CN115521928A - Ace2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用 - Google Patents
Ace2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用 Download PDFInfo
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Abstract
本申请属于生物医药技术领域,尤其涉及ACE2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用。本申请提供了ACE2功能结构域肽段,ACE2功能结构域肽段具有SEQIDNO:1~6任一项所示的氨基酸序列,或者具有与SEQIDNO:1~6任一项所示的氨基酸序列存在至少80%同源性的序列。本申请提供了ACE2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用,有效解决现有ACE2蛋白与SARS‑CoV‑2结合力弱,免疫原性高的技术问题。
Description
技术领域
本申请属于生物医药技术领域,尤其涉及ACE2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用。
背景技术
新型冠状病毒(SARS-CoV-2)是一种单正链RNA病毒,可引起人类严重的呼吸综合症。随着SARS-CoV-2突变株的不断出现,限制了当前疫苗的有效性并导致感染激增,因此,亟需研发用于防治SARS-CoV-2及其突变体的技术。
SARS-CoV-2的基因组与SARS-CoV的基因组具有约80%的同一性。就 SARS-CoV而言,病毒体表面的Spike糖蛋白(S蛋白)介导受体识别和膜融合。在病毒感染期间,三聚体S蛋白被切割成S1和S2亚基,并且在过渡到融合后构象的过程中释放了S1亚基。S1包含受体结合结构域(RBD),它直接与 ACE2的肽酶结构域(PD)结合,而S2负责膜融合。当S1与宿主受体ACE2 结合时,S2上的另一个切割位点被暴露,并被宿主蛋白酶切割,这一过程对于病毒感染至关重要。
ACE2有两种形式,存在于细胞膜上或者以分泌型的形式存在于体液中。提示递送分泌型的ACE2(sACE2)治疗COVID-19可发挥双重功能,sACE2与病毒S蛋白结合,减缓或者阻止病毒进入细胞,ACE2对肾素-血管紧张素系统(RAS)产生负性调节作用,从而保护肺免受伤害。过量表达sACE2能够与 S1结合,阻止病毒附着在细胞表面,切断病毒进入宿主的可能性。并在时间上和空间上使得S2介导的膜融合无法发生,因此,基于ACE2与S1的结合部位设计药物,从而有效的遏制病毒的感染。并且,无论SARS-CoV-2如何变异,这些变异体能否感染细胞仍取决于ACE2受体介导的细胞进入,降低病毒与sACE2结合亲和力的SARS-CoV-2突变株同时也降低其与宿主细胞上 ACE2受体的亲和力,从而降低传染性和毒力。
但是,现有技术的人可溶性胞外区ACE2的基因片段可通过形成sACE2-S-加压素复合物介导SARS-CoV-2进入细胞,导致严重的副作用,此外较长的胞外段ACE2可能引发机体过敏反应。
发明内容
鉴于此,本申请提供了ACE2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用,有效解决现有ACE2蛋白与SARS-CoV-2结合力弱,免疫原性高的技术问题。
本申请第一方面提供了ACE2功能结构域肽段,ACE2功能结构域肽段具有SEQ IDNO:1~6任一项所示的氨基酸序列,或者具有与SEQ ID NO:1~6任一项所示的氨基酸序列存在至少80%同源性的序列。
本申请第二方面提供了ACE2功能结构域肽段在制备预防和/或治疗新型冠状病毒药物中的应用;所述ACE2功能结构域肽段具有SEQ ID NO:1~6任一项所示的氨基酸序列,或者具有与SEQ ID NO:1~6任一项所示的氨基酸序列存在至少80%同源性的序列。
本申请提供的ACE2功能结构域肽段为分泌型可溶性ACE2功能结构域肽段的基因片段。
本申请的SEQ ID NO:1~6为截取ACE2有效与S蛋白结合的功能结构域肽段,本申请的ACE2功能结构域肽段序列短,其编码的蛋白的较小,使之既可以结合S蛋白中和病毒,又可以避免过敏反应的发生,也不会干扰常规的生理反馈系统。经过分析,本申请的SEQ IDNO:1~6与SARS-CoV-2具有高效亲和力,提高抑制SARS-CoV-2病毒进入能力。
本申请的SEQ ID NO:2截取的是全长人ACE2的基因片段,SEQ ID NO:2 保留了与S蛋白结合的片段和维持ACE2二聚体形态的片段,稳定性较好。此外,SEQ ID NO:2还保留了全长ACE2的酶活性,有助于同时发挥抗新型冠状病毒的作用和治疗心血管疾病的作用,同时SEQ ID NO:2的序列长短比全长人ACE2短,可减少过敏反应等副反应的发生。
本申请第三方面提供了ACE2功能结构域核酸,其编码权利要求1所述的ACE2功能结构域肽段。
本申请第四方面提供了一种表达载体,包含所述ACE2功能结构域核酸。
另一实施例中,所述表达载体包括ACE2功能结构域核酸和十号血清型腺相关病毒AAVrh10载体;
