CN110426514B - 肽酰基精氨酸脱亚胺酶(pad)的测活法 - Google Patents

肽酰基精氨酸脱亚胺酶(pad)的测活法 Download PDF

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CN110426514B
CN110426514B CN201910801766.2A CN201910801766A CN110426514B CN 110426514 B CN110426514 B CN 110426514B CN 201910801766 A CN201910801766 A CN 201910801766A CN 110426514 B CN110426514 B CN 110426514B
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来棽棽
姚晓晖
谢志伟
严俊
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Abstract

本发明公开了肽酰基精氨酸脱亚胺酶（PAD）的测活法，包括试剂，所述试剂包括链霉亲和素，FBNA‑2肽的水溶液，PAD酶样品，胰蛋白酶，润洗液，PAD缓冲液，胰蛋白酶缓冲液，HRP标记的抗6xHIS抗体，四甲基联苯胺（TMB）底物溶液，2M硫酸，所述链霉亲和素包被8孔条板，所述FBNA‑2肽的水溶液为0.5mg/ml，所述胰蛋白酶，10mg/ml溶解于低酸度储存液中；本发明结合了第一部分中列举的几乎所有当前PAD测活方法的优势，尤其是免疫分析法的高灵敏度，易操作性，与胰蛋白酶法的低成本优势，除了方案设计中的底物，其它所有试剂均为生物实验室常用试剂，因而很容易获取。

