cathepsin H和Cathepsin L无交叉反应,具有较好的特异性。
(3)本发明杂交瘤细胞LGMN-1和LGMN-2分泌的单克隆抗体亲和常数分别为1.48×107和1.32×107,可广泛应用于人Legumain抗原的检测,鉴别,筛查,癌症诊断以及预后评价中。
附图说明
图1为重组质粒pCDNA3.1-tpa-LGMN的酶切鉴定结果图;其中1为tpa-hLGMN PCR片段,2为pcDNA3.1-tpa-hLGMN双酶切,M为DNA分子量标准Marker DL2000(Takara,CatNo.3427A)。
图2为重组人Legumain纯化抗原的SDS-PAGE图;其中,P1为亲和层析纯化得到的Legumain(pH 8.0),MW为蛋白分子量标准。
图3为Western blot检测重组人Legumain的免疫原性图;其中P1为人Legumain,MW为蛋白分量标准。
图4为杂交瘤细胞分泌的单克隆抗体的免疫印迹图;其中P1为单克隆抗体LGMN-A,P2为单克隆抗体LGMN-B,MW为蛋白分子量标准,C为阳性对照。
具体实施方式
实施例1、制备抗人Legumain蛋白的单克隆抗体
一、Legumain抗原制备
1)Legumain基因序列的获得
Legumain编码基因位于人的第14号染色体上(14q32.12),在NCBI数据库中的序列号为CCDS:CCDS9904.1。携带人Legumain基因的质粒pLEXSY-sat2-hLGMN由奥地利萨尔兹堡大学的Elfriede Dall博士惠赠,记载过真核表达质粒pLEXSY-sat2-hLGMN的非专利文献是E.Dall and H.Brandstetter,Activation of legumain involves proteolytic andconformational events,resulting in a context-and substrate-dependent activityprofile.,Acta Crystallogr Sect F Struct Biol Cryst Commun.2012Jan 1;68(Pt 1):24–31.
2)重组质粒的构建及酶切鉴定
为了使人的Legumain在表达后能够分泌到细胞外,克隆过程中在hLGMN前端加入组织型纤溶酶原激活物(tpa)信号肽序列,后端加入6×His标签。
PCR上游引物:
5’-NheI-CCAGCTAGCATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGATCC ATGGTTTGGAAAGTAGC-3’
下游引物:
PCR反应体系:
PCR反应程序:
PCR片段纯化经PCR产物快速纯化试剂盒(Qigen)纯化后,经NheI和HindIII酶切,酶切完成后在反应体系中加入1μL DnpI,于37℃反应30min,消化掉模板DNA。
pcDNA3.1(+)载体(购自Invitrogen,Cat No.V790-20)也由NheI和HindIII酶切。
酶切体系:
酶切产物经1%的琼脂糖凝胶电泳分析,切取目的片段,经凝胶纯化试剂盒(Qigen,Cat No.20021)提取目的片段,之后进行连接反应。
连接反应体系为:约0.1pmol tpa-hLGMN片段,约0.01pmol pcDNA3.1载体DNA片段,1μL T4DNA连接酶缓冲液(NEB),1μL T4DNA连接酶(NEB),加去离子水补足到10μL体系,16℃连接过夜。取3μL反应产物转化至100μL感受态细胞XL-1blue(Agilent)中。
挑取阳性克隆,经37℃培养12h后,用质粒小提试剂盒(Qigen)提取质粒,并对提取的质粒进行酶切鉴定。
使用NheI和HindIII对所提质粒进行酶切鉴定,酶切体系为:
酶切鉴定结果见图1。由图1可见,Lane 2pcDNA3.1-tpa-hLGMN经酶切后得到的较小的片段与Lane 1的PCR片段大小一致,说明重组质粒构建成功。
3)转染及蛋白纯化
提前一天用无血清培养基F17Medium(Invitrogen,cat.0050092DK)悬浮培养HEK293 6E细胞(购自National Research Council Canada),细胞密度为1.0×106/ml,培养条件为130rpm,37℃.and 5%CO2。
采用上述构建的pCDNA3.1-tpa-LGMN质粒,通过线性PEI瞬时转染的方法(JagerV.et al.,High level transient production of recombinant antibodies andantibody fusion proteins in HEK293 cells,BMC Biotechnology 13(1):52·June2013.)