所述ACE2功能结构域核酸位于所述AAVrh10载体的启动子和所述 AAVrh10载体的polyA尾巴之间。
具体的,本申请中AAVrh10载体从十号血清型自恒河猴体内分离得到的衣壳,该载体介导的体外转导效率更高,并且在细胞和组织类型中具有更广泛且更高的感染率。使用AAVrh10载体可减少发生宿主血清学免疫反应的机会,同时使其携带的目的基因在特定组织长效表达,简化蛋白制备流程,经济,有助于大规模应用。
具体的,本申请的表达载体具有rAAV衣壳,所述rAAV衣壳具有包装在其中的载体基因组,所述载体基因组包含rAAV反向末端重组序列和编码 ACE2功能结构域核酸的核酸序列,可溶性ACE2从所述rAAV表达,利用 AAVrh10作为载体,表达分泌型ACE2进行中和疫苗的研制具有高效性和安全性,对防治SARS-CoV-2感染具有重要意义。
另一实施例中,所述AAVrh10载体的两端具有反向终端重复序列ITR。
另一实施例中,所述AAVrh10载体的启动子为人巨细胞病毒启动子 CMV。
另一实施例中,所述AAVrh10载体的polyA尾巴为SV40。
本申请第五方面提供了一种宿主细胞,其包含所述表达载体。
本申请第六方面提供了一种用于治疗或预防新型冠状病毒感染的药物组合物,其包括所述表达载体。
具体的,本申请的表达载体是基于十号血清型腺相关病毒AAVrh10载体编码ACE2功能结构域肽段的基因片段,该序列编码的蛋白质可分泌到胞外,且具有与S蛋白特异结合的功能域。利用AAVrh10载体将编码ACE2功能结构域肽段蛋白的基因序列导入靶细胞,使靶细胞分泌ACE2功能结构域肽段蛋白到细胞外以便与新型冠状病毒的S蛋白结合,达到拮抗SARS-CoV-2感染的目的。
另一实施例中,所述药物组合物包括所述表达载体以及药学上可接受的辅料。
本申请通过筛选不同物种的ACE2蛋白,得到六条分泌型可溶性ACE2 功能结构域肽段,这几条ACE2功能结构域肽段长度较短,具有比人全长ACE2 更高的结合力,使之更有效的结合新型冠状病毒及其突变体,从而阻止病毒进入宿主细胞;而且这些肽段的免疫原性较低,具有更高的安全性。
此外,本申请使用这些分泌型可溶性ACE2功能结构域肽段作为诱饵阻断SARS-CoV-2及其突变株感染具有很大的发展潜力;本申请提供的治疗新型冠状病毒药物,基于腺相关病毒十号血清型AAVrh10载体编码本申请的可溶性胞外区非全长ACE2的蛋白基因片段,该序列编码的蛋白质可分泌到胞外,且具有与S蛋白特异结合的功能域。利用AAVrh10载体将编码可溶性胞外区ACE2蛋白的基因序列导入靶细胞,使靶细胞分泌可溶性ACE2到细胞外以便与冠状病毒的S蛋白结合,达到拮抗SARS-CoV-2感染的目的。而且本申请中AAVrh10血清型自恒河猴体内分离,人类感染机会较少,且AAVrh10 载体介导的体外转导效率更高,并且在细胞和组织类型中具有更广泛更高的感染率,本申请使用AAVrh10载体可有效减少发生宿主血清学免疫反应的机会,并提高转导效率。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本申请实施例提供的ACE2氨基酸序列的相似性分析,图1A是使用Simplot软件分析多个物种的ACE2氨基酸序列与人ACE2(Q9BYF1.2)在各个氨基酸位置的相似性;图1B热图代表17个物种的ACE2氨基酸序列的百分比相似性矩阵;
图2为本申请实施例提供的不同生物体中SARS-COV-2病毒RBD与 ACE2界面之间的重要氨基酸残基。突变残基用蓝色或绿色标记。蓝色残基代表具有相似性质的突变(极性到极性或非极性到非极性),绿色残基代表导致残基性质发生变化的突变(极性到非极性或非极性到极性);
图3为本申请实施例提供的SARS-CoV-2S蛋白RBD(紫色)与人ACE2 结合域(蓝色)结合的示意图,图3B和图3C是图3A的特写视图,SARS-CoV-2 S蛋白RBD(紫色)与人ACE2结合域(蓝色)的界面区域。界面上的重要残基已在相应位置标出。包括氢键、静电相互作用和疏水相互作用在内的分子间相互作用分别以黄色、蓝色和绿色显示;
图4为本申请实施例提供的设计的不同物种ACE2肽与SARS-CoV-2 RBD复合物的关键分子间相互作用力和结合亲和力的比较,绿色的√号和红色的×号分别代表相互作用的存在和不存在。ΔG和Kd值分别代表结合自由能和结合亲和力;
图5为本申请实施例提供的基于AlphaFold2和分子对接的截短ACE2 蛋白复合物的结合自由能和分子间相互作用分析。图5A是人类和非人类物种的ACE2-RBD复合物之间的RMSD值。图5B-图5D是不同物种蛋白质三维结构的比较。图5B是猫和人类,图5C是蝙蝠和人类,图5D是鸡和人类的截短ACE2蛋白复合物的结构比对。重要的突变残基以横杆显示;
图6为本申请实施例提供的不同物种的截短型ACE2小片段与 SARS-CoV-2野生型(wuhan-Hu-1)的结合自由能和结合亲和力的预测。ΔG和 Kd值分别代表结合自由能和结合亲和力;
图7为本申请实施例提供的不同物种的截短型ACE2小片段与 SARS-CoV-2B.1.351株的结合自由能和结合亲和力的预测。ΔG和Kd值分别代表结合自由能和结合亲和力;
图8为本申请实施例提供的不同物种的截短型ACE2小片段与 SARS-CoV-2P.1株的结合自由能和结合亲和力的预测。ΔG和Kd值分别代表结合自由能和结合亲和力;
