CN107312096B - 用于检测组蛋白位点三甲基化修饰的重组蛋白及其应用 - Google Patents
用于检测组蛋白位点三甲基化修饰的重组蛋白及其应用 Download PDFInfo
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Abstract
本发明提供用于检测组蛋白位点三甲基化修饰的重组蛋白及其应用。本发明重组蛋白的氨基酸序列中包括串联的N端GST融合蛋白序列和C端Tudor结构域序列。本发明的重组蛋白对H3K4和H4K20的亲和力强,可用于替代商业化抗体来进行Western Blot实验,灵敏度高,没有交叉结合发生,且生产成本显著降低,批次质量十分稳定,可以快速实现批量化生产。
Description
技术领域
本发明涉及蛋白修饰化检测技术领域,更具体地,涉及一种用于检测组蛋白位点三甲基化修饰的重组蛋白及其应用。
背景技术
组蛋白是染色体的重要组成部分。组蛋白会经历100多种不同的翻译后修饰,比如磷酸化、乙酰化、甲基化、泛素化和糖基化。这些翻译后修饰是调控染色体的结构和功能的重要方式,对发育、代谢、疾病等众多生理过程均起到关键的调控作用。研究这些组蛋白修饰具有重要意义,系统地定位这些组蛋白修饰已成为众多国际性大课题的焦点。这些组蛋白修饰在基因组范围的定位需要借助针对这些组蛋白修饰的特异性抗体。
组蛋白的甲基化修饰方式主要有赖氨酸或精氨酸位点上的甲基化修饰,组蛋白上赖氨酸的甲基化是一种很广泛的修饰,包括了单甲基化,双甲基化,和三甲基化3种不同修饰方式。不同程度的甲基化修饰在基因转录和调节中具有重要的生物学功能。甲基化发生在位于氨基末端的组蛋白尾部的五个主要的赖氨酸残基(H3K4,H3K9,H3K27,H3K36,H4K20),以及一个球状蛋白结构域(H3K79)内的赖氨酸残基,这些赖氨酸可发生单甲基化、二甲基化和三甲基化。
针对不同位点的甲基化,通常购买商业化抗组蛋白特定修饰位点的多抗,进行蛋白质印迹检测组蛋白修饰的程度。目前,针对组蛋白三甲基化修饰位点的商业化抗体价格昂贵,而且批次质量不稳定,特定位点修饰的抗体制备周期很长,产量低,抗体对不同甲基化修饰程度的识别反应容易发生交叉。
发明内容
(一)要解决的技术问题
本发明要解决的技术问题是针对组蛋白三甲基化修饰位点的商业化抗体价格昂贵,各批次质量不稳定,交叉反应明显,特定位点修饰的抗体制备周期长,产量低。
(二)技术方案
为了解决上述技术问题或者至少部分地解决上述问题,本发明提供了一种重组蛋白,该重组蛋白的氨基酸序列中包括串联的N端的GST融合蛋白序列和C端的Tudor结构域序列。
其中,Tudor结构域是一个由约60个氨基酸残基组成的可以特异性识别甲基化赖氨酸或精氨酸的结构域。
本发明的重组蛋白包含了N端的GST融合蛋白序列和C端的Tudor结构域序列,两者串联,采取了融合表达,即可增加Tudor蛋白的可溶性表达,也十分方便纯化和检测,在实验规模即可方便的纯化得到大量且质量稳定的重组蛋白。
在本发明一个优选实施方式中,为了提高对三甲基化修饰的识别能力,Tudor结构域序列,记为Tud01,为:
a)SEQ ID NO.1所示的氨基酸序列;或
b)SEQ ID NO.1所示的氨基酸序列经替换、缺失和/或添加一个或几个氨基酸残基形成的具有同等功能的氨基酸序列。
应当理解,本领域技术人员可根据本发明公开的SEQ ID NO.1片段,在不影响其活性的前提下,本发明的Tudor结构域序列还包括SEQ ID NO.1所示氨基酸序列取代、缺失或增加一个或几个氨基酸,具有与该结构域序列同等活性的由该Tudor结构域序列衍生得到的蛋白质。
