CN102827869A - 一种p2y122稳定表达细胞系的建立及其在药物筛选中的应用 - Google Patents
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Abstract
本发明提供的P2Y122稳定表达细胞系的制备方法，从而提供一种筛选预防和/或治疗高血压、缺血性心脏病的药物的细胞模型。本发明提供的P2Y122稳定表达细胞系的制备方法，包括以下步骤：1)将P2Y122基因的编码序列插入到真核表达载体的多克隆位点，得到重组表达载体；2)将步骤1得到的重组表达载体导入宿主细胞中，筛选得到稳定表达P2Y122的细胞系。
Description
技术领域
本发明属于生物工程领域和医药技术领域，具体涉及一种P2Y122稳定表达细胞系及其在药物筛选中的应用。
背景技术
人类血小板包括三种不同的ADP受体：P2Y12、P2Y122和P2X1。P2X1是配体门控离子通道，P2Y12、2Y12是与不同的两种G蛋白偶联受体(GPCR)。P2Y122属于G蛋白偶联受体中的Gi类，其中P2Y12、2Y12是ADP作用的受体，也是ADP受体阻滞剂作用靶点。P2YI2受体与Gi蛋白偶联，当与其激动剂ADP结合通过激活Gi信号传导抑制腺昔酸环化酶(AC)，使细胞内ATP不能有效地转化为的cAMP，从而使血小板内的cAMP升高的幅度下降，在P2Y12受体的协同作用下，传导信号被放大，促进血小极分泌大量ADP，形成正反馈加速血小板聚集。此外，通过ADP激动该受体，易化了纤维蛋白原绑定糖蛋白(GP)IIbIIIa受体，最终导致部分可逆的血小板初次聚集与不可逆的血小板再次聚集。P2Y122是ADP诱导血小板聚集反应中的主要受体，也是噻氯吡啶、氯吡格雷等药物的作用靶点。
发明内容
本发明的目的是提供一种P2Y122稳定表达细胞系的制备方法。
本发明提供的P2Y122稳定表达细胞系的制备方法，包括以下步骤：
1)将P2Y122基因的编码序列插入到真核表达载体的多克隆位点，得到重组表达载体；
2)将步骤1得到的重组表达载体导入宿主细胞中，筛选得到稳定表达P2Y122的细胞系。
上述真核表达载体具体可为pTagLite-Snap、pEGFP-N3等。
为了便于对表达的P2Y122进行细胞定位，可在真核表达载体上加入能于宿主中表达并产生颜色变化的酶或发光化合物的基因，如SNAP、GFP、YFP等。
上述宿主细胞可为Hela细胞、MDA-MB-231细胞、MCF-7细胞等。
利用上述方法制备的P2Y122稳定表达细胞系也属于本发明的保护范围。
本发明的另一个目的是提供一种筛选预防和/或治疗缺血性脑卒中、心肌梗死等药物的细胞模型。
实验证明，本发明的P2Y122稳定表达细胞株对P2Y122阻断剂和P2Y122抗体敏感，作用显著。本发明的P2Y122稳定表达细胞系为深入探索P2Y122在血栓形成的作用提供实验技术平台，可用于筛选预防和/或治疗缺血性脑卒中、心肌梗死等药物。
附图说明
图1为重组表达载体pTagLite-Snap-P2Y122的构建流程图
图2为各稳定表达P2Y122细胞系的P2Y122表达量实验结果
图3为P2Y122稳定表达细胞系对P2Y122抑制剂的敏感性分析实验结果
具体实施方式
下面结合具体实施例对本发明作进一步说明，实施例仅为解释性的，绝不意味着以任何方式限制本发明的范围。
本发明的P2Y122稳定表达细胞系的具体构建流程如图1所示。
实施例1.
重组真核表达载体pTagLite-Snap-P2Y122的构建
下述实验过程中所用的DNA引物由上海生工生物技术有限公司合成。
根据P2Y122蛋白编码框的cDNA序列，设计PCR引物如下：正向引物：
5’-ACTGATATCAGAAATGCAAGCCGTCGACAACCTC-3’(下划线处为限制性内切酶的识别位点)；反向引物：
5′-AACGAGCTCCATTGGAGTCTCTTCATTTGGGTCA-3′(下划线处为限制性内切酶的识别位点)。
取人脐静脉内皮细胞HUVEC并提取总RNA，将其反转成cDNA，以该cDNA为模板，利用上述引物进行PCR扩增。将PCR扩增产物进行琼脂糖凝胶电泳纯化和回收，用限制性内切酶EcoRV和Xho I对纯化回收后的产物进行酶切，酶切产物再次进行琼脂糖凝胶电泳纯化和回收，得到P2Y122蛋白编码框的cDNA序列。同时，将真核表达载体pTagLite-Snap(购自Cisbio公司)也用EcoRV和Xho I进行双酶切，酶切产物进行琼脂糖凝胶电泳纯化和回收。用DNA连接酶连接上述扩增得到的P2Y122蛋白编码框的cDNA序列和真核表达载体pTagLite-Snap酶切后的产物，连接产物转化大肠杆菌(E.coli Top 10)感受态细胞，并涂布于含有氨苄青霉素的LB平板上，挑取单克隆进行PCR检测。对PCR产物进行测序，以检测阳性克隆中插入序列的正确。测序结果表明，插入的1026bp的P2Y122蛋白编码框的cDNA序列与GenBank Accession Number NM 022788.3的自5′端第245位-第1270位的序列一致。将得到的重组表达载体命名为pTagLite-Snap-P2Y122。
实施例2.