图9为本申请实施例提供的不同物种的截短型ACE2小片段与 SARS-CoV-2B.1.617株的结合自由能和结合亲和力的预测。ΔG和Kd值分别代表结合自由能和结合亲和力;
图10为本申请实施例提供的不同物种的截短型ACE2小片段与 SARS-CoV-2B.1.1.529株的结合自由能和结合亲和力的预测。ΔG和Kd值分别代表结合自由能和结合亲和力;
图11为本申请实施例提供的不同物种的截短型可溶性ACE2片段的理化性质、致敏性和免疫原性分析;
图12为本申请实施例提供的表达载体的结构示意图;
图13为本申请实施例提供的AAVrh10递送截短型人可溶性ACE2阻断 SARS-CoV-2感染过程,图13A说明将AAV-shACE2、AAV-shACE2PD和 AAV-shACE2(PD+Neck)质粒转染到Hela细胞中后,通过蛋白质免疫印迹法检测到的细胞内(中)和上清液(下)中的蛋白质表达情况。图13B是流式细胞仪检测AAV-shACE2、AAV-shACE2PD和AAV-shACE2(PD+Neck)与S蛋白的结合能力,利用分泌型ACE2蛋白和S抗体竞争结合S蛋白原理,通过检测与S抗体结合的表达S蛋白的细胞数量反应ACE2竞争结合效果,结合S抗体的细胞数量越少,ACE2蛋白与S蛋白的结合能力越强。图13C表示AAV-shACE2、 AAV-shACE2PD和AAV-shACE2(PD+Neck)抑制SARS-CoV-2假病毒感染细胞的能力,检测荧光素酶的表达,表达越少,说明假病毒越少,ACE2抑制能力越强。图13D表示CACO-2-N细胞在被AAVrh10-shACE2或 AAVrh10-shACE2(PD+Neck)病毒感染24小时后,通过RT-qPCR检测到 CACO-2-N细胞的SARS-CoV-2trVLP RNA水平,越低说明抑制能力越好。图13E表示PBS、AAVrh10-shACE2或AAVrh10-shACE2(PD+Neck)病毒处理的小鼠血清对SARS-CoV-2假病毒感染的抑制百分比,百分比越高,抑制效果越好;
图14为本申请实施例提供的AAVrh10递送的截短型ACE2多肽在体外阻断SARS-CoV-2感染,图14A是用表达AAV-shACE2、AAV-hACE224-83、 AAV-cACE224-83、AAV-mACE224-83和AAV-dACE224-83质粒转染CACO-2-N细胞后,加入SARS-CoV-2trVLP病毒颗粒感染细胞,之后通过荧光定量PCR 检测SARS-CoV-2trVLP RNA水平,越低抑制效果越好。图14B是用MOI=106的AAVrh10-shACE2、AAVrh10-shACE2(PD+Neck)、AAVrh10-hACE224-83和 AAVrh10-dACE224-83感染BHK-ACE2细胞24小时以表达ACE2可溶性片段,然后加入SARS-CoV-2假病毒感染细胞,检测荧光素酶的表达,计算病毒抑制率,抑制率越高,说明该ACE2片段的抑制能力越强;
图15为本申请实施例提供的AAVrh10递送的截短型ACE2可溶性片段在体外抑制表达S蛋白的细胞和表达ACE2受体的细胞之间的融合,在荧光显微镜下观察AAVrh10-shACE2、AAVrh10-shACE2(PD+Neck)、AAVrh10-hACE224-83和AAVrh10-dACE224-83处理后细胞的融合情况,比例尺为2000μm。
具体实施方式
本申请提供了ACE2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用,用于解决现有技术中ACE2蛋白与SARS-CoV-2结合力弱,免疫原性高的技术缺陷。
下面将对本申请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申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,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,都属于本申请保护的范围。
其中,以下实施例所用原料或试剂均为市售或自制。
SARS-CoV-2病毒样颗粒(trVLP):是一种细胞培养系统,由报告基因(GFP) 取代病毒核衣壳基因(N)的重组SARS-CoV-2病毒和表达SARS-CoV-2N蛋白的CACO-2细胞(CACO-2-N)组成,在此系统中,重组SARS-CoV-2病毒具有复制能力,可以在P2实验室进行实验,具有良好的安全性。
Simplot分析:Simplot是SimilarityPlotting简写形式,意即序列相似性作图,是一款非常流行的重组分析工具。
GenBank:是美国国家生物技术信息中心(National Center for BiotechnologyInformation,NCBI)建立的DNA序列数据库,从公共资源中获取序列数据,主要是科研人员直接提供或来源于大规模基因组测序计划。
ggplot2:R语言里面一个比较重要的绘图包,核心理念是将绘图与数据分离,数据相关的绘图与数据无关的绘图分离,是按图层作图,分为数据层、几何图形层和美学层。
R-Studio:是一个功能强大、节省成本的反删除和数据恢复软件系列。
