CN102375061B - 一种检测前列腺癌的elisa试剂盒 - Google Patents
一种检测前列腺癌的elisa试剂盒 Download PDFInfo
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Abstract
本发明采用双向电泳方法比较了前列腺增生细胞BPH-1、雄激素依赖性前列腺癌细胞LNCaP及雄激素非依赖性前列腺癌细胞PC-3三种细胞蛋白的组成。通过经质谱分析及数据库检索,确定2个差异蛋白GRP78和Annexin A2。通过Western Blotting检测上述三种细胞中2个差异蛋白的表达。最终确定GRP78和Annexin A2作为检测前列腺癌的标志蛋白,并公开了检测前列腺癌ELISA试剂盒的制备和使用方法。
Description
技术领域:
本发明涉及一种检测试剂盒,具体而言,涉及一种能快速、有效的检测出前列腺癌的ELISA试剂盒。
背景技术:
近年来随着人们生活水平的提高,人口结构的老龄化、饮食结构的改变,前列腺癌(prostate cancer,PCa)的发病率和死亡率呈上升趋势,并且发病年龄日趋年轻化。流行病学数据显示,中国前列腺癌发病率从1993年的1.71人/10万男性人口增加到2005年的7.9人/10万男性人口,而且在以每年10%的速度攀升,成为威胁男性健康的主要因素[ALVAREZA,LOKESHWAR V B.Bladder cancer biomarkers:current developments and future implementation.Curr Opin Urol,2007,17(5):341-346.]。由于PCa发病机理复杂、易恶化转移,大多数诊断时已是进展期或晚期,最终转变为雄激素非依赖型PCa,临床缺少有效的治疗方法。因此,进行PCa的筛查,早期诊断和治疗,成为提升男性生殖健康的关键点。
临床早期诊断方法主要是通过前列腺特异性抗原(prostate specific antigen,PSA)筛查,截至目前PSA是最为成熟和广泛的血清肿瘤标志物,然而近年来发现PSA存在着明显缺陷:在前列腺增生、前列腺炎、尿路感染、导尿、前列腺按摩以及膀胱镜检均可导致PSA升高,有较大的重叠,且检测受多因素影响。当PSA水平位于4~10ng/ml时,鉴别良性前列腺增生和前列腺癌困难。由此可见PSA仅对前列腺组织有特异性,但对前列腺癌特异性极低,其升高仅能提示前列腺癌的可能性。在美国经直肠超声引导穿刺活检证实约1/3PSA筛查结果为假阳性。更有研究表明PSA与前列腺癌的关联性已经大大下降[Na YQ,Li M.Attach importance to the research of criterion of treatment in prostate cancer.Nat Med J China,2005,85:3169.Stamey TA,Caldwell M,McNeal JE,et al.The prostate specific antigen era in the United States is over for prostate cancer:what happened in the last 20years?J Urol,2004,172:1297-1301.]。
鉴于上述现状,人们着力寻找更有效的新的肿瘤标记物的探索工作始终未曾停止,相关蛋白、基因、酶等在前列腺癌组织中的表达逐渐被人们重视。为此,不断有学者提出各种肿瘤标志物,试图寻找敏感性、特异性更高的生物标志物。
本发明依据实验研究,筛选出了同时多个指标进行前列腺癌的检测,从而大大提高了前列腺癌的诊断率。
发明内容:
本发明提供了一种检测前列腺癌的ELISA试剂盒,包括:抗人AnnexinA2抗体、抗人GRP78抗体、辣根过氧化物酶底物缓冲液、蛋白标准品AnnexinA2与GRP78。
本发明所述的抗人AnnexinA2抗体和抗人GRP78抗体为辣根过氧化物酶标记的抗体。
本发明所述的辣根过氧化物酶底物缓冲液为TMB、H2O2和磷酸柠檬酸底物缓冲液的所组成溶液。
本发明的ELISA试剂盒可以应用于前列腺癌的临床检测。
本发明所要解决的问题是,提供一种特异性的快速检测前列腺癌的试剂盒,以及对应的方法和应用。
本发明采用双向电泳方法比较了前列腺增生细胞BPH-1、雄激素依赖性前列腺癌细胞LNCaP及雄激素非依赖性前列腺癌细胞PC-3三种细胞蛋白的组成。并通过质谱分析和数据库检索锁定了2个差异蛋白Annexin A2(SEQ ID NO:1)和GRP78(SEQ ID NO:2)。
本发明通过Western Blotting方法检测了BPH-1、LNCaP和PC-3细胞中GRP78和Annexin A2蛋白的表达,结果显示GRP78在PC-3中缺失表达,Annexin A2在PC-3特异性表达。
本发明还公开了所述试剂盒的组装方法与使用方法。
附图说明:
图1正常良性前列腺增生细胞系BPH-1的双向电泳图固相胶条等点聚焦及12%SDS-PAGE分离显示的蛋白成分。
图2前列腺癌雄激素非依赖性细胞系PC-3的双向电泳图固相胶条等点聚焦及12%SDS-PAGE分离显示的蛋白成分。
图3前列腺癌雄激素依赖性细胞系LNCaP的双向电泳图固相胶条等点聚焦及12%SDS-PAGE分离显示的蛋白成分。