Description

肽酰基精氨酸脱亚胺酶（PAD）的测活法
技术领域
本发明属于技术领域，具体涉及肽酰基精氨酸脱亚胺酶（PAD）的测活法。
背景技术
肽酰基精氨酸脱亚胺酶(Peptidylarginine deiminases，PADs)是一类依赖于Ca2+而催化肽酰基精氨酸转化为肽酰基瓜氨酸的酶，该家族在人类细胞中由5个同工酶组成，PAD1-4和PAD6，PAD的酶活力通常是以底物的转化作为读出而进行测定的。
现有的PAD测活方法具备着一定的问题，例如比色分析法只可对纯化的PAD蛋白进行快速酶活力测定，HPLC荧光分析法繁琐耗时，不适宜同时检测多份样品，PAD免疫分析法获得特异抗体成本高，具有灵敏度低，操作性差，成本高等问题，为此我们提出肽酰基精氨酸脱亚胺酶（PAD）的测活法。
发明内容
本发明的目的在于提供肽酰基精氨酸脱亚胺酶（PAD）的测活法，以解决上述背景技术中提出的现有的PAD测活方法具有灵敏度低，操作性差，成本高等问题。
为实现上述目的，本发明提供如下技术方案：肽酰基精氨酸脱亚胺酶（PAD）的测活法，包括试剂，所述试剂包括链霉亲和素，FBNA-2肽的水溶液，PAD酶样品，胰蛋白酶，润洗液，PAD缓冲液，胰蛋白酶缓冲液，HRP标记的抗6xHIS抗体，四甲基联苯胺（TMB）底物溶液，2M硫酸。
进一步的，所述链霉亲和素包被8孔条板，所述FBNA-2肽的水溶液为0.5mg/ml。
进一步的，所述FBNA-2肽的分子序列为: Biotin-GGSGG SYSAQ FTSST SYNRGDSTFE SHHHH HH。
进一步的，所述胰蛋白酶，10mg/ml溶解于低酸度储存液中。
进一步的，所述润洗液，1x TBS (25mM Tris，150mM NaCl，pH 7.2)，0.1% BSA和0.05% Tween-20。
进一步的，所述PAD缓冲液，0.1M Tris-HCl (pH 7.4)，10mM CaCl2，使用前加入0.5mM DTT及1mM PMSF，所述胰蛋白酶缓冲液，50mM Tris-HCl (pH 8.0)，2.5mM EDTA。
进一步的，所述测活步骤包括：
S1：用润洗液稀释FBNA-2肽至0.5 ug/ml作为包被液；
S2：在ELISA板架中安装8孔条板，润洗条板3次后将板倒置于洁净吸水纸上并拍干；
S3：往每孔中加入100ul包被液，室温下孵育1小时；
S4：弃去条板中的溶液，用润洗液润洗4次后将板倒置于洁净吸水纸上并拍干；
S5：用PAD缓冲液将酶稀释至所需浓度，加入50ul至每孔中，将条板置于37℃孵育30分钟；
S6：重复步骤S4；
S7：每孔中加入100ul胰蛋白酶缓冲液，平衡后弃去溶液；
S8：将胰蛋白酶以缓冲液稀释1000倍后，加100ul至条板的每个反应孔中，置于37℃孵育1小时；
S9：重复步骤S4；
S10：将HIS检测抗体以润洗液稀释10倍后，加100ul至条板的每个反应孔中，置于室温孵育1小时；
S11：重复步骤S4；
S12：将TMB溶液平衡至室温后，加100ul至条板的每个反应孔中，置于室温，避光环境中孵育20分钟；
S13：加50ul 2M硫酸至反应孔以终止显色反应，轻微震动条板，此时溶液颜色由蓝色转变为黄色，在30分钟内于450nm和540nm分别读取每孔的吸光度。
与现有技术相比，本发明的有益效果是：
1.本发明结合了第一部分中列举的几乎所有当前PAD测活方法的优势。尤其是免疫分析法的高灵敏度，易操作性，与胰蛋白酶法的低成本优势。除了方案设计中的底物，其它所有试剂均为生物实验室常用试剂，因而很容易获取。本方案已被证实可用于测定PAD酶家族所有6种人类基因编码蛋白的活性。
附图说明
图1为本发明的结构示意图；
图2为本发明实施例中PAD酶家族编码蛋白的活性检测结果图。
实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
请参阅图1至图2，本发明提供一种技术方案：肽酰基精氨酸脱亚胺酶（PAD）的测活法，包括试剂，试剂包括链霉亲和素，FBNA-2肽的水溶液，PAD酶样品，胰蛋白酶，润洗液，PAD缓冲液，胰蛋白酶缓冲液，HRP标记的抗6xHIS抗体，四甲基联苯胺（TMB）底物溶液，2M硫酸。
优选的，链霉亲和素包被8孔条板，FBNA-2肽的水溶液为0.5mg/ml。
优选的，所述FBNA-2肽的分子序列为: Biotin-GGSGG SYSAQ FTSST SYNRG DSTFESHHHH HH。
优选的，胰蛋白酶，10mg/ml溶解于低酸度储存液中。
优选的，润洗液，1x TBS (25mM Tris，150mM NaCl，pH 7.2)，0.1% BSA和0.05%Tween-20。
优选的，PAD缓冲液，0.1M Tris-HCl (pH 7.4)，10mM CaCl2，使用前加入0.5mMDTT及1mM PMSF，胰蛋白酶缓冲液，50mM Tris-HCl (pH 8.0)，2.5mM EDTA。
优选的，测活步骤包括：
S1：用润洗液稀释FBNA-2肽至0.5 ug/ml作为包被液；
S2：在ELISA板架中安装8孔条板，润洗条板3次后将板倒置于洁净吸水纸上并拍干；
S3：往每孔中加入100ul包被液，室温下孵育1小时；
S4：弃去条板中的溶液，用润洗液润洗4次后将板倒置于洁净吸水纸上并拍干；
S5：用PAD缓冲液将酶稀释至所需浓度，加入50ul至每孔中，将条板置于37℃孵育30分钟；
S6：重复步骤S4；
S7：每孔中加入100ul胰蛋白酶缓冲液，平衡后弃去溶液；
S8：将胰蛋白酶以缓冲液稀释1000倍后，加100ul至条板的每个反应孔中，置于37℃孵育1小时；
S9：重复步骤S4；
S10：将HIS检测抗体以润洗液稀释10倍后，加100ul至条板的每个反应孔中，置于室温孵育1小时；
S11：重复步骤S4；
S12：将TMB溶液平衡至室温后，加100ul至条板的每个反应孔中，置于室温，避光环境中孵育20分钟；
S13：加50ul 2M硫酸至反应孔以终止显色反应，轻微震动条板，此时溶液颜色由蓝色转变为黄色，在30分钟内于450nm和540nm分别读取每孔的吸光度。
本发明的工作原理及使用流程：PAD的酶活性使结合在8孔条板亲和素包被上的底物FBNA-2肽中的精氨酸转化为瓜氨酸，后者不被胰蛋白酶水解，从而能使C末端的6组氨酸结构保留在孔板中。该6组氨酸结构继而与特异性抗体结合。该抗体因被HRP标记，所以可以通过ELISA方法测定。因此，最终读出的吸光度应该与PAD活性呈正比线性关系。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (1)