以在125ml培养瓶中悬浮培养的细胞为例:转染前一天调整细胞密度为1.0×106/ml,培养体积为22.5ml,24h后,细胞密度为1.5-2.0×106/ml,此时可以进行转染实验。
首先在25-37℃预热传染培养基,并且解冻DNA和PEI(Polyethylenimine(linear,25kDa),Polysciences,CatNo.23966):
准备DNA溶液:
在15ml离心管中加入1.5ml转染培养基F17
加入25μg待转染DNA
涡旋混匀
准备PEI溶液:
在15ml离心管中加入1.5ml转染培养基F17
加入50μl PEI solution(1μg/ul)(50μg)
涡旋混匀
准备DNA-PEI复合物:
将PEI溶液加入到DNA溶液中
涡旋混匀
室温静置15min
转染:
将DNA-PEI复合物加入培养细胞中,并迅速转动培养瓶使之混匀,将培养瓶放回到5%二氧化碳培养箱中继续悬浮培养。24h后加入25ml新鲜的Freestyle F17expressionmedium(购自Invitrogen,Cat No.A1383501),使培养基体积加倍,120h后收获细胞培养基,10000rpm,30min,离心去除细胞,收获细胞上清进行蛋白纯化。
使用Ni-NTA agarose(Qigen,Cat No.30230)对hLGMN-his进行亲和层析,.细胞上清首先通过渗滤法交换为缓冲液(50mM NaHPO4,pH 7.6,300mM NaCl),使用1ml Ni-NTA琼脂糖凝胶柱进行亲和层析。
使用30ml洗涤液B(50mM NaHPO4,pH 7.6,300mM NaCl,30mM咪唑)洗涤,5ml洗脱液C(50mM NaHPO4,pH 7.6,300mM NaCl,300mM咪唑)洗脱。取少量洗脱蛋白液做电泳分析和Western blot鉴定。电泳分析结果如图2所示,由图2可见,纯化后的目的片段大小为56kD,与理论值相符。
Western blot流程:
使用表达的重组人Legumain蛋白,进行SDS-PAGE凝胶电泳,并将蛋白转移至硝酸纤维素膜上,5%脱脂奶粉4℃封闭过夜后,加1:1000倍稀释的山羊抗Legumain多克隆抗体(Santa Cruz Biotechnology,Inc.,Cat No.sc-47105),室温孵育1h,洗膜3次,加入HRP-山羊抗小鼠IgG(H+L)(Invitrogen,Cat No.62-6520),最后加入DAB显色试剂盒(上海生工,Cat No.PW017)中的反应液和溶液A和溶液C的混合液,避光显色10min后,加入终止液,拍照记录实验结果(图3)。由图3可见,表达纯化的蛋白在56kD处有显色条带,说明重组legumain蛋白具有良好的免疫原性。
剩余蛋白用0.85%的NaCl溶液在4℃透析48h。用30kD的超滤膜浓缩至5mg/ml,即制备得到人Legumain抗原。
二、动物免疫
将人Legumain抗原与弗氏佐剂(0天免疫为弗氏完全佐剂(Sigma,Cat No.:F5881),14天、28天、35天免疫为弗氏不完全佐剂(Sigma,Cat No.:F5506))等体积混合乳化后于0天、14天、28天、35天背部皮下多点免疫BALb/c小鼠(购自北京维通利华实验动物技术有限公司),0.2mg/只。
末次免疫一周后采血,间接ELISA检测抗体效价,血清稀释104倍时OD值为1.116,于末次免疫一周后采用人Legumain抗原腹腔冲击选定的小鼠,3天后取小鼠脾脏,进行细胞融合。
三、细胞融合及建株
(1)细胞融合前复苏培养SP2/0细胞株(购自Sigma Aldrich,Cat
No.85072401)需在含10%胎牛血清(Gibco,Cat No.10099133)的RPMI 1640培养液(Gibco,Cat No.11875093)中,置5%CO2培养箱中37℃培养,每2~3天换培养液一次,3~5天换液一次。在倒置显微镜下观察为圆形明亮、排列整齐、形态完整、密度适宜(0.1~1×106/ml)、存活率大于95%时,既可供细胞融合使用。融合前3天扩大培养,融合前1天去除RPMI 1640细胞培养液,重新添加培养液,准备SP2/0细胞。
(2)制备脾细胞悬液:取3天前加强免疫的小鼠,断颈处死,浸泡于75%乙醇中3~5min。无菌操作取出脾脏,制备脾细胞悬液,计数活细胞。
(3)根据脾细胞与SP2/0细胞计数结果分别加入适量RPMI-1640培养液,SP2/0细胞晃动混匀,脾细胞用移液管吹打均匀。然后将脾细胞与SP2/0细胞按1:5合于50ml离心管中,混匀。