包含了上述序列的重组蛋白对H3K4和H4K20上的三甲基化修饰均有很好的识别能力,特别是H3K4。
在本发明一个优选实施方式中,由该结构域与融合蛋白一起表达的重组蛋白GST-Tudor(记为GST-Tud01)的氨基酸序列为:
a)SEQ ID NO.2所示的氨基酸序列;或
b)SEQ ID NO.2所示的氨基酸序列经替换、缺失和/或添加一个或几个氨基酸残基形成的具有同等功能的氨基酸序列。
应当理解,本领域技术人员可根据本发明公开的重组蛋白GST-Tudor的氨基酸序列SEQ ID NO.2片段,在不影响其活性的前提下,本发明的重组蛋白还包括SEQ ID NO.2所示氨基酸序列取代、缺失或增加一个或几个氨基酸,具有与重组蛋白GST-Tudor同等活性的由GST-Tudor衍生得到的蛋白质。
使用该氨基酸序列的重组蛋白对H4K20me3的亲和力降低约200倍,显著提高了对H3K4me3的识别能力,有效地减少了交叉反应。
在本发明一个优选实施方式中,为了提高对H4K20me3的识别能力,Tudor结构域序列,记为Tud02,为:
a)SEQ ID NO.3所示的氨基酸序列;或
b)SEQ ID NO.3所示的氨基酸序列经替换、缺失和/或添加一个或几个氨基酸残基形成的具有同等功能的氨基酸序列。
应当理解,本领域技术人员可根据本发明公开的SEQ ID NO.3片段,在不影响其活性的前提下,本发明的Tudor结构域序列还包括SEQ ID NO.3所示氨基酸序列取代、缺失或增加一个或几个氨基酸,具有与该结构域序列同等活性的由该Tudor结构域序列衍生得到的蛋白质。
包含了上述序列的重组蛋白对H3K4和H4K20上的三甲基化修饰均有很好的识别能力,特别是H4K20。
在本发明一个优选实施方式中,由该结构域与融合蛋白序列的重组蛋白GST-Tudor(记为GST-Tud02)的氨基酸序列为:
a)SEQ ID NO.4所示的氨基酸序列;或
b)SEQ ID NO.4所示的氨基酸序列经替换、缺失和/或添加一个或几个氨基酸残基形成的具有同等功能的氨基酸序列。
应当理解,本领域技术人员可根据本发明公开的重组蛋白GST-Tudor的氨基酸序列SEQ ID NO.4片段,在不影响其活性的前提下,本发明的重组蛋白还包括SEQ ID NO.4所示氨基酸序列取代、缺失或增加一个或几个氨基酸,具有与重组蛋白GST-Tudor同等活性的由GST-Tudor衍生得到的蛋白质。
使用该氨基酸序列的重组蛋白对H3K4me3的亲和力降低约200倍,显著提高了对H4K20me3的识别能力,有效地减少了交叉反应。
根据本发明的一个方面,本发明还提供了编码上述重组蛋白的基因。
根据本发明的另一个方面,本发明还提供了提供含有上述基因的表达载体。
根据本发明的另一个方面,本发明还提供了含有上述表达载体的宿主细胞。
根据本发明的另一个方面,本发明还提供了含有上述重组蛋白的抗体。
根据本发明的另一个方面,本发明还提供了上述重组蛋白的制备方法,包括:
1)全基因优化合成Tudor序列;
2)构建表达质粒pGEX-4t-1-Tudor;
3)用步骤2)中pGEX-4t-1-Tudor转化大肠杆菌感受态细胞BL21,活化,诱导培养,即得。
其中,在一个优选实施方式中,步骤2)具体为:
根据步骤1)的Tudor序列合成引物,引物两端引入BamHI和XhoI限制性内切酶,PCR扩增,双酶切后,连接到亚克隆到pGEX-4t-1载体中,经筛选鉴定得到正确的重组后载体编码pGEX-4t-1-Tudor。