图4Western Blotting检测三种细胞GRP78的表达Lane M:分子量标准;LaneB:BPH-1细胞;Lane L:LNCaP细胞;Lane P:PC-3细胞;箭头示被抗GRP78抗体识别的蛋白条带。
图5:GRP78蛋白Western Blotting检测各细胞NMPs吸光度值1为BPH-1细胞;2为LNCaP细胞;3为PC-3细胞。
图6:Western Blotting检测三种细胞Annexin A2的表达Lane M:分子量标准;Lane B:BPH-1细胞;Lane L:LNCaP细胞;Lane P:PC-3细胞;箭头示被抗GRP78抗体识别的蛋白条带。
图7:Annexin A2蛋白Western Blotting检测各细胞NMPs吸光度值。1为BPH-1细胞;2为LNCaP细胞;3为PC-3细胞
具体实施方案:
实施例一肿瘤标志物的筛选
一.实验材料与设备
1.主要实验材料
BPH-1细胞、LNCaP细胞、PC-3细胞:购于北京协和医学院细胞中心;RPMI-1640培养基:GIBCO公司;胎牛血清:GIBCO公司;青霉素、链霉素:Hyclone公司;胰蛋白酶:GIBCO公司;25cm2培养瓶、75cm2培养瓶:Corning公司;15ml离心管:Corning公司;5ml、10ml吸管:Corning公司;过氧核糖核酸酶I(DNAI):上海生物工程有限公司;氧化核苷复合物(VRC)、Leupetin、Aprotinin:北京天恩泽基因有限公司;固相pH梯度胶条(ImmobilineTM DryStrip,pH 3-10L,24cm):GE公司;蛋白酶混合抑制剂(Protease Inhibitor Cocktail SetIII):Calbiochem公司;PVDF膜:Millipore公司;碱性磷酸酶标记羊抗兔LaminA/C抗体购自Pierce公司;兔抗人AnnexinA2多克隆抗体(sc-9061)抗体购自Santa Cruz公司;兔抗人GRP78多克隆抗体(sc-13968)均购自Santa Cruz公司;羊抗兔Mucin1单克隆抗体购自北京中杉金桥生物技术有限公司;SDS-6H分子量标志:购自Sigma公司;预染蛋白质分子量标志:Fermentas原产;尿素、硫脲、琼脂糖、三羟甲基氨基甲烷(Tris)、IPG buffer(pH 3-10)、蛋白定量试剂盒(2D Quant Kit)、2-D Clean-Up Kit均购自Pharmacia生物技术公司;丙烯酰胺、甲叉双丙烯酰胺、TX-100、SDS购自Sigma公司;CHAPS、二硫苏糖醇(DTT)、碘代乙酰胺、过硫酸铵、TEMED购自Merck公司;BCIP、NBT购自Bicmol公司;其余试剂均为国产或进口分析纯。
2、主要实验仪器
等电聚胶电泳仪(IPGphorTM Isoelectric Focusing System)Amersham Pharmacia Biotec公司产品;Power PAC 3000型电泳仪、Minigel电泳槽及电转移系统:Bio-Rad公司产品;组织匀浆器(Ultra-turrax T25型):德国Janke&Kunkel-IKA-Labortechnik公司产品;pH计(pH211型):HANNA公司产品;分光光度仪(756MC型):上海精密科学仪器有限公司产品;超净台:北京半导体一厂产品;培养箱:Thermo公司;小型台式离心机(TGL-16G型):上海医用仪器厂产品;小型台式冷冻离心机(1K15型)为Sigma公司;恒温水浴箱:北京市长风仪器仪表公司产品;高压灭菌锅:上海医用仪器厂;小型反转摇床(MutiliTube-Rotator):Barnstead公司;平转摇床(Orbital Shaker 51300):Cloe-Parmer公司;全自动化学发光凝胶成像系统:北京赛智公司;常规外科手 术器械购自北京王府井医药公司。
二.实验方法及操作步骤
1.前列腺癌细胞系培养与收集
1)细胞复苏
取出保存于液氮或-80℃冰箱的细胞,将含有细胞的冻存管迅速放入37~42℃的水浴中,轻摇使之融化;用少量含10%GIBCO胎牛血清1640培养基轻轻悬起细胞,转移到离心管里;1000rpm离心,弃上清;加入5ml含10%GIBCO胎牛血清1640培养基,转入细胞培养瓶中,于37℃、5%CO2培养箱中培养,每一至两天更换新鲜培养液一次。
2)细胞传代
先用PBS清洗细胞2~3遍,而后用0.25%胰酶(含0.02%EDTA)消化细胞约1-3分钟,使之皱缩变为圆形;用含10%胎牛血清的1640培养基轻轻悬起细胞,转移到离心管里;1000rpm离心,弃上清;加入10ml含10%胎牛血清的1640培养基,按1∶2~1∶4分入细胞培养瓶中,37℃、5%CO2培养箱中继续培养,每一至两天更换新鲜培养液一次。
3)细胞冻存
需冻存的细胞在前一天换为新鲜培养基;先用PBS清洗细胞2~3遍,而后用0.25%胰酶(含0.02%EDTA)消化细胞约1-3分钟,使之皱缩变为圆形;用含10%胎牛血清的1640培养基轻轻悬起细胞,转移到离心管里;1000rpm离心,弃上清;加入3-5ml冻存液(10%二甲基亚砜、20%GIBCO胎牛血清和70%的完全培养基),重悬后放入冻存管中,4℃放置15分钟,-20℃放置60分钟后转入-80℃,如需长期保存可再转入液氮中。
2.前列腺癌细胞系蛋白组分的提取