以下实施例5所用的细胞为Hela细胞,293T细胞,BHK-ACE2细胞, CACO-2-N细胞。以下实施例所用的试剂:胎牛血清FBS(品牌:GIBCO,货号:10099-141);青霉素/链霉素(品牌:GIBCO,货号:15140122); DMEM培养基(品牌:GIBCO,货号:DMEM-c1995500BT);lipo2000转染试剂(品牌:Invitrogen,货号:11668019);opti-MEM培养基(品牌:GIBCO,货号:31985070);DMEM/High Modified去酚红培养基(品牌:HyClone,货号:SH30604.01);AmiconUltra超滤管(品牌:Merckmillipore,货号: UFC801008);RIPA裂解液(品牌:biosharp,货号:RIPA-BL504A);5×SDS 蛋白缓冲液(品牌:Dingguo,货号:WB-0091);脱脂奶粉(品牌:BD,货号:232100);Anti-DYKDDDDKTag抗体(品牌:abcam,货号:ab49763);GAPDH抗体(品牌:CST,货号:97166S);鼠二抗(品牌:Beyotime,货号:P0625);兔二抗(品牌:Beyotime,货号:P0628);ECL发光液(品牌:biosharp,货号:ECL-BL520A);抗S蛋白抗体(品牌:CHAMOT,货号:CM002-0.1A1CoV);Anti-human IgG二抗(品牌:ThermoFisher,货号:MA514728);荧光素酶检测试剂盒(品牌:Promega,货号:N1610);RNA 提取试剂盒(品牌:Yeasen,货号:19221ES08);HifairTM III 1st Strand cDNA Synthesis SuperMixforqPCR(品牌:Yeasen,货号:11141ES60);PerfectStart SYBR Green qPCR supermix(品牌:Transgen,货号:AQ601)。
以下实施例5所用的细胞为CACO-2-N细胞,BHK-ACE2细胞。
以下实施例5所用的试剂:胎牛血清FBS(品牌:GIBCO,货号: 10099-141);青霉素/链霉素(品牌:GIBCO,货号:15140122);DMEM 培养基(品牌:GIBCO,货号:DMEM-c1995500BT);lipo2000转染试剂(品牌:Invitrogen,货号:11668019);opti-MEM培养基(品牌:GIBCO,货号: 31985070);RNA提取试剂盒(品牌:Yeasen,货号:19221ES08);HifairTMIII 1st Strand cDNA Synthesis SuperMix for qPCR(品牌:Yeasen,货号: 11141ES60);PerfectStart SYBR Green qPCR supermix(品牌:Transgen,货号:AQ601);荧光素酶检测试剂盒(品牌:Promega,货号:N1610)。
以下实施例6所用的细胞为HEK293T细胞、Hela细胞、VeroE6细胞。
以下实施例6所用的试剂:胎牛血清FBS(品牌:GIBCO,货号: 10099-141);青霉素/链霉素(品牌:GIBCO,货号:15140122);DMEM 培养基(品牌:GIBCO,货号:DMEM-c1995500BT);lipo2000转染试剂(品牌:Invitrogen,货号:11668019);opti-MEM培养基(品牌:GIBCO,货号: 31985070)。
实施例1
本申请实施例提供了不同物种的ACE2氨基酸序列的相似性以及ACE2 与SARS-CoV-2RBD界面之间的关键分子间的相互作用
1.1不同物种ACE2序列的相似性分析
1)不同物种ACE2序列的相似性分析,根据以下标准选择了17个具有代表性的ACE2同源序列进行进一步分析:与人类持续密切接触的物种,如宠物和牲畜;在生物医学研究中用作动物模型的物种,如雪貂、恒河猴和小鼠;根据以往研究可能作为潜在宿主的物种,如穿山甲、果子狸、蝙蝠等。2) 从国家生物技术信息中心(NCBI)获得上述多个物种的ACE2氨基酸参考序列和相应的登录号;以人ACE2(Q9BYF1.2)作为参考株。
17个ACE2序列分别为:人ACE2(编号:Q9BYF1.2)、恒河猴ACE2(编号:NP_001129168.1)、仓鼠ACE2(编号:XP_027288607.1)、猫ACE2 (编号:Q56H28.1)、老虎ACE2(编号:XP_042830021.1)、雪貂ACE2 (编号:XP_004758942.1)、麝猫ACE2(编号:NP_ 001034545.1)、牛 ACE2(编号:Q58DD0.1)、羊ACE2(编号:XP_042098229.1)、兔ACE2 (编号:XP_002719891.1)、狗ACE2(编号:NP_001158732.1)、猪ACE2 (编号:NP_001116542.1)、穿山甲ACE2(编号:XP_036768816.1)、蝙蝠ACE2(编号:XP_032963186.1)、鼠ACE2(编号:Q8R0I0.1)、鸡 ACE2(编号:XP_416822.3)、鸭ACE2(编号:XP_012949915.3); SARS-CoV-2S蛋白的编号为YP_009724390.1。
