CN105695418B - 一种结构特异性核酸酶fen1的单克隆抗体及应用 - Google Patents
一种结构特异性核酸酶fen1的单克隆抗体及应用 Download PDFInfo
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Abstract
本发明涉及一种结构特异性核酸酶FEN1的单克隆抗体及应用，还提供了分泌该单克隆抗体的杂交瘤细胞株。所述的单克隆抗体可以用于检测癌细胞的试剂盒，所述癌细胞优选为前列腺癌、乳腺癌、胃癌细胞、神经母细胞瘤、胰腺癌或肺癌。
Description
技术领域
本发明涉及一种结构特异性核酸酶(FEN1)的单克隆抗体、分泌该单克隆抗体的杂交瘤细胞株以及所述的抗体的应用。
背景技术
基因组稳定性的维持，以及DNA的复制和修复，都需要一系列的核酸内切酶和外切酶的参与，结构特异性核酸酶1(flap endonuclease 1,FEN1)就是其中之一。FEN1能识别特定的DNA分叉结构，并切除含有游离5’端的单链核酸。在DNA复制过程中，FEN1通过其外切酶、内切酶活性去除了冈崎片段前端RNA引物的最后一个核糖核苷，在DNA修复中，FEN1以其内切酶活力参与了损伤碱基的修复过程(1)。由于FEN1在DNA复制和修复中的重要性，一旦编码FEN1的基因有所缺陷就会导致FEN1在体内含量的不正常增多或者减少，或发生功能性变异，从而引起基因组的不稳定，导致肿瘤或者癌症的发生。
FEN1属于XPG/Rad2核酸家族中的一个成员，拥有5’分叉内切酶(FEN)，5’外切酶(EXO)和切口依赖核酸内切酶(GEN)等3种结构特异的核酸酶活性。FEN1基因在人类基因组中被定位在染色体的11q12.2上(2)，包含了2个外显子与1个内含子。其中外显子的1141个碱基编码FEN1的380个氨基酸残基，总分子量约42KD(3)。
FEN1作为一种结构特异性核酸酶，主要参与了DNA复制时冈崎片段成熟过程中RNA引物的切除，长片段碱基切除修复，DNA二级结构的分解，DNA复制叉的维持和促进细胞凋亡诱导的DNA片段生成等过程。在这些过程中会形成很多类似皮瓣的瓣状结构，而FEN1主要识别这种结构，并通过其活性将其切除，以维持DNA的稳定性。在FEN1发挥功能的过程中，可与不同的蛋白质结合，从而影响其参与不同的DNA复制和修复途径。迄今为止，已发现至少20多种蛋白质与FEN1之间存在着相互作用。PCNA是一个DNA聚合酶的附属因子，它在细胞S期DNA复制过程中是必需的，对损伤DNA的NER过程中的碱基重新合成也有作用。而对于FEN1的功能，PCNA与它的结合十分重要。FEN1的28个氨基酸区域(AA328-355)有与PCNA结合活性(4)。FEN1在有PCNA刺激的条件下可提高5-50倍的剪切效率，PCNA能增加FEN1到剪切位点后的结合稳定性，也许会有更强的剪切效率(5)。FEN1突变会改变它与PCNA的结合，减少它在复制中的活性，会造成DNA复制扩增，从而引起癌症与遗传性疾病(6)。
FEN1在核酸或蛋白质水平上的高表达会对肿瘤的发生产生影响。在转移性前 列腺癌细胞、乳腺癌、胃癌细胞、神经母细胞瘤、胰腺癌、肺癌细胞株中均可见其表达的上调(7)。通过研究FEN1在前列腺癌中的表达情况，并比较原发性前列腺癌、前列腺上皮内肿瘤、良性前列腺增生和正常前列腺上皮内FEN1的表达情况发现FEN1在前列腺癌中的平均表达水平为36.7％，远高于正常水平13.2％，而在良性前列腺增生中为14.5％，前列腺上皮内肿瘤为15.4％，这说明FEN1在前列腺癌中过高表达，而且其表达增高的程度与细胞的去分化有关。其结果暗示FEN1可能会做为前列腺癌的一种诊断标志。
