CN106636007B - 抗USP2a蛋白单克隆抗体杂交瘤细胞及其产生的抗USP2a单克隆抗体和应用 - Google Patents
抗USP2a蛋白单克隆抗体杂交瘤细胞及其产生的抗USP2a单克隆抗体和应用 Download PDFInfo
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Abstract
本发明公开了一种抗USP2a蛋白单克隆抗体杂交瘤细胞及其产生的抗USP2a单克隆抗体和应用。本发明公开的一株杂交瘤细胞株，命名为杂交瘤细胞株3D9，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：C2016186；该杂交瘤细胞株3D9分泌产生了抗USP2a的单克隆抗体；该抗USP2a的单克隆抗体可以应用在肿瘤组织切片免疫组织化学检测中。本发明的杂交瘤细胞株3D9分泌产生的抗USP2a的单克隆抗体具有效价高、性质稳定和特异性强等特点。
Description
一、技术领域：
本发明涉及一种抗USP2a单克隆抗体及其应用，属于分子免疫学技术领域；具体涉及一种抗USP2a蛋白单克隆抗体杂交瘤细胞及其产生的抗USP2a单克隆抗体和在肿瘤组织切片免疫组织化学检测中的应用。
二、背景技术：
USP2又称泛素特异性蛋白酶2，是去泛素化酶家族，泛素特异性蛋白酶的成员，USP2基因定位于11号染色体长臂(11q23.3)，最早是在大鼠睾丸中经克隆鉴定出来的。USP2基因经过5’末端的选择性剪切可产生两个不同的亚基USP2a(USP2-69；UBP-t2)和USP2b(USP2-45；UBP-t1)，但它们都拥有基因3’末端的催化结构域。
研究证明，当USP2a在未转变的细胞中超表达时，显示出致癌性，并且能阻止化疗因子诱导的凋亡；而在多种肿瘤细胞株中，USP2a基因沉默能诱导细胞凋亡。USP2a在人前列腺癌细胞中高表达，高表达的USP2a能够稳定脂肪酸合成酶(fatty acid synthase:FAS)，FAS在包括前列腺癌在内的许多恶性肿瘤中超表达，并能促进细胞增生，并且FAS的表达与前列腺癌的级别密切相关。通过siRNA作用下调USP2a的表达后发现，FAS含量降低，导致细胞凋亡。另外USP2a的异常表达能够使MDM2以剂量依赖形式聚集，同时能促进MDM2介导的p53降解，而抑制USP2a则能使MDM2不稳定，从而引起p53蛋白的聚集和激活。除此之外，在膀胱癌、卵巢癌和胃癌等的研究中均发现USP2a与癌细胞增生和迁移密切相关。这些实验结果都证明USP2a与肿瘤的发生、发展有重要的联系，USP2a是治疗肿瘤的又一个重要靶点。
USP2a单克隆抗体药物具有广泛的应用前景，可用于多个类型肿瘤的检测和治疗，主要包括：前列腺癌、膀胱癌、卵巢癌和胃癌等。因此，开发与USP2a具有高亲和力的抗体药物，用于癌症的检测和免疫治疗，使其具有更好的治疗效果、更低的毒副作用具有重要的意义。然而，目前现有的USP2a抗体药物亲和力低，尚缺少一种具有高亲和力的USP2a抗体。
三、发明内容：
本发明要解决的技术问题是：提供一种抗USP2a蛋白单克隆抗体杂交瘤细胞株(命名为杂交瘤细胞株3D9)及其产生的抗USP2a单克隆抗体和应用。本发明的杂交瘤细胞株3D9分泌产生的抗USP2a的单克隆抗体具有效价高、性质稳定和特异性强等特点。
本发明要解决的技术问题是采用如下技术方案实现的：
本发明提供了杂交瘤细胞株3D9，该杂交瘤细胞株3D9已于2016年11月1日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址：中国，武汉，武汉大学，保藏编号为CCTCCNO：C2016186；经检测为存活。
本发明提供了抗USP2a的单克隆抗体，它是由保藏编号为CCTCC NO：C2016186的杂交瘤细胞株3D9分泌产生。