3)由具有自动策略的MAFFT(amultiple sequence alignment program,多序列比对程序)软件(版本7.453)执行多序列比对。
4)使用Simplot软件(版本3.5.1)分析多个物种的ACE2氨基酸序列与人ACE2参考株在各个氨基酸位置的相似性,该分析基于Kimura 2参数模型,将窗口宽度和步长分别设置为200bp和20bp进行。
5)为了分析多个物种的ACE2氨基酸序列与人ACE2参考株的总体相似性,将比对序列导入BioEdit软件(版本7.0.9)计算序列相似性矩阵;使用 ggplot2包在R studio(版本4.0.2)中绘制相似性矩阵热图。
1.2ACE2-RBD复合体结合界面分子间相互作用力分析
1)SARS-CoV-2 spike-ACE2(PDB ID:6LZG)复合物的晶体结构来自蛋白质数据库(https://www.rcsb.org/)用于后续分析。
2)将复合物的晶体结构上传至Protein Interactions Calculator (http://pic.mbu.iisc.ernet.in/job.html)计算蛋白质与蛋白质之间的相互作用力,相互作用是根据标准氢键的标准确定的。疏水侧链之间的相互作用是使用的距离截止值确定的。此外,将复合物的晶体结构上传至PRODIGY网络服务器(https://bianca.science.uu.nl/prodigy/),选择A链为SARS-CoV-2RBD,B 链为ACE2,作用温度为37℃,计算分子间作用力及结合自由能和亲和力。
3)使用PyMOL软件(2.3.0版)制作了6LZG的晶体结构图。重要的残基在晶体结构中显示为stick,而其他残基显示为cartoon。使用PyMOL软件的measurement工具来确定分子间相互作用的两个原子之间的距离,包括氢键、静电相互作用和疏水相互作用在内的分子间相互作用分别以不同颜色显示。
2.实验结果
2.1不同物种的ACE2氨基酸序列的相似性分析
结果如图1~图3所示和表1。图1是不同物种的ACE2氨基酸序列的相似性分析以及不同物种的ACE2与SARS-CoV-2RBD界面之间的关键分子间相互作用。图1A是人ACE2(Q9BYF1.2)对多个物种的Simplot分析。不同的颜色对应人类ACE2与不同物种之间的氨基酸相似性。图1A的X轴表示多重比对中的氨基酸位置,图1A的Y轴表示查询序列和参考株之间的氨基酸同一性。图1B热图代表17个物种的ACE2氨基酸序列的百分比相似性矩阵。热图右侧的注释表示氨基酸同一性的值。如图1A所示,不同颜色对应人类ACE2与不同物种之间的氨基酸相似性,与人ACE2相比,其他物种的ACE2在氨基酸序列的位置上保持80%以上的相似性。此外,图1B表示这 17个物种的ACE2氨基酸序列的百分比相似性矩阵。图1B右侧的注释表示氨基酸同一性的值,结果显示在主要负责与SARS-CoV-2的RBD结合的PD 片段中的α1螺旋、α2螺旋以及β3和β4发夹之间的连接处(位于人ACE2蛋白序列的19-355)与人类ACE2的同一性超过90%,不同物种之间多次比对的氨基酸序列显示出超过64%的整体相似性。恒河猴ACE2序列与人类ACE2 的相似度最高(95%),而鸡和鸭的相似度约为65%(图1B)。
本申请实施例还比较不同物种与SARS-CoV-2RBD直接连接的20个重要残基的变异,以更好地了解SARS-CoV-2的跨物种传播。侧链生物物理特性相似的残基用蓝色标记(极性到极性或非极性到非极性),而绿色残基表示突变引起的生物物理特性变化(极性到非极性或非极性到非极性)(图2)。它表明不同物种的这些关键位置存在ACE2蛋白序列的突变,这可能会影响它们与SARS-CoV-2RBD的结合亲和力。图2表示不同生物体中SARS-COV-2 病毒RBD与ACE2界面之间的重要氨基酸残基。突变残基用蓝色或绿色标记。蓝色残基代表具有相似性质的突变(极性到极性或非极性到非极性),绿色残基代表导致残基性质发生变化的突变(极性到非极性或非极性到极性)。图3A表示SARS-CoV-2S蛋白RBD(紫色)与人ACE2结合域(蓝色)结合的示意图。图3B和图3C是图3A的特写视图,SARS-CoV-2S蛋白RBD (紫色)与人ACE2结合域(蓝色)的界面区域。界面上的重要残基已在相应位置标出,其包括氢键、静电相互作用和疏水相互作用在内的分子间相互作用分别以黄色、蓝色和绿色显示。该图是在PyMOL中使用PDB文件 6LZG创建的。
2.2ACE2与SARS-CoV-2RBD界面之间的关键分子间的相互作用