FEN1作为一种结构特异性核酸酶，参与了众多的生命过程，对其结构与功能的研究一直是DNA复制和损伤研究领域的热点。对于FEN1与肿瘤的关系的研究表明它很可能作为一种肿瘤的标记物用于肿瘤的早起诊断。
发明内容
本发明一方面提供了一种杂交瘤细胞系，其特征在于，所述杂交瘤细胞系的保藏号为CGMCC No.12013。
本发明另一方面提供了一种由前述的杂交瘤细胞系分泌的结构特异性核酸酶1的单克隆抗体，其特征在于，其能够特异地结合结构特异性核酸酶FEN1。
本发明再一方面提供了前述的杂交瘤细胞及前述的单克隆抗体在检测靶结构特异性核酸酶1中的应用。
本发明再一方面提供了包含前述的杂交瘤细胞和/或前述的单克隆抗体的试剂盒。
本发明再一方面提供了前述的杂交瘤细胞及前述的单克隆抗体在检测癌细胞的试剂盒中的应用，所述癌细胞优选为前列腺癌、乳腺癌、胃癌细胞、神经母细胞瘤、胰腺癌、肺癌、宫颈癌。
附图说明
图1、FEN-1抗体十二烷基硫酸钠聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测。
图2、免疫印迹检测不同裂解液(CHO-K1、Raji、Raw264.7、C6、PC-12、COS7、3T3、Hela、Jurkat)中FEN-1蛋白的表达(抗体稀释倍数1:1000)。
图3、FEN-1抗体的免疫荧光测试(所用细胞系：HeLa，抗体稀释比1:400)。
具体实施方式
下述实施例中所用方法如无特别说明均为常施方法。
试剂、酶、载体及细胞株：
(1)细胞株：HeLa(CCL-2TM)、CHO-K1(CCL-61TM)；Raji(CCL-86TM)、Raw264.7(TIB-71TM)、C6(CCL-107TM)、PC-12(CRL-1721TM)、COS7(CRL-1651TM)、 3T3(CRL-1658TM)、Jurkat(TIB-152TM)。
(2)实验动物：雌性BALB/c小鼠(湖南斯莱克景达实验动物有限公司)；
(3)载体：pET41a(Novagen)
(4)酶及抗体试剂：构建载体过程和PCR过程的各种酶购自Fermentas，ELISA实验所用抗体anti-mouse IgG/HRP(Jackson ImmunoResearch)，ELISA底物TMB(Sigma)、逆转录试剂盒(Fermentas)，BSA购自(北京元亨金马生物科技有限公司)
(5)其他试剂如无特别说明为国产分析纯。
实施例1：杂交瘤细胞株FEN1及其产生的单克隆抗体的获得
1.抗原制备
(1)获得目的基因
Trizol Reagent试剂提取HeLa细胞总RNA，将总RNA反转录为cDNA并以cDNA为模板PCR扩增FEN1基因。
(2)构建重组表达载体
将步骤(1)获得的PCR产物双酶切后回收，在T4DNA连接酶作用下连接入表达载体pET41a，构建重组质粒pET41a-FEN1。载体经过酶切和测序鉴定后用于转化表达细菌。
(3)获得含重组表达质粒的表达菌种
将步骤(2)获得的重组质粒转化大肠杆菌Rosetta(DE3)感受态细胞，用含有硫酸卡那霉素的LB固体培养基筛选，挑取单克隆用于抗原表达。
(4)诱导表达和纯化
将含有表达质粒的Rosetta(DE3)细菌单克隆接种于10ml含有硫酸卡那霉素的LB液体培养基中，37度，220rpm培养10hr。将菌液按1:100稀释倍数接种于200ml新鲜的LB培养基，培养至OD值为0.6，加入0.1mM IPTG诱导表达，20度5hr，收集菌液超声破碎，从上清中纯化重组抗原FEN1。
2.单克隆抗体的制备和纯化
(1)、免疫动物
一般使用6-8周龄雌性BALB/c小鼠，按照预先制定的免疫方法进行三次免疫注射。