本发明还提供了抗USP2a的单克隆抗体在肿瘤组织切片免疫组织化学检测中的应用。
本发明抗USP2a单克隆抗体的制备方法，主要步骤为：克隆重组USP2a基因，表达USP2a融合蛋白，以USP2a融合蛋白作为抗原免疫BALB/c小鼠，用免疫小鼠的脾细胞与骨髓瘤细胞融合，采用间接酶联免疫吸附法(ELSA)筛选产生USP2a单克隆抗体的阳性杂交瘤细胞株，利用所得阳性杂交瘤细胞株进行克隆化，并采用间接酶联免疫吸附法(ELSA)筛选克隆化后的杂交瘤细胞产生的USP2a单克隆抗体的效价，选取效价最高的杂交瘤细胞株即得到杂交瘤细胞株3D9，将所得的杂交瘤细胞株3D9腹腔注射BABL/c小鼠，收集腹水即得到USP2a单克隆抗体。
本发明对USP2a单克隆抗体制备过程中涉及到的几个方面进行以下详细描述：
1)USP2a具体是指在前列腺癌、膀胱癌、卵巢癌和胃癌等多种肿瘤组织中表达异常的USP2a基因，该基因在未转变的细胞中超表达时，显示出致癌性，并且能阻止化疗因子诱导的凋亡；普通的检测方法速度较慢、耗时较长和特异性不佳。本发明在制备USP2a单克隆抗体的方法中通过PCR扩增获得USP2a基因，构建USP2a重组克隆，并优化表达条件使重组蛋白实现分泌型表达；分泌型表达的USP2a融合蛋白空间结构接近天然蛋白，具有很高的酶活性能够激发免疫细胞产生特异性抗体。
实验证明，通过本发明所述方法制备的USP2a单克隆抗体具有效价高、性质稳定和特异性强等特点。
2)重组蛋白的制备与纯化过程：
按照NCBIGenBank上公布的USP2amRNA编码序列(GenBank ID：BC002854)进行分析，设计引物(上游引物：5’GGAATTCATGAATTCTAAGAGTGCCCAGG3’，下游引物：5’CCGCTCGAGCTACATTCGGGAGGG3’)，在上、下游引物上分别加入EcoRI和XhoI酶切位点，以前列腺癌细胞cDNA为模板进行PCR扩增，获得USP2a基因，采用EcoRI和XhoI分别双酶切PCR产物和载体pET28a(+)，之后进行连接、筛选，获得重组质粒pET28a-USP2a，重组质粒pET28a-USP2a在大肠杆菌BL21(DE3)感受态细胞中进行表达，优化表达条件，在30℃温度条件下进行表达，离心收集细胞菌体，超声破碎，离心弃沉淀，上清经过镍离子亲和层析纯化收集，进一步浓缩，获得纯化的USP2a融合蛋白。
3)USP2a单克隆抗体的制备过程为：
(a)将纯化得到的USP2a融合蛋白作为抗原与福氏完全佐剂充分乳化后皮下注射小鼠，再分别于3周、6周后用USP2a融合蛋白作为抗原与福氏不完全佐剂充分乳化后各免疫1次，8周后用相同剂量的USP2a融合蛋白作为抗原腹腔注射小鼠；
(b)最后一次注射3天后，取上述免疫小鼠的脾细胞进行细胞融合；骨髓瘤细胞与脾细胞按1：10的比例混合在一起，在50mL离心管中用无血清1640不完全培养基洗涤1次，1200rpm离心8min，弃上清，轻轻弹击离心管底，使细胞沉淀略松动；90s内加入37℃、预温的1mL45％聚乙二醇PEG(分子量4000)溶液(PEG的温度为37℃)，边加边轻微摇动；之后在37℃水浴中作用90s；再分别加入37℃预温的1640不完全培养基1mL、2mL、3mL、4mL、5mL和6mL以终止PEG作用，每次加入的时间间隔为2min；800rpm离心6min，弃上清，得到细胞；用含20％小牛血清HAT选择培养基对离心所得细胞进行重悬；将重悬后的细胞，加到已有饲养细胞层的96孔板内，每孔加100μL；将培养板置37℃、5％CO2培养箱中进行培养；
(c)利用间接ELISA检测方法，对步骤(b)中96孔板内每个孔中细胞的培养液中的抗体进行检测，筛选出抗体阳性杂交瘤细胞。