1)将收集的细胞用PBS洗2次,4℃离心5min(1000g)去上清,加入含0.5%Triton-X100的细胞骨架CSK100提取液,0℃放置10min,4℃离心10min(1000g),去上清,细胞膜系统、细胞质随之去除;
2)加入含0.5%TritonX-100的细胞骨架提取液CSK50『10mM PIPES PH6.8、3mM MgCl2、50mM NaCl、300mM蔗糖、4mM氧化核苷复合物(VRC)、3μg/mlLeupetin、3μg/ml Aprotinin、1.2mM PMSF抽提液,洗涤两次,4℃离心5min(1000g),去除上清液,细胞质中的微丝、微管等蛋白被去除;
3)然后加入以CSK50配制的消化液(含300-500U/ml的DNase I)室温消化30min;
4)最后加入1mol/L硫酸铵至终浓度0.25mol/L,室温15min,4℃离心(1000g),去上清,除去细胞中的DNA和组蛋白,剩余不溶性沉淀即为蛋白成分;
5)用CSK50洗一次,4℃离心(1000g)5min,吸出上清,剩余些溶液吹散样品,将悬液移入已称重的1.5mlEP管中,离心(1000g)5min,吸去上清,称重;
6)按1∶5(M/V)比例向蛋白沉淀中加入裂解缓冲液『7mol/L尿素、2mol/L硫尿』溶解,冰浴超声30-45min后,4℃离心(12000g)30min,收集上清,在-80℃储存备用。
3.蛋白质样品除盐处理
使用Amersham公司的2D clean-up试剂盒进行蛋白质提取后的纯化处理;
1)取出样品100μl,加入100μl沉淀剂,涡旋混匀,在冰上孵育15min;
2)在蛋白和沉淀剂混合物中加入100μl共沉淀剂,简单涡旋混匀。以至少12,000g速度离心5min,去除上清,并在简单离心后以移液枪除去剩余上清,至无可见的液体;
3)在沉淀顶端加上40μl共沉淀剂,冰浴放置5min;
4)以至少12,000g速度离心5min,移去上清;
5)吸取25μl去离子水加在每个蛋白沉淀颗粒上,涡旋振荡5-10s,将沉淀打散但不溶解;
6)加入-20℃预冷的洗液1ml和wash additive 5μl,充分混匀使之完全打散;
7)-20℃放置至少30min,每10min涡旋振荡20~30s,以至少12,000g速度离心5min;
8)弃除上清,可见蛋白沉淀,在空气中简单晾干,不超过5min;
9)溶涨液复溶。
4.蛋白质含量的测定
用Amersham公司的2D Quant Kit定量试剂盒对提取的细胞内总蛋白质进行定量
1)准备工作液:B液80μl+A液8m1,配成工作显色剂;
2)按照下表逐一加入BSA标准液和样品于2.0ml Eppendorf管中;
3)表1:蛋白标准品加样表
4)分别于每管中加入500μl沉淀剂,简单混匀,室温孵育2~3min;
5)分别于每管中加入500μl共沉淀剂,使用涡旋或颠倒简单混匀;
6)至少以12,000g离心5min,管底可见沉淀蛋白,弃去上清。并于简短离心后用移液器除去管底上清,至管中无可见液体存在;
7)分别于每管加入100μl铜溶液和400μl去离子水。确保充分涡旋以溶解沉淀的蛋白,使之彻底完全重悬;
8)分别于每管中加入1ml工作液,立即颠倒混匀,室温孵育15~20min;
9)在加入工作液之后40min之内读取480nm处吸光值;
10)绘制标准曲线,确定样品中蛋白质浓度。
5.SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)
1)所需溶液
12%分离胶
4%堆积胶
5×电泳缓冲液(pH 8.3):
Tris Base 9.0g
甘氨酸 43.2g
SDS 3.0g
用ddH2O定容至600ml,常温保存。临用前用ddH2O5倍稀释后使用。考马斯亮蓝(R-250)染液及脱色液体系:
固定液:10%(v/v)甲醇;10%(v/v)冰乙酸;40%(v/v)乙醇。用ddH2O配制。
敏化液:1%(v/v)冰乙酸;10%(v/v)硫酸铵;用ddH2O配制。
染色液:5%(v/v)冰乙酸;40%(v/v)乙醇;0.125%(v/v)考马斯亮蓝R-250。
用ddH2O配制。
脱色液I:5%(v/v)冰乙酸;40%(v/v)乙醇。用ddH2O配制。
脱色液II:3%(v/v)冰乙酸;30%(v/v)乙醇。用ddH2O配制。
5×样品缓冲液(还原型含DTT):
2)操作步骤:
仔细清洗灌胶用的玻璃板,待玻璃板完全干燥后组装好灌胶装置。注意使大小两块玻璃板的下缘与Spacer的下缘平齐;灌制12%分离胶,胶面之上用水封闭。待胶聚合后,吸干水膜,继续灌入4%堆积胶,插好梳子。灌胶过程中注意勿使凝胶中产生气泡;凝胶完全聚合后,小心拔去梳子,勿破坏梳孔,将灌胶装置转移至电泳装置中,倒满1×电泳缓冲液;样品与5×样品缓冲液按4∶1的体积比混合,于95℃℃热变性3min;用微量进样器将分子量标准及样品分别上样于梳孔底部;150V电压恒压电泳至溴酚蓝前沿距胶底部0.5cm时停止电泳;0.1%考马斯亮蓝R-250染液染色约4h;考马斯亮蓝R-250染液脱色液脱色至胶上的蛋白条带清晰。扫描记录图像信息。凝胶在5%(v/v)的乙酸密封袋中保存。
6.免疫印迹(Immunoblot/Western blotting)
1)所需溶液
10×电转移缓冲液(甲醇free)
Tris 30.3g
甘氨酸 144g
