CN106749655B - 一种针对蛋白e2-2的单克隆抗体 - Google Patents
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Abstract
本发明涉及一种针对蛋白E2‑2的单克隆抗体，具体是利用原核表达***表达E2‑2(TCF4)蛋白，免疫Balb/c小鼠，利用ELISA和免疫组化方法，筛选得到一株杂交瘤细胞株，其能够产生特异性结合人E2‑2蛋白的单克隆抗体。在免疫酶学检测(ELISA)、免疫印迹(Western)和免疫组化实验中，该抗体能够特异性的结合E2‑2。所述单克隆抗体可以用于诊断各种pDC相关的疾病，包括Pitt‑Hopkins综合征、精神***症、有pDC浸润的实体肿瘤、母细胞性浆样树突状细胞淋巴瘤、其它与pDC频率变化相关的疾病等。
Description
发明领域
本发明涉及抗体工程领域，具体涉及针对蛋白E2-2的单克隆抗体。
背景技术
E2-2是E蛋白家族的一员，又名TCF4(Transcription factor 4)，从属于bHLH(basic helix-loop-helix)转录因子超家族[1]。
人E2-2基因有41个外显子，在18号染色体18q21.2。作为单体E2-2没有DNA结合活性，但是和其它蛋白形成同源二聚体或异源二聚体，可以结合DNA并发挥转录活性[2]。E2-2有多个可变剪接的转录本，对应编码不同的蛋白质。Sepp等人研究人E2-2基因的可变剪接转录本调节E2-2基因结构、表达和编码潜力的作用。他们发现人E2-2在脑里表达的特别高，通过使用大量的5’端外显子可以形成不同的蛋白亚型，有18种不同的N端。他们也观察到内部外显子的可变剪接，可形成更多的转录本。功能分析显示，不同的E2-2蛋白亚型有不同的亚细胞定位：含有核定位信号(Nuclear localization signal，NLS)的蛋白亚型定位在细胞核中，其它的在细胞质中也能检测到。没有NLS的蛋白亚型可能通过结合一些含有NLS的bHLH蛋白进入细胞核。没有NLS的亚型还可能通过结合一些含有核输出信号(Nuclearexport signal，NES)的蛋白离开细胞核，被运输到细胞质中[3]。E2-2可以和钙调蛋白结合，可能参与到Ca2+信号的调节。在体外和生理条件一致的Ca2+浓度下，E2-2和E2A蛋白碱性区域的N端序列能结合钙调蛋白，其DNA结合的活性将被抑制[4]。增加胞内Ca2+浓度或者过表达钙调蛋白，都可以抑制E2-2的转录活性。Ca2+信号可能通过形成E蛋白-钙调蛋白复合物阻止E蛋白结合目的DNA[5]。
E2-2主要表达在人浆细胞样树突状细胞(pDCs)中，髓样树突状细胞(DCs)和前体细胞均不表达。在人胸腺前体细胞中过表达E2-2可刺激pDCs的发育，通过RNA干扰抑制E2-2表达可有效抑制前体细胞向pDCs发育[6]。在CD34+造血祖细胞中过表达Id2或Id3可通过抑制E蛋白活性进而抑制pDCs发育，不影响DCs发育[7]。E2-2和SpiB可以协同作用，在前体细胞中同时过表达二者可进一步刺激pDCs发育[6]。研究认为Pitt-Hopkins综合征(PHS)和E2-2单等位基因突变有关[3]。检测病人血液可以发现pDC数量减少，BDCA2基因表达下调，而且产生干扰素的能力被阻断，PHS病人持续的呼吸道感染可能就是由pDC的缺陷引起的[1,8]。