(d)采用有限稀释法对步骤(c)筛选出的抗体阳性杂交瘤细胞进行克隆化，先将要克隆的杂交瘤细胞计数，调整细胞浓度为3～10个细胞/mL；取头天准备的具有饲养细胞层的细胞培养板，每孔加入调整浓度后的细胞100μL；孵育于37℃、5％CO2培养箱中；在第7天换液，以后每2～3天换液1次；8～9天可见细胞克隆形成，利用间接ELISA检测方法检测每个孔中细胞培养液中的抗体活性，将阳性孔的细胞移至24孔板中扩大培养；扩大培养后得到杂交瘤细胞；
(e)取小鼠，以0.5mL/只的剂量腹腔注射液体石蜡，1周后以1×106CFU/只的剂量对小鼠腹腔注射步骤(d)得到的杂交瘤细胞，而后从小鼠腹腔采集腹水，离心取上清，即得到USP2a单克隆抗体；
(f)采用Southern biotech小鼠单抗分型ELISA试剂盒测定步骤(e)得到的USP2a单克隆抗体，杂交瘤细胞株3D9产生的单抗为IgG1；利用亲和层析法对产生的单抗进行纯化，获得纯化的USP2a单克隆抗体；免疫印迹和免疫组化实验显示纯化的USP2a单克隆抗体能特异性识别USP2a融合蛋白以及高表达USP2a的前列腺癌组织。
本发明USP2a抗原即USP2a融合蛋白是指由具有序列表中SEQ ID NO.1的核苷酸序列编码；所述的USP2a融合蛋白抗原，该融合蛋白由全长为348个氨基酸的USP2a蛋白以及用于融合蛋白纯化的六个组氨酸标签组成，具体融合蛋白全长为序列表中SEQ ID NO.2所示的氨基酸序列。
本发明上述制备方法中采用的免疫小鼠是6～8周龄，体重18～20g的雌性BALB/c小鼠；采用的完全或者不完全培养基是1640培养基；
采用的间接ELISA检测方法，阳性对照为免疫小鼠的血清，阴性对照为非免疫小鼠的血清。
四、附图说明：
图1USP2a融合蛋白的蛋白表达SDS-PAGE电泳图；
图1中：1、未诱导的菌液，2、诱导后的全菌液，3、超声破碎后的上清液，4：Marker。
图2纯化的USP2a融合蛋白的SDS-PAGE电泳图；
图2中：1、诱导后的全菌液，2、超声破碎的离心的上清液，3、超声破碎的离心的沉淀，4、洗脱液1，5、洗脱液2，6、洗脱液3，7、纯化的USP2a蛋白，8、Marker。
图3：纯化的USP2a融合蛋白的Weston blot分析图；
图4：单克隆抗体USP2a的免疫组化分析图；
图4中，A阴性对照图，B前列腺癌染色结果图。
五、具体实施方式：
下面结合具体实施例对本发明作进一步的说明，但本发明的内容不受以下实施例的限制。
下面实施例中所采用的实验材料、实验试剂和仪器，未经特殊说明，均为本领域中常规的材料、试剂和仪器，均可通过商业途径获得。
实施例1：
本发明一株杂交瘤细胞株3D9的制备方法，该制备方法的详细步骤为：
(一)USP2a基因的克隆、表达和纯化：
1、USP2a基因克隆：USP2a按照NCBIGenBank上公布的登录号BC002854的USP2amRNA编码序列的参考序列所定义的编码区DNA序列，设计引物：(上游引物：5’GGAATTCATGAATTCTAAGAGTGCCCAGG3’，下游引物：5’CCGCTCGAGCTACATTCGGGAGGG3’)；在上、下游引物上分别加入EcoRI和XhoI酶切位点；以前列腺癌细胞cDNA为模板，进行PCR扩增，PCR扩增反应体系如下：采用25μl反应体系：2.5μl10×PCR反应缓冲液，上游引物、下游引物各1μl(100ng)，dNTP1μl(50mM)，cDNA模板1μl(1ng)，TaqDNA聚合酶1μl(2.5U)，加ddH2O17.5μl补至总体积25μl；PCR扩增反应条件如下：第一步预变性：95℃5min；第二步循环扩增：95℃30s、58℃45s、72℃1min，25个循环；第三步延伸：72℃8min；PCR扩增完成后进行琼脂糖凝胶电泳，即可获得USP2a基因；将PCR扩增得到的USP2a基因与载体pET28a(+)用EcoRI和XhoI双酶切，电泳、回收，用T4连接酶对回收片段进行连接；所得连接产物转化大肠杆菌BL21(DE3)感受态细胞，挑取平板上的克隆菌落，进行接种，提取质粒DNA，并对提取的质粒DNA进行PCR和酶切鉴定；对鉴定结果显示阳性的克隆进行测序分析，将测序完全正确的克隆进行菌种保存；
