CN114940715B - 一种能降解新冠病毒n蛋白的短肽及其应用 - Google Patents
一种能降解新冠病毒n蛋白的短肽及其应用 Download PDFInfo
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Abstract
一种能降解新冠病毒N蛋白的短肽,它是通过在一个淀粉样肽中放入两个APR(aggregation‑prone region),在两个APR中间由一个连接物隔开,在两个APR的两端添加有带负电荷的天冬氨酸残基(aspartate,D)来增加多肽的容量,在N‑末端添加色氨酸(tryptophan,W),所述的淀粉样肽,是利用TANGO分析SARS‑CoV‑2病毒N蛋白中的APR(aggregation‑prone region)位置及其TANGO评分,筛选出其中评分高于5分的部分并将其设计为N蛋白特异性串联淀粉沉淀抗原淀粉样肽。本发明副作用小,细胞实验证实该多肽几乎没有细胞毒性;N蛋白高度保守,可以避免因病毒突变产生耐药性;淀粉样沉淀原淀粉样肽特异性好;相比中和抗体,淀粉样沉淀原淀粉样肽分子量小,合成方便;基于病毒蛋白相分离原理设计药物是全新的药物设计思路。
Description
技术领域
本发明属于生物制药领域,具体涉及利用液-液相分离原理,开发一种能降解新冠病毒N蛋白的短肽及其应用。
背景技术
新冠肺炎是一种由新型冠状病毒SARS-CoV-2引起的肺炎。SARS-CoV-2病毒入侵宿主细胞后,病毒基因组中长度约30kb的核酸被翻译成结构蛋白和非结构蛋白,同时复制病毒基因组从而组装更多的病毒颗粒。其中,核衣壳蛋白 (Nucleocapsid,N)可以与病毒基因组RNA结合,并将其包装成大小约100nm 的颗粒,帮助膜蛋白(M)和包膜蛋白(E)有效地组装病毒包膜。
有些蛋白质中含有内在无序区域(Intrinsically Disordered Regions,IDR),而含有IDR序列的蛋白在生物过程中发挥其作用时受到液-液相分离(Liquid-liquid PhaseSeparation,LLPS)机制的调控,,可以进一步凝聚成淀粉样纤维。利用这一特征,研究人员设计串联淀粉样沉淀原-淀粉样肽促进靶蛋白凝聚从而实现抑制蛋白功能。这一设计首先应用于肿瘤治疗靶点VEGFR2(vascular endothelial growth factor receptor 2,VEGFR2),取得良好的抑制效果。最近这一设计应用于抗病毒治疗方面,研究人员设计出了针对甲型流感和寨卡病毒蛋白的病毒特异性淀粉样沉淀原淀粉样肽。实验证明设计的淀粉样肽可以有效结合流感病毒和寨卡病毒,抑制病毒蛋白在细胞内复制。动物实验表明淀粉样肽药物可以有效降低小鼠体内病毒含量,对小鼠脏器没有明显损伤。
这些提示可以利用冠状病毒N蛋白相分离特征设计特异性淀粉样肽促进N 蛋白凝聚从而抑制N蛋白功能。
发明内容
本发明的目的是克服上述技术的不足,提供一种能降解新冠病毒N蛋白的短肽,利用冠状病毒N蛋白相分离特征设计特异性淀粉样肽。
为实现上述目的,本发明采用的技术方案是:一种能降解新冠病毒N蛋白的短肽,它是通过在一个淀粉样肽中放入两个APR(aggregation-prone region),在两个APR中间由一个连接物隔开,在两个APR的两端添加有带负电荷的天冬氨酸残基(aspartate,D)来增加多肽的容量,在N-末端添加色氨酸(tryptophan, W),该短肽的氨基酸序列如SEQ.ID.NO.1、SEQ.ID.NO.2所示。
进一步的,所述的淀粉样肽,是利用TANGO分析SARS-CoV-2病毒N蛋白中的APR(aggregation-prone region)位置及其TANGO评分,筛选出其中评分高于 5分的部分并将其设计为N蛋白特异性串联淀粉沉淀抗原淀粉样肽。
所述的一种能降解新冠病毒N蛋白的短肽,在制备治疗新冠肺炎的药物中的应用。
优选的,所述的短肽在制备治疗新冠肺炎的药物中的应用,其浓度为 10μM~40μM。
本发明的有益效果在于:1)副作用小,细胞实验证实该多肽几乎没有细胞毒性;2)N蛋白高度保守,可以避免因病毒突变产生耐药性;3)淀粉样沉淀原淀粉样肽特异性好;4)相比中和抗体,淀粉样沉淀原淀粉样肽分子量小,合成方便;5)基于病毒蛋白相分离原理设计药物是全新的药物设计思路。
附图说明
图1为使用TANGO算法分析N蛋白的APR及其TANGO评分图。
图2为SARS-CoV-2N蛋白特异性串联淀粉沉淀抗原淀粉样肽示意图。
图3为CCK8检测NSA2A及NSA7A对细胞增殖的影响效果图。
图4为免疫杂交检测NSA2A和NSA7A降解N蛋白的能力效果图。
图5为NSA2A诱导N蛋白形成液滴示意图;
图6为FITC-NSA2A与SARS-CoV-2N蛋白在U2OS细胞中存在共定位测定效果图;
