CN108277214B - 一种应激磷酸化抗原多肽、抗体、制备方法以及应用 - Google Patents
一种应激磷酸化抗原多肽、抗体、制备方法以及应用 Download PDFInfo
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Abstract
本发明提供了一种应激磷酸化抗原多肽、抗体、制备方法以及应用，所述磷酸化抗原多肽，其特征在于所述抗原多肽包含人MMP9蛋白第458位苏氨酸位点的附近15肽作为候选多肽，其中该T184位点的苏氨酸为磷酸化状态，且C端连接一个半胱氨酸的氨基酸序列。本发明还提供了通过上述抗原多肽制备的抗体及其制备方法和应用，包含该抗体的试剂盒和癌症检测方法。本发明制备的抗体能够检测应激环境下MMP9蛋白的磷酸化修饰，以探讨应激条件下MMP9蛋白T458位点磷酸化对肿瘤细胞增殖、迁移等过程中的影响，为临床肿瘤疾病的诊疗提供潜在实用工具。
Description
技术领域
本发明涉及抗体制备技术领域，具体涉及一种针对人MMP9蛋白T458位点的应激磷酸化抗原多肽、抗体、其制备方法以及应用。
背景技术
肿瘤转移被认为是一个多步骤的过程，需要多个基因的共同作用而完成。某些特定的基因可以让肿瘤细胞从原发肿瘤中脱落，粘附内皮细胞，侵入细胞外基质(extracellular matrix, ECM)，肿瘤细胞穿过血管，并且迅速生长，最终在远处器官形成一个新的肿瘤。而基质金属蛋白酶（Matrix metalloproteinases, MMPs）是参与这一进程的重要基因。MMPs是ECM降解的主要生理介质。基质金属蛋白酶是一族锌依赖性内肽酶，其主要功能是降解ECM。MMPs参与多种生理和病理过程，如形态发生，创伤愈合，组织修复和重塑]。此外，MMPs通过发挥增加细胞的生长，迁移，侵入，转移和血管生成的能力，因此在肿瘤的侵袭与转移中起着重要的作用。MMP9为Ⅳ型胶原酶，又称明胶酶，以无活性的酶原形式分泌，经水解激活后可降解ECM中的明胶及多种蛋白，在肿瘤浸润及转移中起着重要作用。
基质金属肽酶9(MMP9)也称为92kDa IV型胶原酶或明胶酶B(GELB)，是MMP家族酶的成员，其负责降解变性和基底膜胶原和通过加工可溶性蛋白质促进炎症，可溶性蛋白质包括蛋白酶抑制剂、趋化因子和细胞因子。MMP9还通过膜结合分子(如生长因子前体和受体、酪氨酸激酶受体(TKR)、细胞粘附分子的蛋白水解来控制肿瘤细胞的迁移、侵袭和转移。在疾病中，MMP9由包括白细胞的许多细胞类型分泌，例如嗜中性粒细胞、单核细胞/巨噬细胞和淋巴细胞，以及成纤维细胞、肌成纤维细胞、上皮细胞、平滑肌细胞、内皮细胞、破骨细胞和肿瘤细胞。
临床和实验证据表明，MMP9水平升高与癌症发展、转移和缩短的患者存活时间相联，因为它通过消化基底膜和细胞外基质组分在肿瘤细胞侵袭和转移中起关键作用。与人嗜中性粒细胞中的MMP9共价连接的中性粒细胞明胶酶相关脂质运载蛋白(NGAL)保护MMP9免受蛋白水解降解并增加MMP9的酶活性和随后增强肿瘤侵袭和扩散。血清中高浓度的MMP9/NGAL复合物与透明细胞肾细胞癌中较短的无进展生存期和较差的总生存期相关。MMP-9的作用与结肠直肠癌、胰腺癌、乳腺癌、肺癌、卵巢癌、膀胱癌和胃癌相关。
发明内容
本发明公开了一种针对MMP9蛋白T458磷酸化位点的抗原多肽，可特异性识别人肿瘤细胞表达的MMP9蛋白T458磷酸化位点的多克隆抗体以及其制备方法。
本发明的第一方面提供了一种磷酸化抗原多肽，其特征在于所述抗原多肽为人MMP9蛋白第458位苏氨酸位点的附近15肽作为候选多肽筛选得到，其中该T458位点的苏氨酸为磷酸化状态，且C端连接一个半胱氨酸的氨基酸序列。