在一个优选实施方式中,步骤3)具体为:
重组载体pGEX-4t-1-Tudor转化大肠杆菌感受态细胞BL21,单克隆活化到LB液体培养基,当OD600=0.6时,加入IPTG,16至20度诱导12-15h,可以得到该重组蛋白的可溶性表达;从1L液体LB诱导表达的发酵液中获得约15-20mg GST-Tudor重组蛋白。
根据本发明的另一个方面,本发明还提供了含有上述重组蛋白的检测试剂盒。
根据本发明的另一个方面,本发明还提供了使用上述重组蛋白、基因或是抗体在检测组蛋白位点三甲基化中的应用。
更选地是,组蛋白位点为组蛋白H3中的位点。所述组蛋白位点更优选为H3K4或H4K20。
当Tudor结构域序列为SEQ ID NO.1所示的氨基酸序列或与其具有同等活性的氨基酸序列,或GST-Tudor为GST-Tud01,即氨基酸序列为SEQ ID NO.2所示的氨基酸序列或与其具有同等活性的氨基酸序列时,所得到的重组蛋白对H3K4me3有特异性的结合,即所检测的组蛋白的位点更优选为H3K4me3。
当Tudor结构域序列为SEQ ID NO.3所示的氨基酸序列或与其具有同等活性的氨基酸序列时,或GST-Tudor为GST-Tud02,即氨基酸序列为SEQ ID NO.4所示的氨基酸序列或与其具有同等活性的氨基酸序列时,所得到的重组蛋白对H4K20me3有特异性的结合,即所检测的组蛋白的位点更优选为H4K20me3。
在本发明中,组蛋白可以取自人、鼠、马、牛、猪、绵羊、山羊、鸡、狗、猫、果蝇、线虫和酵母等细胞中。
根据本发明的另一个方面,本发明还提供了使用上述重组蛋白在检测组蛋白H3K4me3或H4K20me3免疫印迹中的应用。
本发明涉及的SEQ ID No.2,SEQ ID No.4重组蛋白融合的GST蛋白,也可以方便使用抗GST通用的抗体,应用到蛋白免疫印记检测中。
在该应用中,使用上述重组蛋白与抗GST通用单抗以及HRP标记的羊抗鼠多抗,经过结合和信号放大,可得到高灵敏度的检测效果。
在该应用中,检测方法具体为:
1)提取待检测蛋白质,并将其转移至硝酸纤维膜上,封闭,TBST洗涤;
2)使用PBS稀释后终浓度为1-10ug/mL的重组蛋白GST-Tudor孵育,加入抗GST单抗和带HRP的羊抗鼠IgG多抗孵育,观察免疫印迹结果。
其中,以SEQ ID No.2所述的重组蛋白为例,检测方法具体为:
1)提取待检组织或细胞的细胞核蛋白质,先在15%的聚丙烯酰胺凝胶电泳中分离,随后将其转移至硝酸纤维膜(NC)上,封闭,TBST洗涤;
2)使用SEQ ID No.2制备的GST-Tudor重组蛋白孵育NC膜,,37℃孵育,使用TBST和TBS依次洗涤。
3)加入小鼠抗HA单抗,37℃孵育,使用TBST和TBS依次洗涤。
4)加入带HRP的羊抗鼠IgG多抗孵育,加入TMB或ECL底物液,暗室反应或曝光,观察免疫印迹结果。
本发明提出的重组蛋白GST-Tudor优化了Tudor结构域,提高了结构Tudor可溶性表达水平,对H3K4me3或H4K20me3均有较强的亲和力,特异性识别强。更有优势的是,利用大肠杆菌表达系统制备重组蛋白的成本低,一次研发理论上可以无数次重复生产,容易实现标准化工艺和规模化生产,从而保证批次间同质性好,质量稳定。