P2Y122稳定表达细胞系的建立
将上述步骤一得到的重组大肠杆菌利用
Plus SV Minipreps(购自Promega)提取质粒，得到高纯度的重组表达载体pTagLite-Snap-P2Y122，将重组表达载体pTagLite-Snap-P2Y122利用Lipofectamine 2000(购自Invitrogen)转染HeLa细胞，继续培养转染后的HeLa细胞24小时以上。用G418(浓度为1μg/ml)对重组HeLa细胞进行筛选，经多次筛选后，得到P2Y122的稳定表达细胞系。将各株P2Y122稳定表达的Hela细胞株保存于液氮中。
选取四个P2Y122稳定表达细胞株，利用抗P2Y122的抗体(购自AbCam公司)进行蛋白免疫印迹实验，结果如图2所示。其中，A-D分别表示不同的ET2的稳定表达细胞株的P2Y122表达量，Blank为转入真核表达载体pTagLite-Snap的空白对照细胞，anti-Tubulin表示参照。结果表明，各P2Y122的稳定表达细胞株均有不用程度的P2Y122表达。
实施例3.
P2Y122稳定表达细胞系对P2Y122抑制剂(Clopidogrel)的敏感性分析
选用上述实施例2的P2Y122稳定表达细胞、转入真核表达载体pTagLite-Snap的细胞进行P2Y122抑制剂敏感性试验。将P2Y122稳定表达细胞、转入真核表达载体pTagLite-Snap的细胞培养于DMEM+10％胎牛血清的培养基里，将处于旺盛生长期的细胞(一般在传代后20小时左右)经膜蛋白酶消化重悬后，进行细胞计数，以每孔103到104个细胞的量接种于96孔板中进行扩增，且每孔细胞数目均匀。24小时后，分别加入P2Y122抑制剂(Clopidogrel)。抑制剂(Clopidogrel)的浓度分别为10、20、30、40和50nM。而后按照ET cell-basedAssay Kit(购自Cisbio)说明书进行HTRF(均相时间分辨荧光)实验，在加入抑制剂后的细胞孔板中加入标记的一抗和二抗，孵育30min后在sunrise酶标仪(TECAN公司产品)上于665和620nm波长处读取吸光值，进行数据分析，具体结果如图3所示。
Claims (6)
1.一种P2Y12稳定表达细胞系的制备方法，包括以下步骤：
1)将P2Y12的编码基因插入真核表达载体的多克隆位点，得到重组表达载体；
2)将步骤1得到的重组表达载体导入宿主细胞中，筛选得到稳定表达P2Y12的细胞系。
2.根据权利要求1所述的方法，其特征在于，所述真核表达载体为pTagLite-Snap或pEGFP-N3。
3.根据权利要求1所述的方法，其特征在于，所述宿主细胞为HeLa细胞、MDA-MB-231细胞或MCF-7细胞。
4.根据权利要求1-3中任一项所述的方法制备的P2Y12稳定表达细胞系。
5.根据权利要求4所述的P2Y12稳定表达细胞系，其特征在于，所述P2Y12稳定表达细胞系为SNAP-P2Y12稳定表达的HeLa细胞系。
6.权利要求4-5中任一项所述的P2Y12稳定表达细胞系在筛选预防和/或治疗针对此靶点药物中的应用。
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Non-Patent Citations (3)
| Title |
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| D ECKE ET AL: "Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 50-O-(a-boranotriphosphate) analogues", 《BRITISH JOURNAL OF PHAMACOLOGY》 * |
| VINCENT ET AL: "P2Y1, P2Y2, P2Y4, and P2Y6 receptors are coupled to Rho and Rho kinase activation in vascular myocytes", 《AM J PHYSIOL HEART CIRC PHYSIOL》 * |
| 胡晖明等: "P2Y受体与卵巢癌转移相关性初步研究", 《中外医疗》 * |
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