本发明的第二方面提供了一种针对人MMP9蛋白T458位点的应激磷酸化抗体，其特征在于所述抗体是针对人MMP9蛋白T458位点氨基酸磷酸化的抗体，其中所述抗体制备中采用了由以下氨基酸序列组成的抗原多肽，其具体序列为：CEPRPPTTTT(p)PQPT，其中T(p)表示人MMP9蛋白T458位点氨基酸磷酸化苏氨酸，其C端连接一个半胱氨酸。
本发明的第三方面提供了一种针对人MMP9蛋白T458位点应激磷酸化的多克隆抗体的制备方法，其特征在于包括（1）对MMP9蛋白第458位点附近氨基酸序列的二级结构、免疫原性、亲疏水性、表面可及性等进行分析，确定合适的一段肽序列进行人工合成；（2）将合成的多肽与马来酰亚氨活化的载体mcKLH偶联，将此偶联产物进行脱盐柱纯化后免疫新西兰兔；（3）经过四次免疫的兔血清用ELISA法对抗体效价进行检测，效价达理想值后收集免疫兔血清，并用多肽包被的溴化氰活化的琼脂糖亲和纯化柱纯化抗体；（4）对纯化后抗体进行ELISA、western bot鉴定。
本发明的第四方面提供了一种针对人MMP9蛋白T458位点的应激磷酸化抗体的应用，其中所述应用使用根据权利要求2或权利要求3制备的抗体进行特异性识别癌细胞表达的人MMP9蛋白T458位点。其中所述的癌细胞优选为人鼻咽癌、宫颈癌、胃癌、胶质瘤细胞。
本发明第五方面一种癌细胞检测试剂盒，包括商上述的针对人MMP9蛋白T458位点的应激磷酸化抗体、抗原修复液、PBS缓冲溶液、酶阻断剂3%H2O2、辣根酶标羊抗小鼠/兔IgG聚合物、DAB显色剂、苏木素染液、乙醇、环保透明剂、0.5%氨水和超纯水。
本发明还提供了一种癌细胞检测的方法，其特征在于使用上述的癌细胞检测试剂盒，包括以下步骤：
（1）将待检测组织常规石蜡切片，经过环保透明剂，乙醇分别摇床脱蜡，超纯水漂洗；
（2）将抗原修复液加热至沸腾，将石蜡切片放入放入沸腾的抗原修复液中，中高档微波处理，室温冷却后置于超纯水中，浸泡，之后用PBS摇洗3次；
（3）将样本置于内源性过氧化物酶阻断剂3%H2O2，避光室温孵育，PBS缓冲液摇洗；
（4）取出切片，滴加经过稀释的针对人MMP9蛋白T458位点的应激磷酸化抗体，放入孵育盒内，4℃冰箱过夜，放入PBS缓冲液中充分洗涤；
（5）擦干组织周围液体后滴加辣根酶标羊抗小鼠/兔IgG聚合物，室温孵育，PBS缓冲液洗涤，滴加现配适量DAB显色剂，室温显色,自来水终止显色，苏木素染液中染色，水洗后将切片置于0.5%氨水中浸泡，继续水洗；
（6）将切片依次置于乙醇中，取出后将切片置于透明剂，用中性树胶封片，光学显微镜下观察。
根据前期工作结果，发明人预测MMP9蛋白第458位苏氨酸（T458）是一个潜在的磷酸化位点，可能与该蛋白的稳定及激活功能相关。本发明采用人工设计和合成含有该磷酸化位点的一段MMP9蛋白多肽（pT458），与马来酰胺活化的匙孔血蓝载体蛋白（KLH）偶联，经脱盐纯化后免疫新西兰兔，通过四次免疫及ELISA效价检测后，采集兔血清并经多肽包被的溴化氰活化的琼脂糖凝胶纯化柱纯化。该多克隆抗体经过ELISA、western blot等鉴定，可特异性识别人鼻咽癌CNE1细胞表达的MMP9蛋白pT458位点。