2、USP2a蛋白的蛋白表达：
2.1蛋白表达条件的优化：选取步骤1中保存的菌种，用接种针蘸取菌液，接种到3mL含有卡那霉素的LB液体培养基中，共接种4支菌液，标记序号，37℃过夜培养，第二天扩大培养到100mL含有卡那霉素的LB液体培养基中，分别采用25℃、30℃、37℃和42℃四个不同的温度，180rpm继续培养；检测OD＝0.6-0.8时进行IPTG诱导，IPTG浓度为0.8mM，诱导4h后收集菌液；所得菌液在5000rpm条件下离心5min，弃上清，所得沉淀用100mmol/L的PBS缓冲液(PH＝7.4)(PBS缓冲液的配制：0.2mmol/L Na2HPO4溶液12.3mL和0.2mmol/LNaH2PO4溶液的87.7mL混合后调节PH值到7.4，加蒸馏水稀释至200mL即可)洗涤2-3遍，然后加入10mL裂解液(裂解液的成分：50mMTris-HCl，pH8.0，2mM EDTA，100mM NaCl，加溶菌酶至100ug/ml，0.1％TritonX-100)重悬菌体，超声破碎(超声破碎条件：300w，每次10s，间隔10s，共超声10分钟)，超声后的菌液在12000rpm条件下离心10min，离心所得上清转移到新的离心管中，取80μl上清加入20μl5×SDS-PAGE上样缓冲液(上样缓冲液的成分：60mmol/LTris-HCl，pH6.8、2％SDS，0.1％溴酚兰，25％甘油，14.4mmol/Lβ-巯基乙醇)，混匀；所得沉淀用100μl裂解液(同上)重悬，取80μl加入20μl5×SDS-PAGE上样缓冲液，混匀；将加入SDS-PAGE上样缓冲液混匀后的上清和沉淀一起放入100℃沸水浴中煮10min，通过SDS-PAGE电泳检测USP2a融合蛋白的表达量与表达方式；结果显示USP2a蛋白在30℃时主要表达方式为可溶性的分泌型表达，表达量也较高；因此，选用30℃的温度条件，延长诱导时间，进行下一步的大量表达；
2.2蛋白的大量表达：选取步骤1中保存的菌种，用接种针蘸取菌液，接种到5mL含有卡那霉素的LB液体培养基中，37℃过夜培养；第二天扩大培养到500mL含有卡那霉素的LB液体培养基中，30℃、180rpm继续培养，检测OD＝0.6-0.8时进行IPTG诱导，IPTG浓度为0.8mM，诱导6h后收集菌液；所得菌液在5000rpm条件下离心5min，弃上清，所得沉淀用PBS缓冲液(PBS缓冲液的配制同上)洗涤2-3遍，然后加入30mL裂解液(裂解液的成分同上)重悬菌体，超声破碎(300w，每次10s，间隔10s，共超声20分钟)，超声后的菌液在12000rpm条件下离心10min，上清转移到新的离心管中，取80μl上清加入20μl5×SDS-PAGE上样缓冲液，混匀；所得沉淀用500μl裂解液重悬，取80μl加入20μl5×SDS-PAGE上样缓冲液，混匀；将加入SDS-PAGE上样缓冲液混匀后的上清和沉淀一起在100℃沸水条件下煮10min，SDS-PAGE电泳检测USP2a融合蛋白的表达量与表达方式(详见附图1)；结果显示USP2a融合蛋白主要表达方式为分泌型表达，表达量也较高；
3、蛋白纯化：蛋白纯化采用北京索莱宝生物科技有限公司生产的蛋白纯化试剂盒(His-Bind Purification Kit)：
取1.5～2mL His-Beads(组氨酸珠子)用10mL去离子水清洗，10mL1×ChargeBuffer(平衡缓冲液)和10mL1×Binding Buffer(结合缓冲液)处理以平衡层析柱，而后将步骤2中超声破碎后离心后得到的上清液与His-Beads(组氨酸珠子)混合，置于混合器上，4℃、旋转30min使之混匀；将上清与His-Bead的混合液加入到柱子中，并固定在三脚架上，收集穿出液标记为Flow through，之后用10mL1×Binding Buffer洗层析柱，收集穿出液到一新管中，标记为Binding through，10mL1×Wash Buffer(洗涤缓冲液)洗柱，同样收集穿出液到一新管中，标记为Wash through，最后用0.5×Elute Buffer(洗脱缓冲液)洗柱，流出液即为USP2a目的蛋白；用SDS–PAGE蛋白电泳验证纯化的蛋白质量(详见附图2)；