使用商业化抗体时,每100次样品实验使用同样的抗体,在保证实验成功的基础上平均消耗100ug抗体,平均成本为4500元,在成千上万次的检测中,这样的抗体价格就十分昂贵。而使用重组的Tudor结构域作为替代抗体,在一个制备周期内,每mg重组蛋白生产的平均成本不超过150元,加上使用通用抗体的市场价格,每100ug为500元,可以预期检测成本至少降低5倍。因此将上述2种重组蛋白替代商业化抗体,实验检测成本将显著降低。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规技术手段。若未特别指明,实施例中所用的试剂为市售。
实施例1
重组蛋白GST-Tud01的制备
1)全基因优化合成SEQ ID NO.1所述的核苷酸序列,即为Tud01的核苷酸序列,基因两端分别引入了限制性酶切位点BamHI和XhoI,合成的基因克隆到pUC57载体中;
2)提取步骤1)中包含Tud01序列的载体,双酶切,连接到同样双酶切的pGEX-4t-1载体,经筛选鉴定得到重组后载体编码pGEX-4t-1-Tudo01;可以编码SEQ ID No.2所述的氨基酸序列;
3)重组载体pGEX-4t-1-Tud01转化大肠杆菌感受态细胞BL21,单克隆活化到5mlLB液体培养基,当OD600=0.6时,加入0.4mM IPTG,16-20度诱导12-15h,可以得到该重组蛋白的可溶性表达。
4)从1L步骤3)中液体LB诱导表达的发酵液中获得约15-20mgGST-Tud01重组蛋白,该重组蛋白的氨基酸序列为SEQ ID NO.2,即为GST-Tud01重组蛋白。
实施例2
重组蛋白GST-Tud02的制备
1)全基因优化合成SEQ ID NO.3所述的核苷酸序列,即为Tud02的核苷酸序列,基因两端分别引入了限制性酶切位点BamHI和XhoI,合成的基因克隆到pUC57载体中;
2)提取步骤1)中包含Tud02序列的载体,双酶切,连接到同样双酶切的pGEX-4t-1载体,经筛选鉴定得到重组后载体编码pGEX-4t-1-Tud02可以编码SEQ ID No.4所述的氨基酸序列;
3)重组载体pGEX-4t-1-Tud01转化大肠杆菌感受态细胞BL21,单克隆活化到5mlLB液体培养基,当OD600=0.6时,加入0.4mM IPTG,低温16度到20度诱导12到15h,可以得到该重组蛋白的可溶性表达;
4)从1L步骤3)中液体LB诱导表达的发酵液中获得约15-20mgGST-Tud01重组蛋白,该重组蛋白的氨基酸序列为SEQ ID NO.4,即为GST-Tud02重组蛋白。
实施例3
重组蛋白的灵敏度检测
ITC仪器设备:The titration was performed using MicroCal iTC200system(GE Healthcare)
实验方法参考仪器使用说明和标准设置。
主要步骤:确定合适的反应物浓度,准备样品;滴定,收集热量数据;校正数据,拟合回归,计算热力学参数,最后分析模型。
1)所有检测样本置于恒温25℃反应;
2)合成待测的多肽10mg(组蛋白H3第4位赖氨酸上及组蛋白H4第20位赖氨酸的三甲基化修饰),使用ITC基础缓冲液溶解。母液浓度为0.5mg/ml;
多肽序列1为ARTKme3QTARKS
ITC结合实验中,使用SEQ ID No.2和4对应的重组蛋白(即实施例1和2中的重组蛋白)分别测定;
多肽序列2为KRHRKme3VLRDN
ITC结合实验中,使用SEQ ID No.2和4对应的重组蛋白(即实施例1和2中的重组蛋白)分别测定;
稀释溶液:20mM Tris-HCl,50mM NaCl,pH7.5,多肽工作浓度0.8-1.2mM。
3)重组蛋白使用紫外分光光度计测定,计算得到蛋白浓度为0.8mg/ml,使用上述缓冲液稀释,工作浓度0.05-0.1mM
4)收集反应的热量数据,最终结果由Origin7拟合计算.