本发明选择了MMP9蛋白包含第458位苏氨酸位点(T458)的附近15肽作为候选多肽，并用人工方法进行包含pT458的多肽合成及完全抗原制备。对MMP9蛋白第458位点附近氨基酸序列的二级结构、免疫原性、亲疏水性、表面可及性等进行分析，确定合适的一段肽序列进行人工合成；将合成的多肽与马来酰亚氨活化的载体mcKLH偶联，将此偶联产物进行脱盐柱纯化后免疫新西兰兔；经过四次免疫的兔血清用ELISA法对抗体效价进行检测，效价达理想值后收集免疫兔血清，并用多肽包被的溴化氰活化的琼脂糖（CNBr-activatedsepharose）亲和纯化柱纯化抗体；对纯化后抗体进行ELISA、western bot等鉴定。鉴定结果表明该多克隆抗体可特异性识别MMP9蛋白pT458位点，可用于检测肿瘤细胞该位点的磷酸化水平，为探索肿瘤细胞增殖及转移机制研究提供一种工具，也为肿瘤诊断提供帮助，并能指导其临床预后判断。
其中，化学合成的多肽抗原是小分子，本身很难具有好的抗原性，只能诱导动物产生很弱的免疫反应，因而与载体蛋白交联是很重要的。载体蛋白含有很多抗原决定基，能够刺激T辅助细胞，进而诱导 B细胞反应。用于与多肽交联的载体蛋白有多种，其中最普遍使用的载体是匙孔血蓝蛋白（keyhole limpet hemacyanin，KLH)，牛血清白蛋白（bovineserum albumin，BSA），卵清蛋白（ovalbumin，OVA）和牛甲状腺球蛋白（bovinethyroglobulin，THY)。KLH具有更高的抗原性，是最为常用的多肽交联载体。BSA也常用来作为多肽载体，但由于BSA经常被用做检测试验的阻断剂而使得该方法生产的抗体在应用上存在着一定的局限性。
发明的有益效果
本发明采用人工设计和合成含有该磷酸化位点的一段MMP9蛋白pT458位点的抗原多肽，并制备对应的多克隆抗体。该多克隆抗体经过ELISA、western blot等鉴定可特异性识别MMP9蛋白pT458磷酸化位点，并且相对于癌旁组织其在多种癌组织细胞中高表达，其差异具有统计学意义。本发明的磷酸化多克隆抗体可特异性识别MMP9蛋白pT458磷酸化位点，可用于检测肿瘤细胞该位点的磷酸化水平，为探索肿瘤细胞增殖及转移机制研究提供一种工具，也为肿瘤诊断提供帮助，并能指导其临床预后判断。
附图说明
图1是采用DNAstar软件分析人MMP9蛋白特性。框中标注的序列为选定的多肽序列，此多肽序列位于MMP9蛋白第458位苏氨酸附近，其抗原性、亲水性及表面可及性较强。
图2是pMMP9-Thr458抗体与非磷酸化MMP9-Thr458抗体的OD值，ELISA检测pMMP9-Thr458抗体为特异性磷酸化位点抗体（* P＜0.05）。
图3是本发明中抗MMP9(pT458)多克隆抗体的western-blot鉴定结果。上样为CNE1细胞(人鼻咽癌细胞系)裂解液。该细胞分别用pcDNA6.0/myc-His-MMP9野生型及pcDNA6.0/myc-His-MMP9(T458A)突变体质粒进行瞬时转染。
图4是本发明的制备流程。
图5是抗MMP9（pT458）多克隆抗体的细胞免疫组化鉴定结果；细胞为鼻咽癌细胞CNE2、肝癌细胞HepG2、胃癌细胞MGC803。
图6是本发明中抗MMP9(pT458)多克隆抗体的免疫组化鉴定结果。所使用的标本为宫颈癌细胞病理切片。
图7是本发明中抗MMP9(pT458)多克隆抗体的免疫组化鉴定结果。所使用的标本为胃癌和胶质瘤细胞病理切片。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明，而不用于限制本发明的范围。在不背离本发明的技术解决方案的前提下，对本发明所作的本领域普通技术人员容易实现的任何改动都将落入本发明的权利要求范围之内。
实施例1
步骤1：MMP9蛋白pT458多肽的设计及合成
1.1 MMP9氨基酸序列
根据GenBank获得人MMP9蛋白氨基酸序列 (NP_004985)：
1 mslwqplvlv llvlgccfaa prqrqstlvl fpgdlrtnlt drqlaeeyly rygytrvaem