4、USP2a纯化蛋白的western blot检测(检测结果详见附图3)：
A、取少量步骤3纯化后所得的USP2a目的蛋白，先进行SDS–PAGE电泳；
B、电泳结束后取出凝胶制作转膜装置“转膜三明治”，准备转膜，所选用的膜为PVDF膜；
C、将做好的“转膜三明治”放到转膜槽中，加入转膜缓冲液，连接转膜仪160V恒压，转膜60-90min；
D、封闭：取出PVDF膜，放入到预先准备好的PBST洗涤液中(PBST洗涤液0.15mol/LpH7；配制方法：0.2gKH2PO4，2.9gNa2HPO4·12H2O，8.0gNaCl，0.2gKCl，Tween-200.5mL，加蒸馏水至1000mL)，漂洗3次，每次5-10min，以洗去膜上的转膜液；加入Western封闭液(含有5％脱脂奶粉的PBST缓冲液)，在摇床上缓慢摇动，4℃下封闭过夜；
E、一抗孵育：倒去封闭液，用PBST洗涤液清洗3次，每次5-10min，之后加入1:1000被稀释好的一抗，室温孵育1小时；
F、二抗孵育：倒去一抗，用PBST洗涤液清洗3次，每次5-10min，加入稀释好的二抗，室温孵育30min-1h；
G、显色：倒去二抗，用PBST洗涤液清洗5次，每次5-10min，清洗完成后，转入暗室准备显色，用ECL显色液显色；
(二)动物免疫：
1、免疫动物：选取健康的6～8周龄的BALB/c小鼠进行免疫；取实施例1制备的可溶性USP2a目的蛋白作为抗原蛋白与等量福氏完全佐剂乳化均匀后，通过背部皮下注射每只小鼠6点，剂量为50μg/每只；每3周加强免疫一次，免疫剂量同第一次；加福氏不完全佐剂，背部皮下多点注射；第三次免疫10天后取血，以间接ELISA方法检测抗体效价；效价达到要求后进行冲刺免疫，间隔上一次免疫时间为3周后，剂量为100μg/每只，不加佐剂，腹腔注射；3天后，取脾进行细胞融合；
2、骨髓瘤细胞悬液制备：在上述步骤1细胞融合前36-48小时，将NS0骨髓瘤细胞扩大培养，按一块96孔板的融合试验约需2-3瓶100ml培养瓶培养的细胞进行准备；融合前一天传代一次，使融合当天NS0骨髓瘤细胞处于对数生长期，用弯头滴管将细胞从瓶壁轻轻吹下，收集于50ml离心管中，1000r/min离心5-10分钟，弃上清，加入30ml、1640不完全培养基，同法离心洗涤一次，然后将细胞重悬于10ml、1640不完全培养基中，混匀；取骨髓瘤细胞悬液，加0.4％台盼蓝染液做活细胞计数后备用；
3、脾淋巴细胞的准备：取步骤1中3天前冲刺免疫的BALB/c鼠，摘除眼球采血，并分离血清作为抗体检测时的阳性对照血清；颈椎脱位致死小鼠，浸泡于75％酒精中5分钟，于解剖台板上固定后，掀开左侧腹部皮肤，在超净台中用无菌手术剪剪开腹膜，取出脾脏置于已盛有10ml、1640不完全培养基的平皿中，轻轻洗涤，并细心剥去周围结缔组织；将洗涤后的脾脏移入另一盛有10ml、1640不完全培养基的平皿中，用注射器内芯挤压脾脏，使脾细胞分散，用吸管吹打数次，用200目铜网过滤，制成单细胞悬液；1000r/min离心10分钟，用1640不完全培养基离心洗涤2次，然后将细胞重悬于10ml、1640不完全培养基中，混匀，取上述悬液，加台酚蓝染液作活细胞计数后备用；