另外，母细胞性浆细胞样树突状细胞淋巴瘤(BPDCN)是一种罕见的起源于浆细胞样树突状前体细胞的恶性肿瘤。BPDCN细胞表达pDCs特异性分子BDCA2、CD123、CD4、TCL1、Bcl11A、CD2AP和Spi-B[9-11]。因为BPDCN细胞表达髓系分子CD56，BPDCN曾被认为是母细胞性NK细胞淋巴瘤，直到pDCs发现以后，根据BPDCN的分子标志和部分功能，将其归为母细胞性浆细胞样树突状细胞淋巴瘤[9]。有文章报道，Spi-B可以作为BPDCN的诊断标志物[12]。
在中枢神经***里，E2-2主要表达在前脑、间脑和小脑中。E2-2对神经胶质细胞分化，尤其是少突胶质细胞前体的成熟，非常重要[13]。在出生后的脊髓中，E2-2仅表达在少突胶质细胞中，随着髓鞘形成逐渐减少。E2-2可以和一系列bHLH转录因子形成异二聚体参与神经***发育的调节，例如ATOH1(Atonal homolog 1，别名MATH1)，在分化中的神经上皮里表达[14]；ASCL1(Achaete-scute complex homolog 1，小鼠中别名MASH-1，人里别名HASH-1)，对中枢神经***特别是前脑神经元回路的形成是必须的[15]；Id1对正确进行神经元发生是必须的[16]；Olig2调节腹侧神经外胚层前体细胞命运，对少突胶质细胞和运动神经元的发育是必须的[13]；NEUROD2(别名NDRF)，对神经元分化和生存非常重要[17]。通过参与到这样一个交错联合的调节网络，E2-2对中枢神经***的发育进行调节。E2-2突变将导致神经发育失调相关的多种疾病[18]。
Pitt-Hopkins综合征是一种罕见的神经发育失调的疾病，病人的学习和记忆能力有严重缺陷，语言学习能力几乎完全缺失，表现为智力残疾，非典型性自闭和过度换气，被称为“孤儿病”。研究认为PHS和E2-2单等位基因突变有关，PHS病人仅有正常量一半的E2-2，这说明PHS对E2-2剂量很敏感。引起PHS的E2-2基因突变是非常多样的，有整个基因的删除，部分基因的删除，读码框移码(包括终止密码子提前出现)，无义突变，剪接位点突变和错义突变。目前为止，几乎所有的病例都有自己独特的E2-2基因突变，只有极少数病例的E2-2基因突变相同。错义突变一般出现在结合DNA调节元件的bHLH结构域，它是一个突变热点，约15％的PHS病例是这种情况。约40％的PHS病例是点突变导致的终止密码子提前出现[19-21]。
遗传流行病学研究显示，精神***症是遗传的可能性高达80％[22]。基因组关联分析发现，E2-2附近两个遗传位点的变异可能有患精神***症的风险。检查精神病人血液中的E2-2水平，是显著减少的。剖析14个精神***症病人组的小脑皮质区，发现和6个对照组相比，E2-2表达是上调的。但是也有研究事后剖析比较三个脑部区域，发现E2-2的mRNA水平和精神***症没有关联[23]。Navarrete等人检测了106个精神***症病人组和96个对照组的淋巴细胞，发现病人组的E2-2mRNA水平比对照组低约20％[24]。
研究显示，Pitt-Hopkins综合征、精神***症、有pDC浸润的实体肿瘤、母细胞性浆样树突状细胞淋巴瘤等疾病，都与pDCs相关，那么如果要鉴定这些疾病，就需要准确快速鉴定pDCs。pDCs的特异性标志物有CD123、BDCA2、Spi-B、E2-2等，CD123即可以识别pDCs，也识别血管内皮细胞；BDCA2和Spi-B虽然有组织化学抗体，但是并没有被广泛应用；而关于E2-2，目前还没有针对其的单克隆抗体。E2-2是pDCs的转录因子，蛋白表达和筛选单克隆抗体非常有难度，我们根据E2-2蛋白的特点，纯化了非全长E2-2的蛋白，利用免疫组化的方法筛选单克隆抗体。筛选得到E2-2单克隆抗体，不仅有利于基础科学的研究，而且在pDCs相关疾病中的鉴定中发挥重要作用。