61 rgeskslgpa llllqkqlsl petgeldsat lkamrtprcg vpdlgrfqtf egdlkwhhhn
121 itywiqnyse dlpravidda farafalwsa vtpltftrvy srdadiviqf gvaehgdgyp
181 fdgkdgllah afppgpgiqg dahfdddelw slgkgvvvpt rfgnadgaac hfpfifegrs
241 ysacttdgrs dglpwcstta nydtddrfgf cpserlytqd gnadgkpcqf pfifqgqsys
301 acttdgrsdg yrwcattany drdklfgfcp tradstvmgg nsagelcvfp ftflgkeyst
361 ctsegrgdgr lwcattsnfd sdkkwgfcpd qgyslflvaa hefghalgld hssvpealmy
421 pmyrftegpp lhkddvngir hlygprpepe prppttttpq ptapptvcpt gpptvhpser
481 ptagptgpps agptgpptag pstattvpls pvddacnvni fdaiaeignq lylfkdgkyw
541 rfsegrgsrp qgpfliadkw palprkldsv feerlskklf ffsgrqvwvy tgasvlgprr
601 ldklglgadv aqvtgalrsg rgkmllfsgr rlwrfdvkaq mvdprsasev drmfpgvpld
661 thdvfqyrekayfcqdrfywrvssrselnqvdqvgyvtydilqcped（SEQ ID NO：3）
结果：人MMP9蛋白含有707个氨基酸。
1.2用DNAstar软件分析人MMP9蛋白特性（表1）：
表1
结果：人MMP9蛋白分子量为784580.47道尔顿，等电点为5.82，为酸性蛋白。
1.3用DNAstar软件分析人MMP9蛋白免疫原性、亲疏水性及表面可及性（图1）：
结果：人MMP9蛋白第449位至第462位共14个氨基酸抗原性、亲水性及表面可及性较强，且第458位苏氨酸包含其中。
1.4 MMP9合成多肽序列：
经过上述分析，选用的多肽序列为CEPRPPTTTTPQPT（SEQ ID NO：1）。
步骤2：多肽合成及与载体蛋白偶联
2.1 多肽合成
为便于与载体蛋白偶联，合成多肽在C端加入一个半胱氨酸，而且第458位苏氨酸为磷酸化状态：CEPRPPTTTT(p)PQPT(SEQ ID NO：2）。同时合成一段含有非磷酸化的第458位苏氨酸多肽序列：CEPRPPTTTTPQPT。多肽由百奇生物科技(苏州)有限公司合成。
多肽合成流程:
A. 多肽合成：采用固相合成法合成，即先将所要合成肽链的羟末端氨基酸的羟基以共价键的结构同一个不溶性的高分子树脂相连，然后以该结合在固相载体上的氨基酸作为氨基组份经过脱去氨基保护基并同过量的活化羧基组分反应，接长肽链，重复操作，直到达到所要合成的肽链长度为止，最后将肽链从树脂上裂解下来，经过纯化等处理，即得所要的多肽。
B. 纯化：RP-HPLC纯化
1）HPLC条件：
流动相：A）0.1%TFA水溶液；B）0.1%TFA乙腈溶液
梯度：A/ B（90/ 40）到A/ B（40/ 90）30min
流速：1ml/min
温度：室温（23℃）
检测：214nm的紫外
样品：冻干的粗品
2）步骤：
a．将粗品溶解在流动相中