有ITC实验测定和计算的Kd值反映了重组蛋白对相应模拟多肽的特异性结合.Kd越低,结合特异性越好。源于JMJD2A蛋白的Tudor结构域对H3K4me3和H4K20me3的Kd值分别为0.50uM和0.40uM。
其中,实施例1的GST-Tud01对H3K4me3的Kd值为0.2uM,结合特异性提高2倍,相比原始的Tudor结构域序列,对H4K20me3的Kd值为99uM,亲和力降低约200倍,显著降低了对H4K20me3的结合能力,优化后的Tudor结构域可以特异性的识别H3K4me3。
实施例2的GST-Tud02对H4K20me3:Kd值为0.083uM,结合特异性提高约5倍,另外相比原始的Tudor结构域序列,对H3K4me3的Kd值为85uM,亲和力降低约180倍,显著降低了对H3K4me3的结合能力,优化后的Tudor结构域可以特异性的识别H4K20me3。
实施例4
以人肝脏组织为检测样本,进行重组蛋白的蛋白免疫印迹WB(Western Blot)检测
1)使用RIPA裂解液提取人肝组织细胞核蛋白.采用15%SDS-PAGE聚丙烯酰胺凝胶电泳分离样品中的蛋白质,随后电转移蛋白到硝酸纤维素膜(NC)上;
2)使用5%BSA或脱脂奶粉37度封闭2个小时,TBST洗涤5次,每次5min,用TBS洗涤3次,每次5min;
3)使用SEQ ID No.2所述的重组蛋白(GST-Tud01)孵育NC膜,工作浓度为5-50nM或0.2-2ug/ml,37℃孵育1到2个小时,使用TBST和TBS依次洗涤3次,每次5min;
4)加入小鼠抗GST单抗,按说明书稀释抗体(1:1000),孵育2h,TBST洗涤3次,每次5min,用TBS洗涤3次,每次5min;
5)加入带HRP的羊抗鼠IgG多抗(1:2000),按说明书稀释抗体,孵育2h,TBST洗涤3次,每次5min,用TBS洗涤3次,每次5min;
6)加入ECL发光液,依次显影,定影,曝光1-5min。观察免疫印迹结果。
以SEQ ID No.4所述的重组蛋白GST-Tud02可以实施同样的技术方案。
对比的原JMJD2A蛋白的Tudor结构域,同时识别了H3K4me3和H4K20me3对应的条带。
以GST-Tud01重组蛋白进行WB时,灵敏度是原始结构域的2倍。在可控灵敏度范围内,只出现了H3K4me3对应大小的条带,没有发生交叉反应,有效使用浓度为25nM。
以GST-Tud02重组蛋白进行WB时,灵敏度是原始结构域的3.5倍左右,在可控灵敏度范围内,只出现了H4K20me3对应大小的条带,没有发生交叉反应,有效使用浓度为10nM。
由于降低了交叉反应,减少了结合的竞争性反应,有效降低了重组蛋白的使用浓度,进一步降低了检测成本。
最后,本发明的方法仅为较佳的实施方案,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 武汉博欧特生物科技有限公司
<120> 用于检测组蛋白位点三甲基化修饰的重组蛋白及其应用
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Claims (8)
1.一种重组蛋白,其特征在于,所述重组蛋白的氨基酸序列中包括串联的N端的GST融合蛋白序列和C端的Tudor结构域序列;其中,所述Tudor结构域序列为:
a)SEQ ID NO.1所示的氨基酸序列;或
b)SEQ ID NO.3所示的氨基酸序列。
2.根据权利要求1所述的重组蛋白,其特征在于,所述重组蛋白的氨基酸序列为:SEQID NO.2所示的氨基酸序列。
3.根据权利要求1所述的重组蛋白,其特征在于,所述重组蛋白的氨基酸序列为:SEQID NO.4所示的氨基酸序列。
4.权利要求1-3中任一项所述重组蛋白的制备方法,包括:
1)全基因优化合成Tudor序列;
2)构建表达质粒pGEX-4t-1-Tudor;
3)用步骤2)中pGEX-4t-1-Tudor转化大肠杆菌感受态细胞BL21,活化,诱导培养,即得。
5.含有根据权利要求1-3中任一项所述重组蛋白的检测试剂盒。
6.权利要求1-3中任一项所述重组蛋白在制备用于检测组蛋白位点三甲基化修饰的试剂盒中的应用。
7.根据权利要求6所述的应用,其特征在于,所述组蛋白位点为H3K4或H4K20。
8.根据权利要求6所述的应用,其特征在于,所述重组蛋白的氨基酸序列为:SEQ IDNO.2所示的氨基酸序列;所述组蛋白位点为H3K4;
或,所述重组蛋白的氨基酸序列为:SEQ ID NO.4所示的氨基酸序列;所述组蛋白位点为H4K20。
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CN103890587A (zh) * | 2011-08-31 | 2014-06-25 | 昂科赛特公司 | 用于治疗和诊断癌症的方法和组合物 |
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