发明内容
本发明涉及以下各项：
1.一种针对蛋白E2-2的单克隆抗体，所述抗体通过以下步骤制备：
1)重组表达E2-2或其片段；
2)用所述重组表达E2-2或其片段免疫小鼠；
3)将免疫后小鼠的脾细胞与鼠骨髓瘤细胞融合；
4)筛选上清特异识别蛋白E2-2或其片段的单克隆。
2.一种杂交瘤细胞，所述杂交瘤细胞分泌特异识别蛋白E2-2或其片段的单克隆抗体。
3.根据第1项所述的单克隆抗体或权利要求2所述的杂交瘤细胞，其中，所述单克隆抗体利用E2-2片段免疫、筛选得到，所述E2-2片段由SEQ ID NO：1所示核苷酸序列编码。
4.一种抗人E2-2的单克隆抗体杂交瘤细胞株，所述杂交瘤细胞保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.11494，名称为E2-2 2G8。
5.一种针对蛋白E2-2的单克隆抗体，所述抗体由第4项所述的杂交瘤细胞分泌。
6.第1-5任一项所述的抗体或杂交瘤细胞在制备用于检测蛋白E2-2的试剂盒中的用途。
7.第1-5任一项所述的抗体或杂交瘤细胞在制备用于诊断各种pDC相关的疾病的试剂盒中的用途。
8.根据第7项所述的用途，所述疾病包括Pitt-Hopkins综合征、精神***症、有pDC浸润的实体肿瘤、母细胞性浆样树突状细胞淋巴瘤、其它与pDC频率变化相关的疾病。
具体地，本发明利用原核表达***表达E2-2(TCF4)蛋白，免疫Balb/c小鼠，利用ELISA和免疫组化方法，筛选得到一株杂交瘤细胞株E2-2 2G8，能够产生特异性结合人E2-2蛋白的单克隆抗体。在免疫酶学检测(ELISA)、免疫印迹(Western)和免疫组化实验中，E2-22G8抗体能够特异性的结合E2-2。
本发明利用分子克隆方法，克隆得到表达部分基因的E2-2(TCF4)真核表达载体。利用真核表达***表达了部分E2-2(TCF4)的蛋白。用60ugE2-2蛋白联合CpG免疫Balb/c小鼠，获得杂交瘤细胞。利用ELISA和免疫组化方法，筛选得到一株抗人E2-2的单克隆抗体杂交瘤细胞株，为E2-2 2G8，其亚型为IgG2a。该杂交瘤细胞已于2015年11月9日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.11494，名称为E2-2 2G8。通过免疫组化证实其分泌的单克隆抗体E2-2 2G8识别组织中pDCs。
其中本发明人采用了E2-2的片段进行单克隆抗体的筛选，该片段的编码序列如SEQ ID NO：1所示。
CTAGAAATGGAGGACAGGCCTCATCGTCTCCTAATTATGAAGGACCCTTACACTCTTTGCAAAGCCGAATTGAAGATCGTTTAGAAAGACTGGATGATGCTATTCATGTTCTCCGGAACCATGCAGTGGGCCCATCCACAGCTATGCCTGGTGGTCATGGGGACATGCATGGAATCATTGGACCTTCTCATAATGGAGCCATGGGTGGTCTGGGCTCAGGGTATGGAACCGGCCTTCTTTCAGCCAACAGACATTCACTCATGGTGGGGACCCATCGTGAAGATGGCGTGGCCCTGAGAGGCAGCCATTCTCTTCTGCCAAACCAGGTTCCGGTTCCACAGCTTCCTGTCCAGTCTGCGACTTCCCCTGACCTGAACCCACCCCAGGACCCTTACAGAGGCATGCCACCAGGACTACAGGGGCAGAGTGTCTCCTCTGGCAGCTCTGAGATCAAATCCGATGACGAGGGTGATGAGAACCTGCAAGACACGAAATCTTCGGAGGACAAGAAATTAGATGACGACAAGAAGGATATCAAATCAATTACTAGGTCAAGATCTAGCAATAATGACGATGAGGACCTGACACCAGAGCAGAAGGCAGAGCGTGAGAAGGAGCGGAGGATGGCCAACAATGCCCGAGAGCGTCTGCGGGTCCGTGACATCAACGAGGCTTTCAAAGAGCTCGGCCGCATGGTGCAGCTCCACCTCAAGAGTGACAAGCCCCAGACCAAGCTCCTGATCCTCCACCAGGCGGTGGCCGTCATCCTCAGTCTGGAGCAGCAAGTCCGAGAAAGGAATCTGAATCCGAAAGCTGCGTGTCTGAAAAGAAGGGAGGAAGAGAAGGTGTCCTCAGAGCCTCCCCCTCTCTCCTTGGCCGGCCCACACCCTGGAATGGGAGACGCATCGAATCACATGGGACAGATGTAA
利用免疫印迹(Western)方法，证实E2-2 2G8可以特异性结合pDCs细胞系Gen2.2中的E2-2蛋白。
附图说明
图1 E2-2基因表达质粒
图2 E2-2蛋白浓缩的鉴定
上样顺序：
0.蛋白marker
1.BSA 2ug
2.BSA 4ug
3.E2-2half 2ug
4.E2-2half 4ug
图3单克隆抗体E2-2 2G8识别E2-2蛋白
A.Marker
B.E2-2 2G8和内参Tubulin
图4 E2-2 2G8识别扁桃体中的pDC细胞
图5 E2-2 2G8识别银屑病组织中的pDC细胞(IF)
图6 E2-2 2G8识别疾病组织中的pDC细胞
具体实施方式
实施例1质粒构建与蛋白表达
质粒构建与诱导表达
从人脑cDNA文库(Invitrogen)中pCR得到E2-2全长基因，引物序列是5’-GAGTGTCTCCTCTGGCAGC-3’和5’-CCATGTGATTCGATGCGTC-3’，利用XbaI和XhoI内切酶(NEB公司)得到E2-2一段基因(NM_001083962.1片段1700-2628)，克隆至pET28a(invitrogen)中，即pET28a-E2-2half(如图1所示)。。
将构建的质粒转入BL21感受态，涂布平板，挑菌在100ml培养基中摇菌过夜后，接入1L的培养基中(1：100)摇菌3h至OD在0.6左右，降温至16度，加IPTG(1mM)诱导16h收菌。
蛋白的纯化
离心收集诱导好的菌，加入蛋白酶抑制(sigma)，利用超声波破碎，离心收集上清，利用HIS标签镍柱纯化。
对于带His标签的蛋白用镍柱进行纯化，方法如下：(1)组装镍柱：在纯化柱中加入2毫升的纯化介质琼脂糖(GE)。取100uM的硫酸镍10毫升加入介质上层，使液体自然留下。(2)用20毫升的结合缓冲液(20mM Na2HPO4/0.5M NaCl)冲洗镍柱，平衡柱子。(3)将收集上清用结合缓冲液等体积稀释，加入镍柱上，收集流出液体。流出液体可再次加到镍柱上，进行蛋白的再次结合。(4)用100毫升清洗缓冲液(20mM Na2HPO4/0.5MNaCl/10mM咪唑/PH7.4)冲洗镍柱，洗掉非特异性结合蛋白。(5)用10毫升洗脱缓冲液(500mM NaCl/20mM Tris碱/1M咪唑PH 7.9)洗脱蛋白，收集洗脱的流出液，即目的蛋白，作为免疫原的E2-2蛋白。(6)蛋白电泳，考马斯亮蓝染色(如图2所示)检测纯化的蛋白浓度。