4、细胞融合：将骨髓瘤细胞与脾细胞按1：10的比例混合在一起，在50ml离心管中用1640无血清不完全培养基洗1次，1200rpm离心8min，弃上清，轻轻弹击离心管底，使细胞沉淀略松动；在90s内加入37℃预温的1ml45％聚乙二醇PEG(分子量4000)溶液，边加边轻微摇动；之后在37℃水浴中作用90s；作用后分别加入1ml、2ml、3ml、4ml、5ml和6ml37℃预温的1640不完全培养基以终止PEG作用，加入的时间间隔为每2min加一次；全部加完后800rpm离心6min，弃上清，所得沉底用含20％小牛血清的HAT选择培养基重悬；将重悬后的细胞加到已有饲养细胞层的96孔板内，每孔加100μl细胞悬液；将培养板置于37℃、5％CO2培养箱中培养；
(三)杂交瘤细胞筛选：
1、阳性杂交瘤细胞的筛选：HAT选择培养基培养7～10天后，换用HT培养基，培养2周，然后使用1640培养基培养；在上述选择培养期间，杂交瘤细胞布满孔底1/10面积时，采用间接ELISA方法进行抗体检测，以P/N≥2.1作为阳性判断的标准，免疫小鼠的血清为阳性对照，非免疫小鼠的血清做为阴性对照，筛选出阳性杂交瘤细胞；
2、杂交瘤细胞的克隆化：采用有限稀释法对步骤1中筛选出的阳性杂交瘤细胞进行克隆化，克隆前1天制备饲养细胞层，将要克隆的步骤1中筛选出的阳性杂交瘤细胞从培养孔内轻轻吹起，计数，调整细胞浓度为3～10个细胞/ml；取头天准备的有饲养细胞层的细胞培养板，每孔加入调整浓度后的细胞100μl；孵育于37℃、5％CO2培养箱中；在第7天换液，以后每2～3天换液1次，采用的培养液为1640培养基；8～9天可见细胞克隆形成，及时检测细胞克隆产生的抗体活性，将阳性孔的细胞移至24孔板中扩大培养，并及时冻存；
(四)USP2a单克隆抗体效价检测：
取上述步骤得到的在24孔板中扩大培养的杂交瘤细胞，取24孔板中细胞的培养液进行梯度稀释，采用间接ELISA的方法测定OD450值，同时设立阳性和阴性对照，以P/N≥2.1作为阳性判断的标准；经测定获得一株阳性杂交瘤细胞，命名为杂交瘤细胞株3D9(杂交瘤细胞的培养液抗体效价见表1；由表1可知，筛选出的这株杂交瘤细胞的培养液中抗体效价较高为1:3200)。
表1杂交瘤细胞株上清效价表
实施例2：
本发明USP2a的单克隆抗体制备方法，该制备方法的详细步骤如下：
(一)腹水的制备：
腹腔注射0.5ml液体石蜡于BALB/c小鼠，1周后腹腔注射含1×106个/ml实施例1所制备的杂交瘤细胞株3D91ml，7天后开始收集腹水；
(二)采用Southern biotech小鼠单抗分型ELISA试剂盒测定收集的腹水中的USP2a单克隆抗体的亚型，杂交瘤细胞株3D9产生的单抗为IgG1；利用亲和层析法对产生的单抗进行纯化，获得纯化的USP2a单克隆抗体。
实施例3：USP2a单克隆抗体的应用实施：
USP2a单克隆抗体的免疫组化应用检测：
(1)选取成人前列腺癌组织放入福尔马林液中固定12-24h；
(2)将固定好的人前列腺癌组织块进行石蜡包埋、切片，切片厚度为5μm；
(3)脱蜡与水化：将组织切片在室温中放置60分钟，于二甲苯中浸泡10分钟，更换二甲苯后再浸泡10分钟；无水乙醇中浸泡5分钟，更换无水乙醇再浸泡5分钟；95％乙醇中浸泡5分钟；70％乙醇中浸泡5分钟；
(4)抗原修复：电炉水浴加热0.01mol/L枸橼酸钠缓冲溶液(pH6.0)至95℃左右，放入组织切片加热10-15分钟，取出标本，自然冷却至室温，去离子水浸泡5min，重复3次；
(5)PBS缓冲液洗涤3次，每次5min；
(6)封闭：PBS+5％脱脂奶粉，室温20min，甩去多余液体；
(7)滴加USP2a单克隆抗体50μl，37℃1小时；
(8)PBS洗3次，每次5分钟；
(9)滴加Ⅱ抗(兔抗鼠)50μl，37℃1小时；
(10)PBS缓冲液洗涤3次，每次5分钟；
(11)DAB显色5～10分钟，自来水冲洗10分钟；
(12)苏木精复染2分钟，1％盐酸酒精分化，自来水冲洗10分钟；
(13)脱水、透明、封片、镜检(详见附图4)。
SEQUENCE LISTING
<110> 新乡学院
<120> 抗USP2a蛋白单克隆抗体杂交瘤细胞及其产生的抗USP2a单克隆抗体和应用