加ddH2O至终体积1000ml,pH值为8.3。
用前按以下比例配制成1×电转移缓冲液:
10×电转移缓冲液 100ml
甲醇 200ml
加ddH2O定容至1000ml,混合后使用。
10×TBS
Tris 121.1g
NaCl 90.0g
盐酸调至pH 7.5,定容至1000ml
膜封闭液(5%TBS脱脂牛奶)
脱脂牛奶 2.5g
1×TBS 50ml
抗体孵育液(2.5%TBS-脱脂牛奶)
5%TBS脱脂牛奶 10ml
dd H2O 10ml
膜洗涤液[0.05%TBST(pH 7.5)]
吐温-20 100μl
1×TBS 200ml
碱性磷酸酶显色剂显色缓冲液(0.1M Tris-HCl,pH 9.5):
Tris 12.1g
盐酸调pH至9.5
ddH2O定容至1000ml
碱性磷酸酶显色液:
NBT(100mg/ml,70%DMFM溶解)66μl
BCIP(50mg/ml DMFM) 33μl
碱性磷酸酶显色剂显色缓冲液 10ml
氨基黑染液及脱色液
氨基黑染液(0.1%,w/v):
氨基黑 0.1g
ddH2O 100ml
氨基黑脱色液:
甲醇 90ml
冰醋酸 2ml
ddH2O定容至100ml。
2)操作步骤:
①SDS-PAGE操作如前;
②预先将PVDF膜(0.22μm或0.45μm,根据转移靶蛋白的分子量而定。<20kD的蛋白分子用0.22μm的PVDF膜)在甲醇中浸泡5min;
③在倒满1×电转移缓冲液的潜盘中组装凝胶-膜“三明治”的电转移装置。
A.打开转移夹,并放置于浅盘中,用转移缓冲液将海绵垫完全浸透后,将其放在转移夹壁上,海绵上再放置一张浸泡过的Whatman 3MM滤纸;
B.小心将凝胶放置于滤纸上,避免产生气泡;
C.以转移缓冲液润湿胶面,小心将PVDF膜放于胶面上,避免产生气泡;
D.在PVDF膜上放一张浸湿的Whatman 3MM滤纸,用一干净的小试管轻轻滚过以消除所有气泡;
E.将另一块海绵用转移缓冲液浸透后,放在滤纸上,关上转移夹形成凝胶-膜“三明治”并插入转移槽的一侧;
F.转移槽另一侧插入冷却冰盒,并在转移槽中注满转移缓冲液;
④设置电转移条件:4℃,恒流150mA,1h。电转移条件根据转移靶蛋白分子量大小进行调整。分子量较大的蛋白分子一般转移时间较长;
⑤封闭PVDF膜的非特异位点
A.卸转移装置,将滤纸揭下;
B.用镊子将PVDF膜放入平皿中,加入5%TBS-脱脂牛奶(液面需没过膜),置于平转摇床上室温封闭15-30min;
⑥加入一抗:将膜装入一小塑料袋,按适当比例加入第一抗体和2.5%TBS脱脂牛奶,清除气泡,封闭塑料袋,室温摇床孵育2h或4℃过夜;
⑦洗膜:用0.05%TBS-T洗膜,10min/次×3;
⑧加入二抗:将膜装入一小塑料袋,按适当比例加入碱性磷酸酶标记二抗和2.5%TBS-脱脂牛奶,清除气泡,封闭塑料袋,室温摇床孵育2h;
⑨洗膜:用0.05%TBS-T(pH7.5)洗膜10min/次×3;
⑩碱性磷酸酶显色剂显色:将PVDF膜置于一个小平皿中,加入AP显色液至10ml(100mg/ml NBT66μl,50mg/ml BCIP 33μl),轻轻摇晃加速显色,观察显色程度,适时用蒸馏水冲洗终止显色反应;吸水纸吸干膜上水分,扫描记录图像信息。避光干燥保存。
7.双向电泳(Two-dimensional Electrophoresis,2DE)
1)操作所需溶液
①裂解液(Lysis Solution):配置40ml
加dd H2O至40ml,混匀后分装保存于-20℃。
②溶胀液(Rehydration stock solution):配制25ml
Urea(8M) 12g
CHAPS(2%) 0.5g
IPG buffer(用前加)(0.5%)
加双蒸水至25ml,分装为1ml/管,用前每管加DTT 2.5mg。
③SDS平衡缓冲液(SDS equilibration buffer):
含有(50mM Tris-HCl pH 8.8,6M Urea,30%Glycerol,2%SDS,Bromophenol blue,200ml)
上述溶液为一储备液,20ml分装保存于-20℃。用前取出恢复室温以使SDS溶解。第一相平衡时加1%(w/v)DTT;第二相平衡时加2.5%(w/v)碘乙酰胺。
④含0.5%琼脂糖的1×电泳缓冲液:
琼脂糖0.05g,与1×电泳缓冲液10ml混合,临用前加热使琼脂糖溶化,用于封闭预制胶条与聚丙烯酰胺凝胶。
2)操作过程
①-80℃低温冰箱取出提取的蛋白样品,室温融化后于4℃,15000r/min,5min离心。取上清待用;取出1ml溶涨液待融化后加入DTT 2.24mg,震荡使充分溶解,取蛋白量为500μg的总蛋白,加入溶涨液,使总体积为425μl,然后加IPG buffer(0.5%v/v)2.5μl,充分混匀;取一根24cm的洁净Holder,将425μl 样品缓慢加入Holder底槽一端,注意勿产生气泡。轻微倾斜Holder,使样品液均匀布满底槽(注意:所有操作均在操作时均应戴干净的口罩和乳胶手套,并保证双向电泳操作区域洁净无灰尘,以防止外源性蛋白的污染);提前半小时从-20℃冰箱中取出三根24cm长固相pH梯度胶条(IPG Strip),待恢复室温。从正极撕去其上的塑料膜。胶面朝下,从Holder的上样端进入(IPG Strip的阳极和阴极分别与Holder的尖头和平头相对),缓缓向前推动,直至IPG Strip完全进入Holder底槽,两端分别搭在Holder的电极丝上。整个过程注意勿使IPGStrip与Holder底槽间产生气泡;吸约3ml矿物油(Mineral Oil)覆盖在IPG Strip上,盖好Holder盖子;将三根Holder平放在IPGPhor的电极板上,尖头、平头分别与电极板的阳极和阴极相对。两根Holder平行放置,置于IPGPhor电极板的相应标记处,盖好IPGPhor的盖子;