为了进一步阐明关键残基突变如何影响ACE2和SARS-CoV-2RBD之间的分子间相互作用,本实施例重建了SARS-CoV-2RBD-ACE2复合物 (PDB ID:6LZG)的三维结构进行氨基酸替代实验,相对于人ACE2基因将特定氨基酸进行替代突变,研究这些突变对SARS-CoV-2RBD-ACE2复合物的三维结构的影响。结果如图3A和表1所示,ACE2关键残基的突变会破坏关键的分子间相互作用,导致其与SARS-CoV-2RBD的结合亲和力降低。首先,由于ACE2中的以下突变,Gln24被Leu取代,Glu 35被Lys或Arg取代,Tyr41和Lys 353被His取代导致氢键被破坏。其次,Asp 30被Glu、Asn 或Ala取代,Lys31被Glu取代对静电相互作用形成的中心盐桥有一定的影响。由于Asp和Glu带负电且Glu的侧链较大,这会缩短带正电和带负电残基之间的距离,因此SARS-CoV-2与Lys 417的盐桥可以保留并变得更加牢固。然而,带负电残基向不带电残基Asn和Ala的转化导致盐桥的破坏。从Lys 31到Glu,从带正电荷的残基到带负电荷的残基的剧烈突变也引起突变残基和Glu484之间的排斥相互作用。最后,尽管与Asn487形成的氢键被破坏,但Tyr 83被保留的疏水相互作用取代(表1)。因此,鸡和鸭中ACE2 残基30和31、79和82、35和83的突变分别破坏了盐桥、疏水相互作用和氢键,导致结合能增加和结合亲和力降低。对蝙蝠ACE2的结合自由能突变贡献很大的残基41破坏了氢键网络。由于ACE2关键残基的突变导致关键分子间相互作用的破坏,导致蝙蝠、小鼠、鸡和鸭中ACE2与SARS-CoV-2RBD 的结合亲和力较差。至于其他物种,残基30和34的突变可能会增加ACE2 和RBD之间的结合亲和力,这可能是SARS-CoV-2感染易感宿主的原因。综上所述,人ACE2与SARS-CoV-2RBD的结合可以耐受大量的突变残基,从而增强或破坏关键的分子间相互作用并影响结合亲和力。有趣的是,大多数重要的分子间相互作用是由ACE2α1和α2螺旋处的残基形成的。
可见,表1为人ACE2与SARS-CoV-2RBD界面之间的关键分子间的相互作用,了解关键残基的突变如何影响分子间相互作用,如表1所示,由于人ACE2中的以下突变,首先,Gln24被Leu取代,Glu 35被Lys或Arg取代,Tyr41和Lys 353被His取代导致氢键被破坏;其次,Asp 30被Glu、Asn 或Ala取代,Lys31被Glu取代对静电相互作用形成的中心盐桥有一定的影响。由于Asp和Glu带负电且Glu的侧链较大,这会缩短带正电和带负电残基之间的距离,因此SARS-CoV-2与Lys 417的盐桥可以保留并变得更加牢固。然而,带负电残基向不带电残基Asn和Ala的转化导致盐桥的破坏。从Lys 31到Glu,从带正电荷的残基到带负电荷的残基的剧烈突变也引起突变残基和Glu484之间的排斥相互作用;最后,尽管与Asn487形成的氢键被破坏,但Tyr 83被保留的疏水相互作用取代。
表1人ACE2与SARS-CoV-2RBD界面之间的关键分子间相互作用及其由 ACE2突变引起的变化
注:表格中的“/”表示无数据。
实施例2
本申请实施例为预测几种不同SARS-COV-2突变体和不同物种中设计的截短ACE2肽的结合亲和力试验,具体包括:
1.1丙氨酸突变扫描鉴定结合界面热点残基,具体包括:
1)基于PDB文件6LZG,进行了计算界面丙氨酸扫描 (http://robetta.bakerlab.org/alascansubmit.jsp)以了解残基的各个作用并确定哪些残基对结合亲和力的贡献最大。选择A链为SARS-CoV-2RBD,B链为不同物种的ACE2。在与SARS-CoV-2RBD接口的重要残基突变为丙氨酸后计算结合自由能。热点残基被定义为当相应的丙氨酸突变对ΔΔG(complex)产生超过1kcal/mol的不稳定影响。
1.2三维结构预测及分子对接预测不同物种ACE2与RBD结合自由能及结合亲和力,具体包括:
1)将非人类截短ACE2蛋白质序列提交给AlphaFold2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb)以预测和构建三维蛋白质结构。
2)将预测的非人类截短ACE2蛋白三维结构以及不同新冠突变株的RBD 三维结构提交到HDOCK sever(http://hdock.phys.hust.edu.cn/)进行分子对接,它是基于模板建模和abinitio自由对接的混合算法。将非人类截短ACE2蛋白三维结构以配体的形式上传,RBD三维结构以受体的形式上传,以同源分子对接的形式开始预测。选择预测分数最高的模型进行下载分析。
3)利用PyMol软件的align工具,将不同物种截短的ACE2片段的三维结构与截短的人ACE2结构域进行比对,计算蛋白质复合物的RMSD值。
4)将预测的三维结构复合体上传至PRODIGY网络服务器计算结合自由能和亲和力。
2.实验结果
2.1丙氨酸突变鉴定热点残基
除了分析结合界面上重要的分子间相互作用外,我们还需要确定重要残基对结合亲和力的贡献,从而合理设计ACE2截短功能肽。如表2所示,对结合界面处的20个重要ACE2残基进行丙氨酸扫描,涉及用丙氨酸取代氨基酸以测量侧链残基缺失对蛋白质复合物结合亲和力的影响。其中,包括Gln 24、 Tyr41、Gln42、Tyr 83和Asp 355在内的5个残基的ΔΔG(complex)值大于 1kcal/mol,被认为对结合至关重要。此外,ΔG(partner)的值用于预测丙氨酸突变后突变复合伴侣蛋白稳定性的变化。结果表明,Gln 24、Phe 28、Glu 31、Tyr41、Tyr 83、Asp 355和Arg 393对蛋白质复合物的稳定性起重要作用。基于RosettaDock软件的计算分析,从Ser 19到Tyr 83的α1和α2螺旋贡献了大约90%的总结合自由能,因此本申请实施例设计了一系列不同物种中从 Gln 24到Tyr 83的潜在ACE2肽,它可能与SARS-CoV-2RBD结合并抑制 SARS-CoV-2的进入。