第一次免疫 用2ML注射器每只小鼠腹股沟皮下注射0.2ml乳化液(100ul纯化的重组蛋白FEN1(用1×PBS稀释)+100ul弗氏完全佐剂，含100ug抗原)。
第二次免疫 间隔两周，用2ML注射器每只小鼠腹腔注射0.2ml乳化液(100ul纯化的重组蛋白FEN1(用1×PBS稀释)+100ul弗氏不完全佐剂，含100ug抗原)。
第三次免疫 间隔两周，用2ML注射器每只小鼠腹股沟皮下注射注射0.2ml乳化液(100ul纯化的重组蛋白FEN1(用1×PBS稀释)+100ul弗氏不完全佐剂， 含100ug抗原)。
测效价 根据融合时间安排，第三次免疫7-10天后取血检测，用常规的western法检测效价，选择效价达到1:1000的小鼠用于融合。
加强免疫 融合前3天，向待取脾小鼠进行加强免疫，小鼠腹腔注射200ul液体(含60ug抗原，溶剂为1×PBS，pH7.4)。
(2)、细胞融合
采用眼球摘除放血法处死小鼠，无菌操作取出脾脏，放入带200目不锈钢网的平皿中研磨，制备细胞悬液。将准备好的同系骨髓瘤细胞SP2/0与小鼠脾细胞按一定比例混合(1:5-1:10)，并加入促融合剂聚乙二醇(50％)。采用HAT选择培养基，进行杂交瘤细胞的选择性培养和筛选。
用ELISA方法检测杂交瘤细胞培养上清：将抗原用包被液(pH 9.6、0.05M碳酸盐缓冲液)稀释为8ug/ml加入96孔酶标板中，50ul/孔，放入4℃冰柜中包被过夜。弃去包被液，用PBST(磷酸盐缓冲液，pH7.4)洗板三次，加入封闭液3％BSA-PBST(加入3％牛血清白蛋白的磷酸盐缓冲液)，100ul/孔，37℃孵育1小时。弃去封闭液，PBST(磷酸盐缓冲液，pH7.4)洗板一次，甩干板子。将杂交瘤细胞培养上清加入酶标板100ul/well，同时加入PBST(磷酸盐缓冲液)作为阴性对照。37℃孵育1小时。弃上清，用洗板机PBST洗板三次，加入稀释好的anti-mouse IgG/HRP，100ul/well，37℃孵育1小时。弃上清，用洗板机PBST洗板三次，加底物TMB100ul/well，RT，避光37℃，10-15min后加入50ul终止液(2mol/L硫酸)。使用酶标仪测定，酶标仪设置为：450nm比色。和阴性对照相比，如果是OD值2.5倍之上就是阳性。最后筛选得到一株效价最好的抗FEN1的杂交瘤细胞株，命名为7H8，亚型鉴定为IgG1。
将筛选出的阳性杂交瘤细胞株7H8进行单克隆筛选(有限稀释法)，获得能产生高效价(WB稀释比>1:5000)单克隆抗体的杂交瘤细胞克隆。将杂交瘤细胞株扩大培养，并冻存保种。所述的阳性杂交瘤细胞为抗人FEN1杂交瘤细胞系7H8，该细胞系已保存于中国微生物菌种保藏管理委员会普通微生物中心，地址为：北京市朝阳区北辰西路1号院3号，保藏编号为：CGMCC No.12013，保藏日期为:2016年1月15日。
(3)单克隆抗体的制备与纯化
将步骤(2)获得的细胞株7H8接种至BALB/c小鼠腹腔，制备腹水，然后从腹水中纯化抗体(所获抗体命名为anti-FEN1)。
抗FEN1单克隆抗体的纯化：使用Protein G亲和纯化法。将用PB溶液平衡好的Protein G填料装入纯化柱中，加入经PB溶液稀释的单克隆抗体anti-FEN1的腹水过柱。上样结束后，用PB溶液洗柱子至流穿液OD值<0.01。然后用0.1M pH3.0的甘氨酸-盐酸溶液洗脱，收集整个洗脱峰的溶液。洗脱液经过透析浓缩并更换为PBS溶液。SDS-PAGE结果显示，纯化后抗体纯度在95％以上(参见图1)。
实施例2：单克隆抗体鉴定及应用
1.小鼠抗FEN1单克隆抗体(anti-FEN1)的免疫印迹检测鉴定