②等点聚焦仪提前半小时开机预热,设好第一相电泳(等电聚焦电泳,IsoelectricFocusing,IEF)的参数:
按“开始”键,输入实际胶条数;再按一次“开始”键,开始第一相等点聚焦。点聚焦过程中随时观察并记录电压变化情况,并根据记录在后期实验中进行条件调整和优化;灌三块普通12%聚丙烯酰胺凝胶,灌至距短玻璃板上端约1cm时,加正丁醇封闭,放入通风厨,待胶聚合备用。
③第一相电泳结束后,用小镊子从Holder中夹出胶条,蒸馏水反复冲洗掉其上的矿物油,用滤纸轻轻沾掉其上液体(胶面朝上);依次用含1%DTT(w/v)和2.5%(w/v)碘代乙酰胺的SDS平衡缓冲液平衡胶条各20min,每个胶条约10min平衡液。将胶条置于专用的平衡槽中,放置于平转腰床上;平衡完毕后,用两把小镊子小心夹住胶条,并置于滤纸上(注意胶面朝上),吸干其上的液体;小心将胶条竖直置入12%聚丙烯酰胺凝胶表面,与凝胶上表面紧密接触,不留缝隙。胶条一端空隙里置一沾有分子量标记的滤纸片;用热的含0.5%琼脂糖的1×电泳缓冲液覆盖胶条。待琼脂糖凝成胶冻状时,将整个装置转移至第二相电泳(SDS-PAGE)装置中,上槽和下槽均灌适量1×电泳缓冲液,盖好盖子;设好SDS-PAGE参数:10W恒功率电泳,约12h结束,使胶条中的蛋白样品充分进入聚丙烯酰胺凝胶。电泳至溴酚蓝前沿到达胶底端约1cm时停止电泳。
④使用改良的考马斯亮蓝染胶:所有的染胶过程均在室温,平转摇床上进行。固定夜固定1h,敏化液作用2h,染色液染胶4h以上或过夜,脱色液I脱色1-2h,脱色液II脱色至胶上的蛋白质点显现清楚;
⑤显色的凝胶经凝胶扫描仪扫描获取图像,并采用ImageMasterTM 2D Platinum Software(Version 5.0)对图像进行分析,寻找差异蛋白点。
8.质谱分析及数据库检索
1)质谱鉴定前样品处理
从考马斯亮蓝染色的双向电泳(或SDS-PAGE)上找到并定位显示清晰的差异蛋白斑点(或条带);用洁净的切胶刀片小心沿考马斯亮蓝染色的边缘切下,置于一洁净的1.5ml Eppendorf管中;用50%乙腈、25mmol/L NH4HCO3溶液对胶片进行脱色,旋转摇床上脱色过夜,中间换液一次,至胶片变为无色,倒去液体;将胶片用小镊子夹成约1mm3的小块,冷冻真空干燥至成面包渣状;Eppendorf管中加入0.01g/L胰蛋白酶、25mmol/L NH4HCO3溶液50μl,37℃保温20小时;加入5%TFA溶液200μl于40℃保温1小时;吸出上清至另一Eppendorf管中,原管中加入2.5%TFA、50%ACN溶液200μl于30℃保温一小时;合并上清液冰冻干燥成干粉,以上由北京蛋白质研发中心有限公司操作并进行质谱分析。
2)质谱分析
进行基质辅助激光解吸离子化飞行时间质谱仪(matrix-assisted laser desorption ionization-time of flight mass spectrometer,MALDI-TOF-MS)的肽质量指纹图谱(peptide mass fingerprint,PMF)分析。鉴定蛋白质的方法如下:采用美国应用生物系统公司的Voyager DETM PRO BiospectrometryTM workstation飞行时间质谱仪进行肽质量指纹图谱分析,所用参数为:延迟时间提取(delayed extraction,150ns)、反射式模式(reflector mode)、正电荷;加速电压(acceleration voltage)为20000V;Grid voltage为75%;Guide wire为0.02%;采集质量数范围为800-3500Da;基质为肉桂酸(a-Cyano-4-hydroxycinnamic acid.CHCA)。
3)数据库检索
使用互联网上的蛋白质数据库检索程序ExPASy Molecular Biology Server网站提供的PeptIdent、UCSF Mass Spectrometry Facility网站提供的MS-Fit或Matrixscience网站提供的MASCOT Peptide Mass Fingerprint,将所得的肽质量指纹谱(Peptide Mass Fingerprinting,PMF)数据与数据库中的标准数据进行比较,即可鉴别出样品中的蛋白质种类。
4)靶分析蛋白的生物信息学数据检索
根据8.3数据库检索及报告的质谱分析结果,对质谱分析的蛋白质进行文献检索及生物学检索分析。利用互联网上的生物信息数据库NCBI,European Bioinformatics Institute(EBI),Protein Information Resource(PIR),Protein Data Bank(PDB),Protein Families Database(PFam)等生物信息学资源网站及数据库中检索分析相应蛋白的表达、基因编码、氨基酸序列结构等信息。