实施例2单克隆抗体制备与鉴定
小鼠免疫流程
E2-2的免疫原准备好之后，免疫用来制备单克隆抗体的小鼠。过程如下：(1)准备6～7周的BABL/c小鼠(维通利华)三只。(2)取60ugE2-2蛋白与20mg CpG1826(invitrogen)混合，体积调至500uL生理盐水中混匀，腹腔注射至小鼠体内。(3)每四周免疫一次，完成三次免疫。(4)在第三次免疫后一周采血，用E2-2蛋白进行包被96孔板，ELISA的方法检测血清中的E2-2抗体的滴度。(5)用E2-2蛋白进行第四次加强免疫，3天后进行杂交瘤细胞融合。
杂交瘤细胞融合与筛选
杂交瘤细胞的融合方法：(1)将加强免疫后的小鼠，摘眼球采血，在室温放置30分钟后，然后放在4℃静置过夜。待第二天低速离心，取小鼠的血清分装冻存。(2)在10cm的培养皿中，加入10毫升RPMI 1640培养基。取小鼠脾脏用剪刀剪成细小碎片，然后用无菌盖玻片的磨砂面进行充分研磨，得到细胞悬液。用吸管进行反复吹打，尽量使之成为单细胞悬液。用0.45um的滤膜进行过滤至50毫升离心管中。(3)离心后，弃掉上清液，轻弹细胞沉淀使疏松，加入2毫升红细胞裂解液处理。2分钟后加入50毫升RPMI1640培养基，快速离心。用PBS洗两遍后进行细胞计数，约3×108个脾细胞。(4)取4个T75的sp20细胞，细胞计数得到sp20细胞约3×108个。PBS洗三遍，200g/分钟室温离心10分钟。(5)将sp20细胞和脾细胞按1:1～1:3的比例进行混合。1500转/分钟，室温离心10分钟。弃掉上清，轻弹细胞沉淀使疏松。(6)准备37℃水浴，将细胞置于水浴中，边晃动50毫升离心管，边逐滴加入预热的2毫升PEG2000。(7)加入预热的40毫升RPMI 1640培养基重悬细胞。上下颠倒使细胞混匀。1500转/分钟，室温离心10分钟。(8)去掉上清，置细胞于37℃水浴中，加入含HAT的完全培养基10毫升/板，上下颠倒混匀。每孔加入2滴液体约100微升。(9)培养约一周左右，待每个克隆增殖到10个细胞左右，进行ELISA检测。
ELISA方法检测
ELISA方法检测上清中的E2-2抗体，(1)用实施例1中制备的E2-2蛋白包被96孔ELISA板，蛋白用PBS稀释至终浓度5μg/mL，每孔加入100μL，4℃包被过夜。(2)用洗液(PBS/0.05tween20/PH7.5，sigma)清洗一次，每孔加入200μL封闭液(PBS/0.5％BSA/PH7.5)，室温封闭2h。(3)用洗液清洗三次，加入100μL免疫E2-2蛋白的小鼠血清(需要按照10倍稀释，即10000,50000，100000,500000,1000000)和100μL相应的待测样品(即细胞培养上清)，摇床混匀，100转/min室温作用2h。(4)用洗液清洗三次，每孔加入100μL生物素标记的检测山羊抗小鼠IgG的抗体(中杉金桥)，室温作用1h。(5)用洗液清洗三次，每孔加入100μLstreptavidin标记的辣根过氧化物酶试剂(中杉金桥)，室温作用1h。(6)用洗液清洗三次，每孔加入100μL TMB底物，摇床混匀，100转/min室温作用10min。待最高浓度的标准蛋白孔呈现深蓝色，每孔加入50μL终止液(2M硫酸)。(7)分光光度计记录450.1nm波长下的光吸收值，即OD值，选择高于小鼠多抗值用于免疫组化检测(小鼠多抗来源于免疫E2-2蛋白的小鼠血清)。