提取各种肿瘤细胞系的细胞裂解液，总蛋白浓度调整到2mg/ml，上样至12％的SDS-PAGE胶孔中，20ug/孔，电泳后将凝胶中的蛋白转移至PVDF膜上，5％脱脂奶粉封闭后，用实施例1制备得到的anti-FEN1抗体进行孵育：加入实施例1步骤(3)制备并纯化的anti-FEN1，工作浓度分别为1:1000，4度孵育过夜，然后加入HRP标记的山羊抗鼠抗体，工作浓度1:20000，于37度反应1hr，每个浓度梯度均出现45KD的条带(参见图2)，与文献报道相符，证明此抗体为抗FEN1的特异性抗体；抗体稀释梯度达到1:1000(抗体工作浓度能达到0.2ug/ml)有清晰条带，能够检测FEN1在CHO-K1(Lane 1)、HeLa(Lane 2)、Raji(Lane 3)、RAW246.7(lane 4)、C6(Lane 5)、PC-12(Lane 6)、COS7(Lane 7)、3T3(Lane 8)、Jurkat(Lane 9)细胞中的表达。
2.免疫荧光细胞染色鉴定
Hela细胞用PBS洗两次，4％多聚甲醛室温固定30min，PBS冲洗，用0.5％Triton X-100PBS室温透化20min。然后加入5％BSA PBS以封闭非特异性反应。加入实施例1步骤(3)制备并纯化的anti-FEN1单克隆抗体1:400，37度孵育30min。冲洗，加入FITC标记的山羊抗鼠IgG，室温避光孵育1hr。去除游离的二抗，PBS冲洗，荧光封片剂封片，荧光显微镜下观察，用蓝色光激发观察绿色荧光信号。实验结果(图3)清楚观察到细胞核中呈现明显绿色荧光，定位和文献报道一致。说明此抗FEN1的特异抗体可以用于免疫荧光检测观察FEN1的表达和定位。
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Claims (5)
1.一种杂交瘤细胞系，其特征在于，所述杂交瘤细胞系的保藏号为CGMCC No.12013。
2.一种由权利要求1所述的杂交瘤细胞系分泌的结构特异性核酸酶FEN1的单克隆抗体，其特征在于，其能够特异地结合结构特异性核酸酶FEN1。
3.权利要求1所述的杂交瘤细胞系及权利要求2所述的单克隆抗体在制备检测靶结构特异性核酸酶FEN1的试剂盒中的应用。
4.一种包含权利要求1所述的杂交瘤细胞系和/或权利要求2所述的单克隆抗体的试剂盒。
5.权利要求1所述的杂交瘤细胞系及权利要求2所述的单克隆抗体在制备检测癌细胞的试剂盒中的应用；所述癌细胞为前列腺癌、乳腺癌、胃癌细胞、神经母细胞瘤、胰腺癌、肺癌、宫颈癌。
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| Identification of flap-structure specific endonuclease 1 as a factor involved in long-term memory formation of aversive learning;Lorena Saavedra-Rodríguez et al.;《J Neurosci》;20090506;第29卷(第18期);第5726-5737页 * |
| 周婷等.结构特异性核酸内切酶FEN1及其与肿瘤的关系.《南京师大学报（自然科学版）》.2014,第37卷(第2期),第7-14页. * |
| 王越.雌激素对乳腺癌细胞中FEN1表达的影响及其机制研究.《中国优秀硕士学位论文全文数据库医药卫生科技辑》.2011,(第4期),第8页倒数第1行. * |
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