三、实验结果与讨论
1.双向电泳与凝胶图像分析结果
良性前列腺增生细胞BPH-1、雄激素依赖性前列腺癌细胞LNCaP、雄激素非依赖性前列腺癌细胞PC-3提取的核基质蛋白双向电泳重复3次以上,对三种蛋白进行分析。结果如图所示:蛋白主要集中分布在等电点pI 4-9、分子量 10-100kD之间,其中以20kD和100kD之间蛋白含量最高。三种细胞系之间蛋白表达存在明显差异,差异蛋白分布在20kD-100kD之间(图1、图2、图3)。
2.差异表达蛋白Western Blotting结果
进一步确认2种差异表达的蛋白在BPH-1、LNCaP和PC-3细胞中的表达差异。制备三种细胞系蛋白样品,经12%的SDS-PAGE分离,转移至PVDF膜上,一抗为兔抗GRP78和AnnexinA2多克隆抗体(1∶200稀释),碱性磷酸酶标记ProteinA/G为第二抗体(1∶2000稀释),进行Western Blotting鉴定。
1)GRP78在BPH-1、LNCaP和PC-3细胞中的表达
结果如图4示:在70kD左右出现了抗GRP78抗体特异识别的单一条带(条带如箭头所示)。并且该蛋白在BPH-1细胞中高表达,LNCaP细胞中低表达,PC-3细胞中表达缺失(图5)。
2)Annexin A2在BPH-1、LNCaP和PC-3细胞中的表达
结果如图6示:在35kD左右出现了抗Annexin A2抗体特异识别的单一条带(条带如箭头所示)。并且BPH-1细胞和LNCaP细胞中没有检测到特异性条带,只在PC-3细胞中有特异性识别的条带(图7)。
3.讨论
经质谱分析及数据库检索,29个蛋白点中阳性分析结果为28个,1个蛋白点未检索出阳性结果,可能由于该蛋白点蛋白含量过低或质谱仪系统误差引起。通过搜集文献及相关实验结果分析,锁定2个差异蛋白GRP78和Annexin A2做进一步确认分析。
1)关于GRP78的研究
葡萄糖调节蛋白78(glucose regulated protein,GRP78),又名免疫球蛋白重链结合蛋白(the immunoglobulin heavy chain binding protein,Bip),与热休克蛋白70(Hsp70)家族具有高度同源性,属于Hsp70家族的主要成员之一。GRP78的主要功能是作为一种分子伴侣参与蛋白质的折叠和转运过程及内质网应激反应中发挥重要作用。另外,GRP78表达对维持内质网的结构和功能的稳定具有重要意义,尤其在肿瘤组织细胞中。目前研究表明,GRP78在某些肿瘤细胞中呈高表达,对 于肿瘤细胞的抗化疗药物的性质及抗原表达有重要意义。另外,GRP78还可增强肿瘤细胞的增殖、侵袭能力以及对抗癌药物的耐受性,被视为肿瘤治疗反应的一种生物标志。Gazit等研究发现,与正常上皮细胞株相比,乳腺癌细胞株的GRP78蛋白质水平约高1.5-3倍,这表明GRP78大量表达对乳腺癌细胞的增值具有促进作用。Fernandez等也通过对比研究发现,在人类乳腺癌标本中GRP78的表达显著高于癌旁组织;应用Western blotting和Northern blotting检测胸腺肿瘤组织中GRP78在蛋白及其mRNA水平表达明显升高。Reetkookagi等发现在人类非小细胞肺癌中,GRP78的水平明显升高。另外,在人上皮细胞样癌、鼠乳癌、大鼠肝癌、鼠纤维肉瘤、人宫颈癌、骨髓瘤细胞和小鼠骨髓性白血病细胞等肿瘤细胞中均发现有GRP78的高度诱导和合成。
目前,GRP78在前列腺癌中的表达已经进行了相关的研究,证明GRP78与前列腺癌具有密切关系。Daneshmand等对1934例接受前列腺癌根治术并行区域淋巴结清扫的患者进行分析,其中153例患者在后来的病理诊断中发现淋巴结有转移。免疫组化方法分析该153例患者的正常前列腺组织、前列腺癌组织及淋巴结中GRP78的表达水平。研究表明,与正常前列腺组织相比,GRP78在前列腺癌组织中呈显著高表达。GRP78过表达的前列腺癌患者比相对弱表达的患者具有更高的临床复发风险,同时其死亡风险也近似后者的两倍,这提示我们,GRP78的表达水平可作为前列腺癌患者预后和临床复发的预测因子。也有报道前列腺癌患者体内抗GRP78自身抗体的高表达与肿瘤的侵袭行为密切相关。另外,最近一次对激素抵抗型进展期前列腺癌患者GRP78表达的回顾性研究发现,GRP78在前列腺癌从局限性向转移性发展的过程中表达显著增高,因此,在那些未给予治疗的局限性肿瘤患者体内,它可能是一种新的促凋亡抑制物。
在本研究中,双向电泳以及Western Blotting结果显示GRP78在BPH-1细胞中高表达,LNCaP细胞中低表达,而在PC-3细胞中表达缺失;使用兔抗GRP78多克隆抗体进一步确认该蛋白在BPH-1、LNCaP和PC-3三种细胞中的蛋白表达。该结果与双向电泳结果一致。GRP78主要存在内质网内,在蛋白质的折叠和转运过程及内质网应激反应中发挥重要作用。GRP78还可增强肿瘤细胞的增殖、侵袭能力以及对抗癌药物的耐受性。
2)关于AnnexinA2的研究
膜联蛋白2(Annexin A2)是annexins家族的成员之一,是一组钙离子结合蛋白,在细胞质和细胞核中具有非常广泛的生物学功能。目前认为,AnnexinA2可以介导胞吞、胞吐等过程,调节离子通道并影响细胞骨架构建。近年研究发现,Annexin A2与其他annexins不同,其具有RNA结合的特性,与DNA合成、复制有关,其蛋白表达异常和肿瘤的发生发展有密切关系。