表2与SARS-CoV-2RBDa相互作用的重要残基的丙氨酸扫描结果
a:int_id:如果侧链中至少有一个原子与另一个原子的距离在以内,则值为1,否则值为0。ΔG(complex):预测丙氨酸突变时结合自由能的变化。ΔG (partner):预测丙氨酸突变后突变复合伴侣蛋白稳定性的变化。
2.2几种不同SARS-COV-2突变体和不同物种中设计的截短ACE2肽的结合亲和力预测。
结果如图4所示。图4为设计的不同物种ACE2肽与SARS-CoV-2RBD 复合物的关键分子间相互作用力和结合亲和力的比较。结果表明狗(dog)和猪(pig)ACE2肽与RBD的之间的结合亲和力高于人类,而仓鼠、雪貂、果子狸、牛和羊的ACE2肽比人类的ACE2肽亲和力更高。图4中绿色的√号和红色的×号分别代表相互作用的存在和不存在。ΔG和Kd值分别代表结合自由能和结合亲和力。
结果如图5所示。图5是基于AlphaFold2和分子对接的截短ACE2蛋白复合物的结合自由能和分子间相互作用分析。图5A是人类和非人类物种的 ACE2-RBD复合物之间的RMSD值。图5B-图5D是不同物种蛋白质三维结构的比较。图5B是猫和人类,图5C是蝙蝠和人类,图5D是鸡和人类的截短ACE2蛋白复合物的结构比对。重要的突变残基以横杆显示。
如图5A所示,人和非人物种的ACE2-RBD蛋白复合物的RMSD值均小于2,说明非人ACE2-RBD蛋白复合物与人相似。由于ACE2序列相似性高,不同物种具有相同的结合模式,特别是在不同物种中ACE2的蛋白质骨架没有观察到重大的结构变化(图5B-图5D)(图5B-图5D显示的依次是猫、蝙蝠和鸡的截短ACE2蛋白质复合物的结构比对结果,其他物种的比对结果类似)。不同物种中设计的ACE2肽从Gln 24到Tyr 83与SARS-CoV-2RBD 复合物的关键分子间相互作用的比较表明狗和猪与RBD的ACE2肽之间的结合亲和力高于人类,而ACE2仓鼠、雪貂、果子狸、牛和羊的肽具有比人类更高的亲和力。因此,该物种的ACE2肽可能是一种潜在的分子抑制剂,通过竞争SARS-CoV-2RBD与人类ACE2的结合来抑制SARS-CoV-2进入。
结果如图6~图10所示。图6-图10是不同物种的截短型ACE2小片段与SARS-CoV-2野生型(图6)、B.1.351株(图7)、P.1株(图8)、B.1.617株(图9)、 B.1.1.529株(图10)的结合自由能和结合亲和力的预测。ΔG和Kd值分别代表结合自由能和结合亲和力。
如图6~图10所示,本申请实施例预测了设计的ACE2肽与SARS-CoV-2 变体之间的结合自由能和结合亲和力。本申请实施例重点研究了S蛋白RBD 区域的突变,该区域与人ACE2直接相互作用并与之结合。因此基于 AlphaFold2算法构建包括了南非变种B.1.351(K417N、N501Y、E484K)、巴西变种P.1(K417T、N501Y、E484K)、印度变种B.1.617(L452R、E484Q) 和南非变种B.1.1.529(K417N、S477N、T478K、E484A、Q498R、N501Y) 三维结构进入后续研究。由于之前提到的几个物种(蝙蝠、小鼠、鸡和鸭) 的ACE2对spike蛋白不敏感,本申请实施例评估了不同SARS-CoV-2突变体与其余物种ACE2肽之间的结合亲和力。如图6~10所示,除了穿山甲、兔和猪ACE2肽分别与wuhan-Hu-1(图6)、突变体B.1.351(图7)和突变体P.1 (图8)。与其他突变体相比,所有设计的肽对南非变体RBD的结合亲和力都降低。结果说明,雪貂和狗ACE2肽对除南非变体以外的所有SARS-CoV-2 变体的表现都优于人类ACE2。因此,本申请实施例设计的ACE2肽对 SARS-CoV-2突变体保持高亲和力。这并不奇怪,考虑到尽管病毒逃逸突变会使抗体治疗无效,但降低ACE2受体陷阱结合效率的逃逸突变也可能减少病毒入侵,并且特异性消除人类ACE2结合的残基替换在病毒中非常保守。
本实施例中,SEQ ID NO:3为狗ACE2功能结构域肽段,SEQ ID NO:4 为猫ACE2功能结构域肽段,SEQ ID NO:5为鼠ACE2功能结构域肽段,SEQ ID NO:6为人ACE2功能结构域肽段。
实施例3
本申请实施例为预测设计的不同物种的ACE2肽的理化特性、过敏性和免疫原性试验,具体包括:
1.实验方法:将设计的不同的截短型ACE2序列上传到以下网站:
1)使用ProtParam工具(https://web.expasy.org/protparam/)预测设计的候选肽的理化特性。该服务器有助于计算蛋白质序列的各种参数,包括理论pI、分子量、估计半衰期、延伸系数、不稳定性指数、脂肪系数和GRAVY指数等。
2)为了预测设计结构的过敏性,使用了AllerTOP服务器 (https://www.ddg-pharmfac.net/AllerTOP/)。使用五种机器学习方法根据肽的主要化学性质预测过敏性。
3)为了描述设计肽的潜在免疫原性,将这些序列以默认设置提交给IEDB 分析工具(http://tools.iedb.org/immunogenicity/)。该工具可以通过确定与T细胞受体结合的肽-HLA复合物中氨基酸残基的位置和侧链特征来对免疫原性进行评分,那些免疫原性低的肽段具有较低的预测分数。