(4)加RPMI-1640培养液至50ml,1000rpm离心5钟,倒净上清。用手指轻轻敲击融合管底,使沉淀细胞松散均匀,离心管置37℃水浴,准备融合。
(5)吸取1ml 37℃预热的50%的PEG4000,缓慢滴入混合细胞管内,1min内加完,边滴边转动离心管,使细胞保存在混匀状态。
(6)静置90s后立即缓慢加入15ml无血清的RPMI-1640培养基(37℃),800rpm,离心5钟,弃去上清。
(7)加入15ml含10%小牛血清的RPMI-1640培养液,混勾,将混悬液分别加至96孔细胞培养板中,100ul/孔,置于37℃,5%CO2培养箱内培养。
(8)第2天细胞板加HAT培养液(RMPI-1640含1×HAT(Sigma,Cat No.H0262)100ul/孔。
(9)每3天换一次HAT培养液,每次吸出培养孔旧液的1/2换入新液,观察是否出现杂交瘤,两周后换HT培养基(MDM含1×HT(Sigma,Cat No.H0137),观察融合细胞生长状况。
(10)细胞融合后第七天开始观察杂交瘤细胞生长情况,待其长至孔底面积1/10以上时吸出上清利用Fortebio octet 96系统(ForteBio)进行抗体检测。使用HIS2生物传感器(Fortebio,Part No:18-5116)捕获带6×His标签的重组人Legumain抗原(用PBS稀释5μg/ml);0.1mg/ml BSA封闭;吸取细胞培养上清加入待检孔中,如果上清中含有抗人Legumain抗体与重组人Legumain抗原结合,系统捕捉到的光信号会发生改变,根据光信号判断是否阳性。将阳性孔细胞转入24孔板扩大培养,及时做亚克隆。
ForteBio Octet系统所运用的生物膜干涉技术,是一种免标记技术。利用ForteBio Octet系统做单克隆抗体筛选的优势是无需对抗体进行标记液无需使用标记的二抗,精密度高于ELISA,测试速度更快,无需人工洗涤步骤,是一种全自动的操作系统。
(11)经3次亚克隆得到2株稳定分泌抗体的细胞系,分别将杂交瘤细胞株进行保藏,保藏号分别为CGMCC NO.12994和CGMCC NO.12995,分类命名分别为LGMN-1和LGMN-2。Legumain抗原单克隆抗体杂交瘤细胞株,保藏时间:2016年10月24日;保藏单位: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:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101)。
四、杂交瘤细胞株的冷冻保存和复苏
将鉴定为阳性的杂交瘤细胞,加入细胞冻存液(30%小牛血清(Gibco,CatNo.16010159),60%RPMI-1640,10%DMSO),调整细胞浓度为1~5×106个/ml。将细胞悬液分装到冻存管中,先4℃预冷10min,再置于-20℃冰箱中30min,再移至-80℃冰箱16~18h(或过夜),最后投入液氮中长期保存。
复苏杂交瘤细胞时,将杂交瘤细胞冷冻管从液氮中取出,立即投入38~40℃水浴中,待细胞悬液解冻后,加入10ml RPMI-1640培养液,将细胞移至25ml培养瓶中,置37℃CO2培养箱中培养。过3~4h,待细胞贴壁后,倒掉旧培养基,加入新鲜的RPMI-1640培养基继续培养。
五、单抗细胞株腹水制备及抗体效价检测
单抗腹水制备:待细胞覆盖25ml的细胞培养瓶瓶底50%以上时,选择健康的SPF级BALB/c小鼠(购自北京维通利华实验动物技术有限公司),先于小鼠腹腔注射0.3ml石蜡油,7~10天后于每只小鼠腹腔注射杂交瘤细胞0.5~5×108个。待小鼠腹部明显膨大时,用9号针头抽取腹水或用16号针头放腹水,可反复抽取多次。腹水经12000rpm,10min离心后,取上清液分装,置-20℃保存备用。
单抗效价检测:
单抗效价检测:利用Fortebio Octet 96系统对单抗细胞株腹水效价进行测定,使用HIS2生物传感器捕获重组人Legumain抗原(用PBS稀释5μg/ml),作用时间10min;PBST洗涤2min;0.1mg/ml BSA封闭5min;PBST洗涤2min;加入梯度稀释的LGMN-A和LGMN-B腹水,作用10min。使用未免疫过重组人Legumain蛋白的小鼠腹水作为阴性对照。结果显示,两株单抗细胞株腹水抗体效价分别为1:2.05×107,1:2.56×108,效价较高。
