CN117430688A - 一种nfe2l1蛋白的ntd多肽及其在制备抗肿瘤药物中的用途 - Google Patents
一种nfe2l1蛋白的ntd多肽及其在制备抗肿瘤药物中的用途 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及一种NFE2L1蛋白的NTD多肽及其在制备抗肿瘤药物中的用途。所述NTD多肽单体的氨基酸序列如SEQ ID NO:1所示。本发明通过构建NFE2L1的NTD氨基酸序列的多肽，意外发现NTD多肽具有良好的抗癌功能，并经过免疫缺陷小鼠荷瘤实验证实，本发明提供的NTD多肽具有显著抑制肿瘤生长的作用。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种NFE2L1蛋白的NTD多肽及其在制备抗肿瘤药物中的用途。
背景技术
NFE2L1(Nuclear Factor,Erythroid 2Like 1)蛋白是一种转录因子，属于NFE2L家族，参与了细胞内抗氧化应激、细胞周期和胰岛素稳态等重要生物学过程。全长NFE2L1蛋白约120-140kDa，氨基酸共772个。NFE2L1蛋白含有N端区域(NTD)、转录激活域1(AD1)、转录激活域2(AD2)、DNA结合区域(DBD)、C端区域(CTD)等主要的功能结构区域。
而NFE2L1的NTD结构域是其锚定于细胞膜系统的结构，将NTD结构切除有利于NFE2L1蛋白进入细胞核，而残留的NTD结构的功能目前是未知的。NFE2L1基因在人类各组织器官中普遍表达且对环境因素十分敏感，辐射、营养、代谢产物、各种药物或重金属的氧化刺激乃至环境温度都能引起NFE2L1基因的差异表达。NFE2L1的分子功能涉及调控氧化还原稳态、糖脂代谢稳态、蛋白质周转，生理功能涉及抗氧化解毒、细胞增殖与凋亡、组织器官发育与功能特化、癌症发生、炎症反应和免疫应答等，其在慢性系统性疾病如慢性炎症、癌症、糖尿病、肥胖、神经退行性疾病等疾病中的功能提示该蛋白具有潜在的临床应用价值。然而由于转录因子直接调控大量下游基因表达，涉及到的生物学功能广泛，往往不能作为临床应用研究的靶标分子。同样，单纯对NFE2L1的过表达或敲低(激活或抑制)都会引起系统性疾病的发生，如组织器官发育不良、神经退行性疾病、糖尿病等。
已有的NFE2L1蛋白修饰切割调控理论、多亚型模型等分子理论模型都基于其抗氧化转录因子功能，在可查阅范围未见对NFE2L1可能存在的非转录因子功能的研究。
发明内容
针对上述技术问题，本发明的发明人通过构建NFE2L1的NTD氨基酸序列的多肽，并通过蛋白质免疫印迹方法验证了NTD多肽能够正常表达，进而意外发现了NFE2L1的NTD结构域多肽SEQ ID NO:1所示的多肽能够显著抑制肿瘤的生长。
为了实现上述发明目的，本发明采用的技术方案为：
第一方面，本发明提供了一种NFE2L1蛋白的NTD多肽，所述NTD多肽单体的氨基酸序列如SEQ ID NO:1所示。
在本专利中，发明人独辟蹊径，通过构建NTD氨基酸序列的多肽(等同于NFE2L1蛋白的第1-130氨基酸区域，即NFE2L1全长蛋白的NTD结构域)，并通过蛋白质免疫印迹方法验证了NTD多肽能够正常表达，进而原始创新性发现了NFE2L1的NTD结构域的抗癌功能。发明人将NTD多肽稳定过表达的HepG2细胞系进行免疫缺陷小鼠荷瘤实验，结果发现NTD多肽具有显著抑制肿瘤生长的作用。
进一步地，所述NTD多肽还包括将SEQ ID NO:1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加，且具有抗肿瘤功能的由SEQ ID NO:1衍生的多肽。
衍生NTD多肽通过对氨基酸序列为SEQ ID NO:1的NTD多肽的序列进行修改、取代、缺失和添加，以此维持甚至增强其针对抑制肿瘤细胞的活性和特异性，使其适合作为潜在的抗肿瘤药物或治疗方法。
第二方面，本发明还提供了上述多肽的编码基因。
进一步地，所述编码基因的核苷酸序列如SEQ ID NO:2所示。
第三方面，本发明提供了上述编码基因的重组载体、转基因细胞系或重组菌。
进一步地，所述重组载体为将上述氨基酸序列为SEQ ID NO:1的NTD多肽的编码基因插入pLVX-EGFP载体中，得到表达所述多肽的重组载体。
进一步地，所述转基因细胞系是将所述的重组载体导入到宿主细胞中得到转基因细胞系，其中所述宿主细胞为肝癌组织细胞。
进一步地，所述肝癌组织细胞为HepG2细胞。
发明人经试验探究发现，通过将编码本发明所述NTD多肽的编码基因插入pLVX-EGFP载体中制备得到表达所述多肽的重组载体后，将其感染肝癌组织HepG2细胞，检测发现NTD多肽在细胞中能正常表达，且将感染后的HepG2细胞种植在小鼠体内形成人工的肝细胞癌，发现表达生成的NTD多肽能显著抑制肿瘤的生长。
第四方面，本发明还提供所述多肽、所述多肽的编码基因、所述重组载体或转基因细胞系在制备抗肿瘤药物中的用途。