b．注入20-30mg（2-2.5ml）样品
c．将主峰收集到50ml管中
d．冻干
3）鉴定：LC/MS条件：
流动相：A）0.05%TFA水溶液B）0.1%TFA乙腈溶液
梯度：A/ B（90/ 10）到A/ B（40/ 60）15min
流速：1ml/min
温度：室温（23℃）
检测：214nm的紫外
MS API：ESI
表2 多肽合成的结果
| 多肽合成号 | 多肽序列 | 多肽纯度(%) |
| 150803001 | NH2-CEPRPPTTTp[Thr]PQPT-CONH2 | >90% |
| 150803002 | NH2-CEPRPPTTTTPQPT-CONH2 | >90% |
2.2 多肽与载体蛋白偶联
化学合成的多肽抗原是小分子，本身很难具有好的抗原性，只能诱导动物产生很弱的免疫反应，因而与载体蛋白交联是很重要的。载体蛋白含有很多抗原决定基，能够刺激T辅助细胞，进而诱导 B细胞反应。用于与多肽交联的载体蛋白有多种，其中最普遍使用的载体是匙孔血蓝蛋白（keyhole limpet hemacyanin，KLH)，牛血清白蛋白（bovine serumalbumin，BSA），卵清蛋白（ovalbumin，OVA）和牛甲状腺球蛋白（bovine thyroglobulin，THY)。KLH具有更高的抗原性，是最为常用的多肽交联载体。BSA也常用来作为多肽载体，但由于BSA经常被用做检测试验的阻断剂而使得该方法生产的抗体在应用上存在着一定的局限性。
多肽偶联流程：
a. 溶液准备：偶联缓冲液包括Na2HPO4，NaH2PO4，NaCl，EDTA，调pH至7.2；
b. 实验步骤：
1）柱床准备：纯水及偶联缓冲液洗涤柱床；
2）多肽准备：少量DMF溶解多肽，静置半小时，待溶液中无颗粒状不溶物，加适量AH液配制成6mg/ml的多肽溶液，从多肽溶液中分出需要偶联的量；
3）KLH、Sulfo-SMCC准备：根据质量比，偶联多肽总量：纯KLH=1：1，计算出纯KLH的量，按照质量比，纯KLH：Sulfo-SMCC=10：1，计算出Sulfo-SMCC的量；
4）KLH和Sulfo-SMCC反应与反应物收集：将称取的KLH溶于适量的AH液配置成终浓度10mg/ml，Sulfo-SMCC用DMSO溶解成100mg/ml的溶液，将二者混合摇匀，室温反应4h并间断混摇使其充分反应，用层析柱分离样品；
5）KLH与Sulfo-SMCC的反应物与多肽偶联：向每一管需偶联多肽中加入相应量KLH与Sulfo-SMCC反应物，室温反应2h或室温过夜，并用垂直混合仪混匀，将偶联好的多肽至于-20℃保存。
注：使用KLH载体蛋白偶联合成多肽，所得偶联肽作免疫抗原用。
步骤3：抗MMP9多肽兔多克隆抗体制备
3.1 免疫与采血：
3.2抗体效价ELISA检测方法
3.2.1 血清ELISA检测：
a.溶液准备：
包被液：50mM Na2CO3 ( pH9.6 )，20mM Tris-HCl ( pH8.5 ) 或10mM PBS (pH7.4 )
封闭液：一般封闭用BSA、脱脂奶粉、酪蛋白、明胶等
洗涤液：PBST或纯水
b.实验步骤：
1）将抗原按适当浓度溶解于包被液中；
2）在对应的孔中加入100ul抗原，4℃过夜；
3）倒空液体并拍干残留液体，洗涤液冲洗3次；
4）每孔加200ul封闭液，37℃孵育1小时；
5）倒空液体并拍干残留液体，洗涤液冲洗3次；
6）每孔加100ul一抗，37℃孵育1小时；
7）倒空液体并拍干残留液体，洗涤液冲洗3次；
8）每孔加100ul二抗，37℃孵育1小时；
9）倒空液体并拍干残留液体，洗涤液冲洗5次；