已有研究证实,其与多种肿瘤的发生发展、浸润、远处转移均有不同程度相关,且不同肿瘤中表达各异。Annexin A2在肿瘤组织中表达显著增高已有多项研究证实。1990年,Frohlich M等人首次报道Annexin A2的mRNA及蛋白水平在肝癌组织中表达量明显高于癌旁正常组织,提示Annexin A2与恶性转化有关。与正常食管黏膜细胞相比,AnnexinA2在食管癌变细胞系中表达缺失,可能充当抑癌基因的角色。食管黏膜细胞癌组织中AnnexinA2在蛋白质和mRNA水平上均低表达,且表达水平随癌细胞分化程度下降而降低,表明Annexin A2与食管鳞状细胞癌的分化程度相关。已多次报道胰腺癌中存在AnnexinA2的过度表达。最近,有学者采用蛋白质印迹和免疫组化方法确认Annexin A2在胰腺癌中过度表达(增加2.6倍),提示其有望成为胰腺癌的候选癌基因。有研究表明:Annexin A2在胰腺癌通过参与tPA信号转导途径促进肿瘤细胞增殖。tPA特异性结合胰腺癌细胞膜外的Annexin A2,激活局部纤溶酶的产生和促进肿瘤细胞入侵。Esposito等发现,从胰腺导管上皮内瘤变到胰腺导管腺癌,Annexin A2和Tenascin-C的表达水平逐渐升高,两者在浸润性胰腺癌中同时高表达,暗示其与肿瘤的侵袭性相关。此外,在妇科肿瘤、部分急性早幼粒细胞性白血病患者、头颈部鳞状细胞癌、肾癌以及甲状腺癌等肿瘤中Annexin A2的表达量均有不同程度的升高。
大量研究证明,前列腺癌中AnnexinA2蛋白表达缺失,其mRNA水平也很低,Annexin A2基因没有缺失或突变,去甲基药物作用可恢复AnnexinA2mRNA表达,推测高甲基化可能是基因沉默的原因。约2/3前列腺上皮内瘤变(PIN)组织中,存在Annexin A2表达缺失,确认其缺失是前列腺癌发生的早期和频发事件。Stewart等用免疫组化方法发现,98%的良性前列腺组织表达Annexin A2,88%的前列腺癌组织中Annexin A2表达缺失。免疫组化染色AnnexinA2弥散且强阳性表达于良性腺体基底细胞的胞质,前列腺癌中无这种表达,高分化PIN显示基底细胞染色中断。Annexin A2弥散且浓重的基底细 胞胞质染色为基底膜完整的标志。因此,annexin A2在基底细胞层中不连续着色的特征,可用于高分化癌与PIN的鉴别诊断。可以说AnnexinA2免疫组化染色如存在于前列腺上皮基底部细胞,是良性分泌腺上皮的肯定生物标记,侵袭性前列腺癌的否定生物标记;因此Annexin A2可能成为前列腺癌组织病理学诊断的辅助标记。
本发明通过抗Annexin A2抗体检测Annexin A2在BPH-1、LNCaP和PC-3三种细胞中的蛋白表达水平。结果发现,Annexin A2在BPH-1和LNCaP细胞中不表达,而在PC-3细胞中表达(见图6)。该结果与双向电泳结果一致。与文献报道一致。另外,人类血浆蛋白质组计划(Human Plasma Proteome Project,HPPP,http://www.peptideatlas.org)高可信度数据集已经成功鉴定到该蛋白。同时,利用生物信息学软SecretomeP2.0(http://www.cbs.dtu.dk/services/SecretomeP/)预测,结果发现它很有可能通过非经典途径被细胞分泌。此外,文献报道它可以定位于外体(exosome)中这些证据都表明,Annexin A2可能通过非经典分泌途径分泌入血,在很可能在PCa患者血清/血浆中检测到,并可能发展为一种比较有潜力的血清标志物。本研究发现Annexin A2是一个核基质结合蛋白,尽管还不知道Annexin A2在细胞核内的作用机制,但从我们的实验结果并结合前人的研究结果可以推测Annexin A2在PC-3细胞中表达上调并被运输进入细胞核内结合到核基质网络上,以一种我们不了解的方式参与细胞增殖与分化的调控,促进PCa细胞恶性转化。该蛋白由表达缺失到再次表达并且表达升高,判断其在PCa恶性转化过程中的诊断和治疗方面具有重要价值。另一方面,由于AnnexinA2通过非经典途径分泌入血,很可能成为PCa候选的血清学标志物。
综上所述,本发明采用双向电泳方法比较了前列腺增生细胞BPH-1、雄激素依赖性细胞LNCaP及雄激素非依赖性细胞PC-3三种细胞蛋白的组成,结果表明在三种细胞中表达改变。通过进一步鉴定分析结果提示,在2个锁定差异蛋白中AnnexinA2与GRP78在恶性前列腺癌雄激素非依赖性细胞PC-3中具有相对特异性,可以成为前列腺癌恶性发展的候选标志物。
实施例二ELISA试剂盒的组装与使用
一、实验材料与仪器
0.1M NaIO4、1mM PH4.4醋酸钠缓冲液、0.2M PH9.5碳酸盐缓冲液、NaBH4溶液、Tween-20、牛血清白蛋白。大肠杆菌BL21,原核表达载体PET32。
酶标仪(ELX800Universal Microplate Reader)、摇床、电子秤、搅拌器、冰箱。
二、实验方法
1.抗体与辣根过氧化物酶的偶联
1)称取4mg辣根过氧化物酶(HRP)溶于1ml去离子水中,加200ul新配制的0.1M NaIO4,室温避光轻搅20min,直到溶液呈棕绿色。
2)在PH为4.4的1mol/L醋酸钠缓冲液中低温(4℃)透析过夜。