进一步地，所述肿瘤为肝癌。
第四方面，本发明还提供一种抗肿瘤的药物，其活性成分为所述多肽、所述编码基因、所述重组载体或转基因细胞系。
进一步地，所述肿瘤为肝癌细胞，所述肝癌细胞尤其优选为HepG2细胞。
进一步地，所述抗肿瘤的药物为冻干粉剂或液体型剂。
进一步地，所述药物还包括药学上可接受的载体。本领域的技术人员可根据药物的剂型等对药学上可接受的载体进行常规选择。
与现有技术相比，本发明具有以下有益效果：
本发明通过构建NFE2L1的NTD氨基酸序列的多肽，意外发现NTD多肽具有良好的抗癌功能，并经过免疫缺陷小鼠荷瘤实验证实，本发明提供的NTD多肽具有显著抑制肿瘤生长的作用，因此该NTD多肽可能成为良好的低副作用的抗肿瘤药物。
附图说明
图1为本发明NFE2L1蛋白的NTD多肽表达效果检测图；
图2为本发明NFE2L1蛋白的NTD稳定过表达对肿瘤细胞皮下成瘤能力的影响结果图；其中，图2A为荷瘤体积对比图；图2B为荷瘤36天时取出荷瘤组织并拍照对比图，图中标尺为1cm；图2C表示荷瘤36天时肿瘤质量统计图。
具体实施方式
以下通过实施例形式的具体实施方法，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。
实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施例提供NFE2L1蛋白的NTD多肽表达质粒构建以及表达验证过程，具体步骤如下：
1、使用总RNA提取试剂盒提取人源细胞系总RNA，使用逆转录试剂盒将总RNA逆转录为cDNA作为PCR反应的模板；
2、用NTD扩增引物(NTD-F:5'-CTCGAGGAATTCATGCTTTCTCTGAAGA AATAC-3'；NTD-R:5'-TGTCACGGATCCGAGGCCACTGGAAC-3')扩增出NFE2L1基因中编码NTD结构域的核酸序列，并插入pLVX-EGFP载体中，得到NTD过表达质粒pLVX-NTD::EGFP。
3、将pLVX-EGFP、pLVX-NTD::EGFG质粒转染入HepG2细胞中，24小时后收取总蛋白，并采用蛋白质免疫印迹方法对提取的总蛋白进行检测。
结果如图1所示，本发明扩增出的NFE2L1基因中编码NTD结构域的核酸序列能正常表达生成NTD多肽。
实施例2
本实施例探究了NTD过表达对肿瘤细胞在裸鼠皮下荷瘤的影响，具体步骤如下：
1、使用慢病毒包装系统包装出NTD过表达慢病毒及空载体慢病毒，并感染HepG2细胞，通过使用含嘌呤霉素培养基培养，筛选出对照组细胞系pLVX、NTD过表达细胞系pNTD；
2、将pLVX、pNTD细胞系消化后用PBS洗2次，再用PBS重悬至1000万细胞每毫升的密度；
3、将8周龄裸鼠随机分为pLVX和pNTD两组，每组4只，每只裸鼠皮下注射150万细胞；荷瘤后第2天开始隔天记录荷瘤组织的长度(a)和宽度(b)，按照以下公式计算并记录小鼠肿瘤体积变化，结果如图2A所示：
V(体积)＝(a×b2)/2
4、待最大的肿瘤的体积达到1cm3左右时，处死荷瘤小鼠，取出肿瘤拍照(如图2B所示)并称取重量(如图2C所示)。
图2A-C结果充分证明NFE2L1基因中编码NTD结构域的核酸序列过表达生成的NTD多肽能显著抑制肿瘤的生长。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所述技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种NFE2L1蛋白的NTD多肽，其特征在于，所述NTD多肽单体的氨基酸序列如SEQ IDNO:1所示。
2.一种编码基因，其特征在于，编码权利要求1所述NTD多肽的编码基因。
3.根据权利要求2所述的编码基因，其特征在于，所述编码基因的核苷酸序列如SEQ IDNO:2所示。
4.含有权利要求2或3所述编码基因的重组载体、转基因细胞系或重组菌。
5.根据权利要求4所述的重组载体，其特征在于，所述重组载体为将权利要求1所述NTD多肽的编码基因插入pLVX-EGFP载体中，得到表达所述多肽的重组载体。
6.根据权利要求4所述的转基因细胞系，其特征在于，所述转基因细胞系是将权利要求5所述的重组载体导入到宿主细胞中得到转基因细胞系，其中所述宿主细胞为肝癌组织细胞。
7.一种如权利要求1所述多肽、权利要求2或3所述编码基因、权利要求4所述重组载体或转基因细胞系在制备抗肿瘤药物中的用途，其特征在于，所述肿瘤为肝癌。
8.根据权利要求7所述在制备抗肿瘤药物中的用途，其特征在于，所述肿瘤为肝癌。
9.一种抗肿瘤的药物，其特征在于，其活性成分为权利要求1所述多肽、权利要求2或3所述编码基因、权利要求4所述重组载体或转基因细胞系。
10.根据权利要求9所述抗肿瘤的药物，其特征在于，所述肿瘤为肝癌。
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