Fortebio octet 96系统(ForteBio)进行抗体检测。使用HIS2生物传感器(Fortebio,Part No:18-5116)捕获带His×6标签的重组人Legumain抗原(用PBS稀释5μg/ml);0.1mg/ml BSA封闭;吸取细胞培养上清加入待检孔中,如果上清中含有抗人Legumain抗体与重组人Legumain抗原结合,系统捕捉到的光信号会发生改变,根据光信号判断是否阳性。将阳性孔细胞转入24孔板扩大培养,及时做亚克隆。
六、Western检测单克隆抗体与抗原的结合
使用表达的重组人Legumain蛋白,进行SDS-PAGE凝胶电泳,并将蛋白转移至硝酸纤维素膜上,5%脱脂奶粉4℃封闭过夜后,加1:1000倍稀释的含有LGMN-1和LGMN-2单克隆抗体的腹水,阳性对照为购买的山羊抗Legumain多克隆抗体(Santa Cruz Biotechnology,Inc.,Cat No.sc-47105),室温孵育1h,洗膜3次,二抗分别为HRP-山羊抗小鼠IgG(H+L)(Invitrogen,Cat No.62-6520)和HRP-兔抗山羊IgG(Invitrogen,Cat No.61-1620),最后加入DAB显色试剂盒(上海生工,Cat No.PW017)中的反应液和溶液A和溶液C的混合液,避光显色10min后,加入终止液,拍照记录实验结果(图4)。由图4可见,C为购买的山羊抗Legumain多克隆抗体阳性对照,P1、P2为两株单克隆抗体与重组Legumain蛋白的反应,说明两株单克隆抗体与重组Legumain蛋白有良好的免疫原性反应。
六、抗体亚类测定
将人Legumain抗原采用0.01M PBS稀释至5μg/ml,100ul/孔包被酶标板,置于4℃过夜,以5%脱脂奶粉37℃封闭1h,然后采用鼠单抗分型试剂盒(Sigma,IS02-1KT)按照单抗亚类试剂盒说明书进行试验,结果显示本发明单克隆抗体LGMN-A为IgG2a型,单克隆抗体LGMN-B为IgG2b型。
七、杂交瘤细胞系分泌单抗的稳定性检测
分别在3个月和9个月后,从液氮中取出冻存的杂交瘤细胞株LGMN-1和LGMN-2进行复苏、扩大培养后,制备腹水,进行间接ELISA检测抗体效价,以前期制备的腹水,LGMN-A和LGMN-B单抗腹水(0天)为对照同时进行检测。结果表明,本发明的杂交瘤细胞株制备的单克隆抗体腹水效价达到107以上,与前期腹水效价无差异,表明细胞保藏后制备的腹水的效价未下降。因此单克隆细胞株分泌抗体的活性没有降低,稳定性良好。
八、亲和常数测定
将本发明的杂交瘤细胞株分泌的单抗LGMN-A和LGMN-B分别纯化后检测其蛋白质含量。采用1:5,1:10,1:20,1:40稀释的不同浓度的Legumain抗原横向包被酶标板,100ul/孔,4℃包被过夜。第二天洗板后封闭2小时,拍干待用。将纯化后的单抗LGMN-A和LGMN-B分别2倍梯度稀释,纵向加入包被后的酶标板,采用间接ELISA法检测抗原抗体反应的OD值。以各抗原浓度下的曲线平坦段的OD值计为100%,计算50%OD值,考察50%OD值所对应点的单抗浓度[Ab]t,再根据亲和常数计算公式K=(n-l)/2(nAb'-Ab)可以得到本发明单抗LGMN-A的亲和常数为1.48×107。LGMN-B的亲和常数为1.32×107(表1)。
表1亲和常数检测结果
九、单克隆抗体的特异性
将人Legumain抗原,Cathepsin B(Sigma Aldrich,Cat No.SRP0289),CathepsinH(Biovision,Cat No.1023-5)和Cathepsin L(Sigma Aldrich,Cat No.SPR0291)采用0.01M PBS稀释至5μg/ml,100ul/孔包被酶标板,每种蛋白包被四个孔,每两个孔作为一个重复,置于4℃过夜,5%脱脂奶粉37℃封闭1h,洗涤三次,然后分别加入单克隆抗体LGMN-A和LGMN-B 37℃孵育1h,洗涤三次,加入1:1000倍稀释的HRP-山羊抗小鼠IgG(H+L),洗涤三次后,加入TMB显色液显色,2M硫酸终止,在450nm测定吸光度。
添加Cathepsin B,Cathepsin H和Cathepsin L孔的OD450值与标准曲线上空白孔OD450值相近,与Legumain蛋白孔差异性显著(P>0.05),故该方法的特异性较好。
序列表
<110> 李伟,南京拂晓生物科技有限公司
<120> 抗人Legumain蛋白的单克隆抗体、杂交瘤细胞株LGMN-2及其用途