图7为Th-T染色发现SARS-CoV-2N蛋白在细胞内出现β聚集测定效果图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,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的实施例,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,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象,而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
1.利用TANGO设计SARS-CoV-2病毒N蛋白特异性淀粉样肽
如图1、图2所示,利用TANGO分析SARS-CoV-2病毒N蛋白中的APR (aggregation-prone region)位置及其TANGO评分,筛选出其中评分高于5分的部分并将其设计为N蛋白特异性串联淀粉沉淀抗原淀粉样肽。设计原则如下:我们在一个淀粉样肽中放入了两个APR,中间由一个坚硬的脯氨酸-脯氨酸(proline-proline linker,P-P linker)或甘氨酸-丝氨酸(glycine-serine linker,G-S linker)连接物隔开。为了保持序列的胶体稳定性和溶解性,我们通过在APR两侧添加带负电荷的天冬氨酸残基(aspartate,D)来增加多肽的容量。同时在N- 末端添加了色氨酸(tryptophan,W)以实现准确的浓度测定。将设计好的淀粉样肽命名为NSA。
2.体外毒性实验
为了验证淀粉样肽NSA的生物毒性并筛选出适宜的使用浓度,将不同位置设计合成的淀粉样肽NSA命名为NSA2A及NSA7A两组,分别用10μM、20μM和 40μM三个浓度处理HEK293T和U2OS细胞4小时后通过CCK8实验检测了它们对细胞增殖的影响。如图3所示,通过CCK8实验表明,与对照组相比,不管是NSA2A 还是NSA7A所选用的浓度几乎都不影响细胞的增殖。这说明设计的淀粉样肽细胞毒性很小,在接下来的实验中将选用10μM的肽浓度。
3.SARS-CoV-2病毒N蛋白特异性淀粉样肽促进N蛋白降解实验
如图4所示,利用免疫杂交技术检测了用淀粉样肽处理的HEK293T细胞中 SARS-CoV-2N蛋白的降解情况。结果发现这两种肽都能诱导N蛋白降解。
4.体外液体实验
如图5所示,为了探索NSA2A是否可以诱导体外纯化的N蛋白形成凝聚体,为此我们进行了体外液滴试验。将两者混合后在37℃条件下共孵育16小时后,通过相差显微镜观察我们发现在10μM浓度的NSA2A肽的存在下,在40μM N蛋白浓度条件下,淀粉样肽可在体外诱导N蛋白形成液状液滴。
5.淀粉样肽NSA2A与SARS-CoV-2病毒N蛋白在细胞内的相互作用实验
如图6所示,为了探索淀粉样肽NSA2A与SARS-CoV-2病毒N蛋白在细胞内的相互作用,并证实这种相互作用可以诱导N蛋白的聚集。首先将mCherry荧光标记的N蛋白质粒转染进入U2OS细胞内,转染48小时后用10μM的FITC荧光标记的 NSA2A淀粉样肽处理细胞4小时后行免疫荧光实验。通过免疫荧光实验观察到 FITC标记的淀粉样肽NSA2A和mCherry荧光标记的N蛋白在细胞质内存在共定位。
6.淀粉样肽NSA2A降低SARS-CoV-2病毒N蛋白活性实验
如图7所示,硫磺素T(Thioflavin,Th-T)是一种淀粉样染料,它可以选择性的与淀粉样蛋白沉淀结合,其中的β折叠结构是硫磺素T结合的关键分子基础,所以经常被用于检测细胞中的淀粉样纤维。使用Th-T观察到NSA2A淀粉样肽处理的细胞中出现了β聚集,在细胞中发现了强烈的Th-T信号,表明该肽能进入细胞并在细胞质中形成β聚集体。此外,还观察到N蛋白液滴与Th-T信号重叠,这预示着N蛋白出现了构象变化和β聚集形成。
综合以上结果,本发明首次证实通过本发明合成的淀粉样肽可显著降解 SARS-CoV-2病毒N蛋白。继而而通过本发明合成的淀粉样肽制成的药物,能够有效治疗新冠肺炎的病症。
序列表
<110> 河南省人民医院
<120> 一种能降解新冠病毒N蛋白的短肽及其应用
<141> 2022-03-10
<160> 2
<170> SIPOSequenceListing 1.0
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Claims (4)
1.一种能降解新冠病毒N蛋白的短肽,其特征是:该短肽的氨基酸序列如SEQ.ID.NO.1、SEQ.ID.NO.2所示。
2.根据权利要求1所述的一种能降解新冠病毒N蛋白的短肽,其特征是:所述短肽,是利用TANGO分析SARS-CoV-2病毒N蛋白中的APR(aggregation-prone region)位置及其TANGO评分,筛选出其中评分高于5分的部分,并设计获得。
3.根据权利要求1所述的一种能降解新冠病毒N蛋白的短肽,其特征是:其在制备治疗新冠肺炎的药物中的应用。
4.根据权利要求3所述的一种能降解新冠病毒N蛋白的短肽,所述的短肽在制备治疗新冠肺炎的药物中的应用,其浓度为10μM~40μM。
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