10）拍干孔中残留液体，每孔加100ul显色液，37℃避光显色10min；
11）每孔加50ul 2M H2SO4终止显色，并立即读取450nm OD值。
c. 血清ELISA检测结果：
注：RB55631-55632血清ELISA检测均转阳（1:32000，P/N值>2.1），将安排两步亲和纯化并进行ELISA抗体鉴定。
3.2.2 抗体亲和纯化结果：
3.2.3 抗体ELISA结果
结论：
该项目免疫的RB55631-55632血清ELISA检测为阳性；两步亲和法纯化抗体，共获得磷酸化化抗体3.89mg。
步骤4 抗MMP9（pT458）多克隆抗体的鉴定
4.1 抗MMP9（pT458）多克隆抗体的ELISA鉴定
A）ELISA实验步骤
⑴将多肽MMP9-Thr458 和pMMP9-Thr458，用1×CBS包被液溶解成0.2 μg/100 ul，铺至每个酶标板的孔中100 ul,4℃过夜包被。
⑵第二天取出包被好的酶标板，甩干包被液，拍板。
⑶加封闭液（3%BSA），每个孔加200 ul，37℃孵育2 h。
⑷封闭完毕后，取出酶标板，洗板三次，每个孔中加入300 ul的PBST静置2 min，后甩干（避免孔与孔之间的污染），将板拍在干净的纱布上，将孔内的液体拍干为佳。
⑸可直接进行下一步实验，也可用自封袋包装好，4℃保存备用。
⑹将MMP9一抗以1:4000的稀释浓度，加入每孔100 ul，37℃孵育1 h。
⑼ 1 h后将酶标板取出，洗板三次后加入HRP二抗，37℃孵育30min。
⑽ 30 min后将酶标板取出，洗板三次后加入TMB显色剂37℃避光孵育15 min。
⑾ 15 min后将酶标板取出（变蓝），立即加入终止液，酶标仪震荡30 s，450 nm读数，分析结果。
B）ELISA实验检测pMMP9-458抗体为特异性磷酸化位点抗体
鉴于MMP9-Thr458位点的磷酸化尚未见相关报道，因此发明人委托公司定制了pMMP9-Thr458抗体，经ELISA实验检测pMMP9-Thr458抗体是否为特异性磷酸化位点抗体。结果显示pMMP9-Thr458抗体与pMMP9-Thr458抗原结合的OD值是明显大于pMMP9-Thr458抗体与非磷酸化MMP9-Thr458抗原结合的OD值（见图2）,并且pMMP9-Thr458抗体与pMMP9-Thr458抗原结合的OD值是随着抗体浓度的稀释而减小的，结果提示该pMMP9-Thr458抗体为特异性的pMMP9-Thr458位点的抗体。
4.2 抗MMP9（pT458）多克隆抗体的Western-Blot鉴定
A）使用Polyplus公司的转染试剂Jetpei进行瞬时转染，以6 cm培养皿为例：
（1）当细胞长至80%-90%，且细胞状态佳时，进行细胞消化后细胞计数，每个6 cm培养皿铺5×105 -1×106 个细胞,细胞悬液和培养基总体积为5 ml
（2）取3 μg DNA，计算所得的质粒体积和NaCl体积总和为 250 ul，然后取6 ul转染试剂jetpei，244 ul NaCl定容到250 ul混匀，将混合好的转染试剂加入到混合好的DNA中，轻轻混匀简单离心后，室温放置15-30 min。
（3）将上述配好的转染体系，轻轻加至做好标记的相应培养皿中，混匀后，放入 37℃，5% CO2恒温培养箱进行细胞培养。
（4）24-48 h后细胞可进行Western-Blot鉴定。
B）抗MMP9（pT458）多克隆抗体的Western-Blot鉴定结果