通过ELISA方法，我们筛选得到E2-2抗体1G4、1G6、2G8、4B5和10F7克隆，OD值明显高于小鼠多抗。为了进一步验证抗体，进行了免疫组化实验。
SDS-PAGE和免疫印迹实验
(1)蛋白样品加入5×SDS buffer，100℃煮样5min，进行SDS-PAGE电泳。
(2)如无需做western实验，电泳完成后蛋白胶直接用考马斯亮蓝染色，脱色后观察实验结果(图2实验)，如需进行western实验，则进入步骤(3)。
(3)转膜：将样品从胶上转移到PVDF膜上，250mA恒流，2h。
(4)封闭：PVDF膜室温下用Blocking buffer(PBS/0.5％BSA/PH7.5)封闭1h。
(5)一抗孵育：加入适量一抗抗体(E2-2 2G8：浓度为1-5ng/mL)，4℃孵育过夜。
(6)漂洗：用TBST清洗PVDF膜三次，每次10min。
(7)二抗孵育：加入对应的二抗(辣根过氧化物酶标记的山羊抗小鼠IgG的抗体，中杉金桥)，室温孵育1h。
(8)漂洗：用TBST清洗PVDF膜三次，每次10min，最后用TBS清洗一次。
(9)洗片：PVDF膜用辣根过氧化物酶的底物(密理博公司)孵育3min，在暗室中经压片，显影，定影，清洗，晾干等步骤得到实验结果(图3)。
免疫组化方法检测
ELISA检测OD值较高的克隆1G4、1G6、2G8、4B5和10F7，用免疫组织化学染色进一步检测。因为pDCs细胞高表达E2-2蛋白，所以选用扁桃体组织检测E2-2。(1)脱蜡和水化：二甲苯Ⅰ、二甲苯Ⅱ、二甲苯Ⅲ、二甲苯Ⅳ中各浸泡5分钟，无水乙醇Ⅰ、无水乙醇Ⅱ、95％乙醇、75％乙醇中各浸泡5分钟。PBS洗2次各5分钟。(2)抗原修复，蒸锅加热40min，室温平衡60分钟。PBS洗2次各5分钟。(3)3％H2O2(80％甲醇稀释)处理，室温静置10分钟。PBS洗2次各5分钟。(4)滴加10％正常山羊血清封闭液，室温30分钟，甩去多余液体。(5)滴加杂交瘤培养上清，4℃过夜。PBS洗3次每次5分钟(如果4℃过夜后需在室温复温30分钟)。(6)滴加二抗(中杉：P6001/6002)，37℃30分钟。PBS洗3次各5分钟。(7)DAB显色1-10分钟，在显微镜下掌握染色程度。自来水冲洗30秒，可以数1至30。(8)苏木精复染30秒。自来水冲洗30秒，可以数1至30。(9)脱水、透明：75％乙醇、95％乙醇、无水乙醇Ⅱ、无水乙醇Ⅰ、二甲苯Ⅳ、二甲苯Ⅲ、二甲苯Ⅱ、二甲苯Ⅰ中各浸泡3分钟。10)中性树胶封片。
结果显示，E2-2 2G8能够识别人扁桃体组织中的E2-2蛋白，并与pDC细胞标志物CD123共染，说明E2-2 2G8能够识别人扁桃体组织中pDC细胞(结果见图4)。
杂交瘤细胞扩增和腹水制备
得到阳性克隆后，对细胞进行亚克隆直到得到单克隆抗体。单克隆的杂交瘤细胞株即可进行大规模培养或免疫小鼠取腹水制备抗体。腹水制备方法如下：(1)取6～8周的NOG(NOD/SCID/IL-2Rγnull)免疫缺陷型小鼠(Jackson Lab)，提前一周腹腔注射0.5毫升降脂烷。(2)筛选得到的阳性杂交瘤克隆，待克隆增殖到50％覆盖率，转移至24孔板中继续培养，扩大培养至T75培养瓶中。取适量的杂交瘤细胞进行冻存。(3)从T75培养瓶中收获杂交瘤细胞，1500转/分钟，室温离心5分钟。(4)弃掉上清，加入PBS重悬细胞，1500转/分钟，室温离心5分钟。PBS重复洗一次，然后用PBS将细胞重悬至4×106个/毫升。(5)将杂交瘤细胞腹腔注射至降脂烷处理一周后的小鼠体内。(6)1～2周后，小鼠腹腔膨大，脱颈处死小鼠，用10毫升注射器抽取腹水，每只小鼠得到2～5毫升腹水。(7)低温高速离心，取上清分装至新的EP管中，-80℃冻存。