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1047
<212> DNA
<213> Artificial Sequence
<220>
<223> 融合蛋白
<400> 1
atgaattcta agagtgccca gggtctggct ggtcttcgaa accttgggaa cacgtgcttc 60
atgaactcaa ttctgcagtg cctgagcaac actcgggagt tgagagatta ctgcctccag 120
aggctctaca tgcgggacct gcaccacggc agcaatgcac acacagccct cgtggaagag 180
tttgcaaaac taattcagac catatggact tcatccccca atgatgtggt gagcccatct 240
gagttcaaga cccagatcca gagatatgca ccgcgctttg ttggctataa tcagcaggat 300
gctcaggagt tccttcgctt tcttctggat gggctccata acgaggtgaa ccgagtgaca 360
ctgagaccta agtccaaccc tgagaacctc gatcatcttc ctgatgacga gaaaggccga 420
cagatgtgga gaaaatatct agaacgggaa gacagtagga tcggggatct ctttgttggg 480
cagctaaaga gctcgctgac gtgtacagat tgtggttact gttctacggt cttcgacccc 540
ttctgggacc tctcactgcc cattgctaag cgaggttatc ctgaggtgac attaatggac 600
tgcatgaggc tcttcaccaa agaggatgtg cttgatggag atgaaaagcc aacatgctgt 660
cgctgccgag gcagaaaacg gtgtataaag aagttctcca tccagaggtt cccaaagatc 720
ttggtgctcc atctgaagcg gttctcagaa tccaggatcc gaaccagcaa gctcacaaca 780
tttgtgaact tccccctaag agacctggac ttaagagaat ttgcctcaga aaacaccaac 840
catgctgttt acaacctgta cgctgtgtcc aatcactccg gaaccaccat gggtggccac 900
tatacagcct actgtcgcag tccagggaca ggagaatggc acactttcaa cgactccagc 960
gtcactccca tgtcctccag ccaagtgcgc accagcgacg cctacctgct cttctacgaa 1020
ctggccagcc cgccctcccg aatgtag 1047
<210> 2
<211> 348
<212> PRT
<213> Artificial Sequence
<220>
<223> 融合蛋白
<400> 2
Met Asn Ser Lys Ser Ala Gln Gly Leu Ala Gly Leu Arg Asn Leu Gly
1 5 10 15
Asn Thr Cys Phe Met Asn Ser Ile Leu Gln Cys Leu Ser Asn Thr Arg
20 25 30
Glu Leu Arg Asp Tyr Cys Leu Gln Arg Leu Tyr Met Arg Asp Leu His
35 40 45
His Gly Ser Asn Ala His Thr Ala Leu Val Glu Glu Phe Ala Lys Leu