CNE1细胞经瞬时转染cDNA、MMP9-WT、MMP9-Thr458Ala后，用pMMP9-Thr458抗体通过Western blot检测Thr458是否磷酸化，结果显示野生型MMP9（WT）的MMP9-Thr458磷酸化水平是明显高于MMP9-Thr458突变点（T458A）的MMP9-Thr458磷酸化水平的（见图3）。结果提示MMP9Thr458Ala是磷酸化的位点。
4.3 抗MMP9（pT458）多克隆抗体的细胞免疫组化鉴定
A）抗MMP9（pT458）多克隆抗体的细胞免疫组化步骤：
（1）将专用细胞爬片置于6孔板培养皿中，按2*104/ml的细胞密度将细胞接种于培养皿中进行细胞爬片，待细胞长满50%-70%后进行免疫细胞组织化学染色鉴定。
（2）吸尽六孔板中的培养基， PBS摇洗标本 3次各 1 min。
（3）固定液中固定 15 min。
（4）PBS摇洗标本1次5min。
（5）0.1%Triton X-100( DPBS配 ) 孵育 1次 20 min。
（6）PBS摇洗标本 3次各 3 min。
（7）2%BSA封闭孵育 30min。
（8）PBS摇洗标本1次5min
（9）一抗（p-MMP9））孵育（PBS配，滴度1：200，湿盒）4OC 过夜或37OC 60 min。阴性对照最好用一抗来源血清，否则用PBS液
（10）PBS清洗标本 4次各 5 min。
（11）二抗工作液孵育（湿盒） 37OC 30 min。
（12）PBS摇洗标本 4次各 5 min。 15 C液（湿盒） 37OC 30 min。
（13）PBS清洗标本 3次各 5 min。
（14）DAB显色（避光，镜下观察至棕色）约1-5min。
（15）蒸馏水洗 2次 1 min。
（16）苏木素复染 0.5~1min。
（17）自来水洗。
（18）8%氨水30s。
（19）甘油或中性树胶转片。
（20）镜下观察。
B）抗MMP9（pT458）多克隆抗体的细胞免疫组化鉴定结果：
选取鼻咽癌细胞CNE2、肝癌细胞HepG2、胃癌细胞MGC803进行细胞免疫组化实验，结果（如图6所示）显示抗MMP9（pT458）实验组在胞浆或胞核中可见棕黄色染色，呈阳性结果，对照组胞浆或胞核见蓝色染色，呈阴性结果。多次重复，结果与之前相符。提示抗MMP9（pT458）多克隆抗体可用于肿瘤细胞水平检测。
4.4 抗MMP9（pT458）多克隆抗体的组织免疫组化鉴定
A）实验步骤：选用宫颈癌、胃癌、胶质瘤病人各50例进行免疫组化，验证P-MMP9（T458）抗体在癌、癌旁组织中的表达差异。
1组织常规石蜡切片厚度3-5um，防脱片捞片后晾干，放入65℃烤箱烤片2小时。
2 切片脱蜡水化程序
⑴环保透明剂Ⅰ、Ⅱ、III摇床脱蜡各12分钟；
⑵100%-95%—80%—70%—50%乙醇各3分钟，摇床。
3超纯水漂洗5分钟，洗涤一定要充分。
4抗原热修复：将EDTA（1X）抗原修复液与微波盒中将热至沸腾，将石蜡切片放入放入沸腾的抗原修复液中，中高档微波处理20-30分钟。
5 停止加热，室温冷却20-30分钟。
6将抗原修复后切片置超纯水，浸泡2次各5分钟，之后用PBS摇洗3次各5分钟。
7将样本置于内源性过氧化物酶阻断剂3%H2O2，避光室温孵育15分钟。PBS缓冲液摇洗3次各5分钟。
8取出切片，滴加一抗（浓度配比,PBS稀释1:200）60ul，滴加一抗后放入专用孵育盒内，4℃冰箱过夜。
9 切片放入PBS缓冲液中清洗3次各5分钟，充分洗涤，防止因洗涤不净引起的非特异性染色。（前两次的PBS倒掉）
10擦干组织周围液体后滴加70 ul辣根酶标羊抗小鼠/兔IgG聚合物（根据实际情况要求覆盖样本），室温孵育30分钟，PBS缓冲液洗涤3分钟×3次。
11 显色，滴加现配适量DAB显色剂，室温显色,5-20分钟。自来水终止显色。第一遍的自来水需集中处理，自来水洗涤3分钟。