单克隆的抗体纯化
用Protein G从腹水中纯化抗体，操作步骤如下：(1)将protein G的预装柱(GE)和纯化所需的缓冲液平衡至室温。(2)将抗体用结合缓冲液(20mM sodium phosophatebuffer,pH 7.0)等体积稀释。取1毫升腹水，加入1毫升结合缓冲液。(3)将protein G的预装柱置于15毫升离心管中，使柱中的存储缓冲液自然流出。加入5毫升结合缓冲液，平衡protein G的预装柱。(4)将稀释后的腹水加入柱上，收集流出液体。(5)加入15毫升结合缓冲液清洗柱子，收集前2毫升清洗液(0.1M glycine,pH 2.7-3.0)和最后2毫升清洗液。(6)取5个EP管，每只管中加入100微升中和缓冲液(1M Tris-HCl,pH 9)。加入5毫升洗脱缓冲液，每管收集1毫升洗脱后液体迅速混匀，防止局部pH过低而导致蛋白失去活性。(7)对纯化后的抗体通过考马斯亮蓝染色检测纯度。
免疫荧光染色
(1)脱蜡和水化：二甲苯Ⅰ、二甲苯Ⅱ、二甲苯Ⅲ、二甲苯Ⅳ中各浸泡5分钟，无水乙醇Ⅰ、无水乙醇Ⅱ、95％乙醇、75％乙醇中各浸泡5分钟。PBS洗2次各5分钟。(2)抗原修复，蒸锅加热40min，室温平衡60分钟。PBS洗2次各5分钟。(3)3％H2O2(80％甲醇稀释)处理，室温静置10分钟。PBS洗2次各5分钟。(4)滴加10％正常山羊血清封闭液，室温30分钟，甩去多余液体。(5)滴加一抗(E2-2抗体，E2-2 2G8),室温静置1小时或4℃过夜。PBS洗3次每次5分钟(如果4℃过夜后需在室温复温30分钟)。(6)滴加荧光二抗(Alesa Fluor 488标记山羊抗小鼠IgG抗体，invitrogen)，37℃30分钟。PBS洗3次各5分钟。(7)DAPI染色1min，PBS洗3次各3分钟。(8)用抗荧光衰减封片剂封片，周围用指甲油在封一圈。4℃保存或荧光显微镜观察。结果显示，克隆E2-2 2G8可以用免疫荧光的方法识别人银屑病皮肤组织中的pDC细胞(结果见图5)。
实施例3单克隆抗体识别组织中pDCs的研究
利用免疫组化方法，验证E2-2抗体E2-2 2G8在各种组织中的pDCs细胞的表达，方法如前。分别在扁桃体组织、肝癌组织、胰腺癌***、乳腺癌***、Peyer’s patches(结肠癌)和BPDCN皮肤组织上鉴定了E2-2阳性pDCs(结果见图6)。
参考文献
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Claims (3)
1.一种抗人E2-2的单克隆抗体杂交瘤细胞株，所述杂交瘤细胞保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.11494，名称为E2-2 2G8。
2.一种针对蛋白E2-2的单克隆抗体，所述抗体由权利要求1所述的杂交瘤细胞分泌。
3.权利要求1-2任一项所述的抗体或杂交瘤细胞在制备用于检测蛋白E2-2的试剂盒中的用途。
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