50 55 60
Ile Gln Thr Ile Trp Thr Ser Ser Pro Asn Asp Val Val Ser Pro Ser
65 70 75 80
Glu Phe Lys Thr Gln Ile Gln Arg Tyr Ala Pro Arg Phe Val Gly Tyr
85 90 95
Asn Gln Gln Asp Ala Gln Glu Phe Leu Arg Phe Leu Leu Asp Gly Leu
100 105 110
His Asn Glu Val Asn Arg Val Thr Leu Arg Pro Lys Ser Asn Pro Glu
115 120 125
Asn Leu Asp His Leu Pro Asp Asp Glu Lys Gly Arg Gln Met Trp Arg
130 135 140
Lys Tyr Leu Glu Arg Glu Asp Ser Arg Ile Gly Asp Leu Phe Val Gly
145 150 155 160
Gln Leu Lys Ser Ser Leu Thr Cys Thr Asp Cys Gly Tyr Cys Ser Thr
165 170 175
Val Phe Asp Pro Phe Trp Asp Leu Ser Leu Pro Ile Ala Lys Arg Gly
180 185 190
Tyr Pro Glu Val Thr Leu Met Asp Cys Met Arg Leu Phe Thr Lys Glu
195 200 205
Asp Val Leu Asp Gly Asp Glu Lys Pro Thr Cys Cys Arg Cys Arg Gly
210 215 220
Arg Lys Arg Cys Ile Lys Lys Phe Ser Ile Gln Arg Phe Pro Lys Ile
225 230 235 240
Leu Val Leu His Leu Lys Arg Phe Ser Glu Ser Arg Ile Arg Thr Ser
245 250 255
Lys Leu Thr Thr Phe Val Asn Phe Pro Leu Arg Asp Leu Asp Leu Arg
260 265 270
Glu Phe Ala Ser Glu Asn Thr Asn His Ala Val Tyr Asn Leu Tyr Ala
275 280 285
Val Ser Asn His Ser Gly Thr Thr Met Gly Gly His Tyr Thr Ala Tyr
290 295 300
Cys Arg Ser Pro Gly Thr Gly Glu Trp His Thr Phe Asn Asp Ser Ser
305 310 315 320
Val Thr Pro Met Ser Ser Ser Gln Val Arg Thr Ser Asp Ala Tyr Leu
325 330 335
Leu Phe Tyr Glu Leu Ala Ser Pro Pro Ser Arg Met
340 345
Claims (3)
1.一种杂交瘤细胞株，其特征在于：命名为杂交瘤细胞株3D9，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：C2016186。
2.一种抗USP2a的单克隆抗体，其特征在于：它由保藏编号为CCTCC NO：C2016186的杂交瘤细胞株3D9分泌产生。
3.根据权利要求2所述的一种抗USP2a的单克隆抗体在制备肿瘤组织切片免疫组织化学检测产品中的应用。
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