12 复染，苏木素染液中染色40秒，水洗1分钟后，将切片置于0.5%氨水中浸泡30秒左右，继续水洗5-10分钟。
13将切片依次置于70%乙醇-80%乙醇-5%乙醇-100%乙醇中各3分钟。
14取出后将切片依次置于透明剂Ⅰ、Ⅱ、III中各5分钟
15 中性树胶封片，光学显微镜下观察。
B）抗MMP9（pT458）多克隆抗体的组织免疫组化鉴定结果
将免疫组化实验过后的宫颈癌、胃癌、胶质瘤切片与镜下观察（如图7所示），比较抗MMP9（pT458）多克隆抗体癌、癌旁组织中的表达，可见癌组织于胞浆或胞核中可见棕黄色，呈阳性。癌旁组织于胞浆或胞核中可见蓝色，呈阴性。结果提示抗MMP9（pT458）多克隆抗体在癌组织中表达、癌旁组织中的不表达，两者存在明显差异。
上述试验结果证明本发明的磷酸化多克隆抗体可特异性识别MMP9蛋白pT458磷酸化位点，可用于检测肿瘤细胞例如胃癌宫颈癌鼻癌等该位点的磷酸化水平，为探索肿瘤细胞增殖及转移机制研究提供一种工具，也为肿瘤诊断提供帮助，并能指导其临床预后判断。
SEQUENCE LISTING
<110> 广东医药大学
<120> 一种应激磷酸化抗原多肽、抗体、制备方法以及应用
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 14
<212> PRT
<213> 人工序列
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<213> 人工序列
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<212> PRT
<213> 人MMP9蛋白
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Gly Asp Leu Arg Thr Asn Leu Thr Asp Arg Gln Leu Ala Glu Glu Tyr
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Pro Pro Gly Pro Gly Ile Gln Gly Asp Ala His Phe Asp Asp Asp Glu
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Claims (5)
1.一种磷酸化抗原多肽，其序列为CEPRPPTTTT(p)PQPT，其中T(p)表示人MMP9蛋白T458位点氨基酸磷酸化苏氨酸。
2.一种针对权利要求1所述的磷酸化抗原多肽的多克隆抗体。
3. 如权利要求2所述的多克隆抗体的制备方法，其特征在于包括：（1）人工合成如权利要求1所述磷酸化抗原多肽；（2）将合成的多肽与马来酰亚氨活化的载体mcKLH偶联，将此偶联产物进行脱盐柱纯化后免疫新西兰兔；（3）经过四次免疫的兔血清用ELISA法对抗体效价进行检测，效价达理想值后收集免疫兔血清，并用多肽包被的溴化氰活化的琼脂糖亲和纯化柱纯化抗体；（4）对纯化后抗体进行ELISA、western bot鉴定。
4.如权利要求2所述的多克隆抗体在制备用于检测发生T458位点磷酸化修饰的人MMP9蛋白的试剂或试剂盒中的应用。
5.一种检测试剂盒，包括权利要求2所述的多克隆抗体、抗原修复液、PBS缓冲溶液、酶阻断剂3%H2O2、辣根酶标羊抗小鼠/兔IgG聚合物、DAB显色剂、苏木素染液、乙醇、环保透明剂、0.5%氨水和超纯水。
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