1.肽酰基精氨酸脱亚胺酶PAD的测活法，包括试剂，其特征在于：所述试剂包括链霉亲和素，FBNA-2肽的水溶液，PAD酶样品，胰蛋白酶，润洗液，PAD缓冲液，胰蛋白酶缓冲液，HRP标记的抗6xHIS抗体，四甲基联苯胺TMB底物溶液，2M硫酸；
所述链霉亲和素包被8孔条板，所述FBNA-2肽的水溶液为0.5mg/ml；
所述FBNA-2肽的分子序列为：Biotin-GGSGG SYSAQ FTSST SYNRG DSTFE SHHHH HH；
所述胰蛋白酶，10mg/ml溶解于低酸度储存液中；
所述润洗液，由25mM Tris、150mM NaCl配制的pH7.2的1x TBS，0.1% BSA和0.05%Tween-20；
所述PAD缓冲液，0.1M pH7.4的Tris-HCl，10mM CaCl2，使用前加入0.5mM DTT及1mMPMSF，所述胰蛋白酶缓冲液，50mM pH8.0的Tris-HCl，2.5mM EDTA；
所述测活步骤包括：
S1：用润洗液稀释FBNA-2肽至0.5 ug/ml作为包被液；
S2：在ELISA板架中安装8孔条板，润洗条板3次后将板倒置于洁净吸水纸上并拍干；
S3：往每孔中加入100ul包被液，室温下孵育1小时；
S4：弃去条板中的溶液，用润洗液润洗4次后将板倒置于洁净吸水纸上并拍干；
S5：用PAD缓冲液将酶稀释至所需浓度，加入50ul至每孔中，将条板置于37℃孵育30分钟；
S6：重复步骤S4；
S7：每孔中加入100ul胰蛋白酶缓冲液，平衡后弃去溶液；
S8：将胰蛋白酶以缓冲液稀释1000倍后，加100ul至条板的每个反应孔中，置于37℃孵育1小时；
S9：重复步骤S4；
S10：将HIS检测抗体以润洗液稀释10倍后，加100ul至条板的每个反应孔中，置于室温孵育1小时；
S11：重复步骤S4；
S12：将TMB溶液平衡至室温后，加100ul至条板的每个反应孔中，置于室温，避光环境中孵育20分钟；
S13：加50ul 2M硫酸至反应孔以终止显色反应，轻微震动条板，此时溶液颜色由蓝色转变为黄色，在30分钟内于450nm和540nm分别读取每孔的吸光度。
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WO2007076302A2 (en) * 2005-12-20 2007-07-05 Boehringer Ingelheim International Gmbh High throughput assay for modulators of peptidylarginine deiminase activity
US20130101611A1 (en) * 2009-11-25 2013-04-25 The Johns Hopkins University Citrullination of human peptidylarginine deiminase 4 (pad-4) regulates its function and immunogenicity
US20120295292A1 (en) * 2011-05-19 2012-11-22 University Of South Carolina Detecting Protein Arginine Deiminase (PAD) Activity in Human Tissues and Sera
CN102445545B (zh) * 2011-09-22 2014-05-14 广东海大蓄牧兽医研究院有限公司 融合有v5标签重组蛋白的elisa定量检测试剂盒和方法
WO2014086365A1 (en) * 2012-12-03 2014-06-12 Rigshospitalet Anti-pad2 antibodies and treatment of autoimmune diseases
CN103869086B (zh) * 2014-04-14 2016-02-17 杭州凯保罗生物科技有限公司 一种血清自身抗体检测试剂盒
CN107271511A (zh) * 2016-12-19 2017-10-20 上海大学 检测肽酰基精氨酸脱亚氨酶用生物传感器及其制备方法和应用
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