3)向上述溶液中滴加20ul 0.2M碳酸盐缓冲液,至PH9~9.5,立即加入5mg兔抗人AnnexinA2单克隆抗体,或者5mg兔抗人GRP78单克隆抗体,室温避光轻搅2h。
4)加0.1ml新配的4mg/ml NaBH4液,混匀,置于4℃反应2小时。
5)将上述液装入透析袋中,在0.15M PH7.4PBS中透析,4℃过夜。
6)10000g离心15min,弃去沉淀的蛋白,上清即为所HRP标记的抗体产物。
7)标记抗体产物的定量其公式如下:
标记HRP的抗体浓度(mg/ml)=(OD280nm-OD403nm×0.3)×0.62当OD403/OD280比值为0.3~0.6时为合格。本实验标记的抗体比值0.4和0.5。
8)将标记的抗体添加25%-50%甘油于-20℃保存。
2.检测前列腺癌的ELISA试剂盒组装
检测前列腺癌的ELISA试剂盒包括:抗人AnnexinA2抗体、抗人GRP78抗体、辣根过氧化物酶底物缓冲液、蛋白标准品AnnexinA2与GRP78。其中辣根过氧化物酶底物缓冲液:称取10mg 3,3′,5,5′-四甲基联苯胺(TMB)溶于5ml无水乙醇中制备成TMB贮存液。待使用时取0.5ml TMB贮存液加到10ml磷酸 柠檬酸底物缓冲液(0.2MNa2HPO4,0.1M柠檬酸)中,再加入32μl 0.75%H2O2混匀,配置成辣根过氧化物酶底物缓冲液。
3.检测前列腺癌标本的ELISA实验
1)ELISA实验中常规试剂的配制:
a)包被缓冲液(PH9.60.05M碳酸盐缓冲液);
NaHCO3 1.59克
NaHCO3 2.93克加蒸馏水至1000ml;
b)洗涤缓冲液(PH7.4PBS):0.15M
KH2PO4 0.2克
Na2HPO4·12H2O 2.9克
NaCl 8.0克
KCl 0.2克
Tween-200.05%0.5ml加蒸馏水至1000ml;
c)样品稀释液:
牛血清白蛋白(BSA)0.1克加洗涤缓冲液至100ml;
d)终止液(2M H2SO4):
蒸馏水178.3ml,逐滴加入浓硫酸(98%)21.7ml;
e)底物缓冲液(PH5.0磷酸枣柠檬酸):
0.2MNa2HPO4(28.4克/L)25.7ml
0.1M柠檬酸(19.2克/L)24.3ml 加蒸馏水50ml;
2)检测前列腺癌标本的ELISA实验步骤如下:
a)将前列腺癌样本的组织进行蛋白质抽提。
b)用包被液稀释抗原至50-10μg/ml,加入酶标板中,每孔100μl,4℃过夜。每个样品包被4个空,其中2个孔用于检测GRP78,2个孔用于检测 AnnexinA2。同时将标准品进行梯度稀释依次为50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、3.125ng/ml、0ng/ml,每孔100μl。
c)洗涤液清洗酶标板3次,每次在摇床上摇5-8min,37℃下用样品稀释液液封闭酶标板4h以上。
d)将辣根过氧化物酶标记的兔抗人AnnexinA2抗体和兔抗人GRP78抗体按1∶3000-1∶5000稀释,分别加入孔中,每孔100μl,37℃孵育2h,再用洗涤液清洗3次。
e)加辣根过氧化物酶底物缓冲液100μl,显色5min后,加入终止液100μl,终止显色。
f)用酶标仪检测OD450的值。
g)结果判断:对GRP78的检测,当样品吸光值是阴性对照吸光值的0.5-2倍时为准阴性,其吸光值和阳性对照吸光值相差不足5%时为准阳性。对AnnexinA2的检测,当样品吸光值和阴性对照吸光值相差不足5%时为准阴性,其吸光值为阳性对照吸光值0.5-2倍时为准阳性。两种蛋白检测结果都为准阳性或准阴性时,确定样品为阳性或阴性。
实施例三ELISA试剂盒的临床检测
取25例前列腺癌阳性组织标本和25例前列腺癌阴性标本,用实施列二所述的试剂盒和使用方法进行检测。所述的阳性和阴性标本是通过前列腺癌穿刺活检组织的病理检测确认的。
用HRP标记的兔抗人GRP78单克隆抗体检测,在25例阳性样品中有21例为阳性,阴性样品中18例为阴性。
用HRP标记的兔抗人AnnexinA2单克隆抗体检测,在25例阳性样品中有22例为阳性,阴性样品中20例为阴性。
采用前列腺特异性抗原试剂盒(PSA试剂盒),当PSA>10ng/ml时,检测25例前列腺癌病人,三次结果平均为15例阳性结果,阴性样品为14例为阴性。
本发明所述的试剂盒与PSA试剂盒对前列腺癌样品检测的准确率见下表:
表1不同试剂盒最前列腺癌样品检测的准确率
联合上述2个抗体所确定的阳性和阴性结果,可以超过与PSA试剂盒检测的结果。说明我们设计的前列腺ELISA试剂盒能够非常准确的检测前列腺癌。
Claims (3)
1.一种检测前列腺癌的ELISA试剂盒,包括:抗AnnexinA2抗体、抗GRP78抗体、辣根过氧化物酶底物缓冲液、蛋白标准品AnnexinA2与GRP78。
2.根据权利要求1所述的检测前列腺癌的ELISA试剂盒,其特征在于,所述的抗人AnnexinA2抗体和抗人GRP78抗体为辣根过氧化物酶标记的抗体。
3.根据权利要求1所述的检测前列腺癌的ELISA试剂盒,其特征在于,所述的辣根过氧化物酶底物缓冲液为TMB、H2O2和磷酸柠檬酸底物缓冲液所组成的溶液。
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