<130> P180849-JFQ
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1302
<212> DNA
<213> Artificial Sequence
<220>
<223> Legumain编码基因
<400> 1
atggtttgga aagtagctgt attcctcagt gtggccctgg gcattggtgc cgttcctata 60
gatgatcctg aagatggagg caagcactgg gtggtgatcg tggcaggttc aaatggctgg 120
tataattata ggcaccaggc agacgcgtgc catgcctacc agatcattca ccgcaatggg 180
attcctgacg aacagatcgt tgtgatgatg tacgatgaca ttgcttactc tgaagacaat 240
cccactccag gaattgtgat caacaggccc aatggcacag atgtctatca gggagtcccg 300
aaggactaca ctggagagga tgttacccca caaaatttcc ttgctgtgtt gagaggcgat 360
gcagaagcag tgaagggcat aggatccggc aaagtcctga agagtggccc ccaggatcac 420
gtgttcattt acttcactga ccatggatct actggaatac tggtttttcc caatgaagat 480
cttcatgtaa aggacctgaa tgagaccatc cattacatgt acaaacacaa aatgtaccga 540
aagatggtgt tctacattga agcctgtgag tctgggtcca tgatgaacca cctgccggat 600
aacatcaatg tttatgcaac tactgctgcc aaccccagag agtcgtccta cgcctgttac 660
tatgatgaga agaggtccac gtacctgggg gactggtaca gcgtcaactg gatggaagat 720
tcggacgtgg aagatctgac taaagagacc ctgcacaagc agtaccacct ggtaaaatcg 780
cacaccaaca ccagccacgt catgcagtat ggaaacaaaa caatctccac catgaaagtg 840
atgcagtttc agggtatgaa acgcaaagcc agttctcccg tccccctacc tccagtcaca 900
caccttgacc tcacccccag ccctgatgtg cctctcacca tcatgaaaag gaaactgatg 960
aacaccaatg atctggagga gtccaggcag ctcacggagg agatccagcg gcatctggat 1020
gccaggcacc tcattgagaa gtcagtgcgt aagatcgtct ccttgctggc agcgtccgag 1080
gctgaggtgg agcagctcct gtccgagaga gccccgctca cggggcacag ctgctaccca 1140
gaggccctgc tgcacttccg gacccactgc ttcaactggc actcccccac gtacgagtat 1200
gcgttgagac atttgtacgt gctggtcaac ctttgtgaga agccgtatcc gcttcacagg 1260
ataaaattgt ccatggacca cgtgtgcctt ggtcactact ga 1302
Claims (3)
1.一种杂交瘤细胞株,名称为LGMN-2,保藏号为CGMCC No.12995,其分泌的单克隆抗体具有的亲和常数在1.12×107-1.39×107之间。
2.一种抗人Legumain蛋白的单克隆抗体,由权利要求1所述的杂交瘤细胞株分泌产生,具有的亲和常数在1.12×107-1.39×107之间,为IgG2b型抗体。
3.权利要求2所述的单克隆抗体在制备识别人Legumain蛋白的产品中的用途。
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