2.实验结果:为了选择合适的肽段,本实施例评估了设计肽段的理化性质、致敏性和免疫原性。结果如图11所示,图11评估的肽为不同物种的截短型 ACE2肽。结果表明,不同的截短型ACE2肽的半衰期为0.8至5.5小时,这意味着细胞中一半的蛋白质在体外哺乳动物网织红细胞中合成后会消失。所有肽段的不稳定指数均大于40,表明它们不稳定。由于肽的物理稳定性受内外因素共同影响,因此应采取预防措施,通过一定的生化分析来稳定具有治疗作用的肽。亲水性大平均值(GRAVY)低于0,将肽归类为亲水性。致敏性预测表明,果子狸、牛、鸡和鸭的ACE2肽可能是过敏原,而雪貂、猪、鸡和鸭的ACE2肽可能被HLA受体识别为免疫原。总的来说,本实施例设计的不同物种的ACE2肽,包括恒河猴、仓鼠、猫、虎、羊和狗,不仅对SARS-CoV-2 RBD变体保持高亲和力,而且显示出良好的安全性,可以用作治疗病毒感染的潜在治疗剂。综合考虑结合亲和力、致敏性和免疫原性,狗ACE2肽表现最好,因此是最有价值的诱饵肽。
上述结果说明,本申请的SEQ ID NO:3~SEQ ID NO:6对SARS-CoV-2 RBD变体具有高亲和力,也有良好的安全性。
实施例4
本申请实施例提供了AAVrh10编码的截短型可溶性人ACE2蛋白的表达试验,具体包括:
1.1构建不同的表达载体,具体包括:
1)请参阅图12,图12为本申请实施例提供的表达载体的结构示意图,按照图12所示的载体结构采用常规方法构建不同的表达载体。
本实施例的载体基因组rAAV包含AAV反向末端重组序列和编码不同物种截短型可溶性ACE2的核酸序列,可溶性ACE2从该rAAV表达,该rAAV 两端具有反向终端重复序列(ITR),择人巨细胞病毒启动子(CMV),选择 SV40的polyA尾,在CMV和SV40的polyA尾中间插入目的基因片段,目的基因片段分别为人shACE2基因、SEQ ID NO:1和SEQ ID NO:2。分别构建对应的载体,图13~15中,AVV空载体(购买自派真生物技术有限公司)标记为vector、含有人shACE2基因的载体标记为shACE2、含有SEQ ID NO:1 的载体标记为shACE2PD、含有SEQID NO:2的载体标记为shACE2(PD+Neck)。
上述人shACE2基因共有740个氨基酸,其氨基酸序列为 MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYN TNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQAL QQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIM ANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYG DYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLM NAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAW DLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANE GFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLP FTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYC DPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEA GQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQN KNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAY AMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVE KAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS。
2.1AAVrh10编码的截短型可溶性人ACE2蛋白的表达,具体包括:
1)第一天,将Hela细胞分别加入4个10cm培养皿中,10ml含有10%胎牛血清和1%青霉素/链霉素的DMEM培养基中培养,3×106细胞/皿。
2)第二天,各取20μl lipo2000转染试剂和500μl opti-MEM培养基于4个 2ml EP管中混合为A液;
3)分别取10μg上述构建的AAV空载体、shACE2、shACE2PD和shACE2 (PD+Neck)质粒和500μl opti-MEM培养基于2ml EP管中混合为B液;
4)A液和B液各静置5min;
5)将各个B液分别加入A液,静置15min;
6)将AB混合液分别加入Hela培养液中,培养6小时;
7)弃去Hela细胞培养基,更换为10ml DMEM/HighModified去酚红培养基,再孵育细胞24小时以表达目的基因;
8)第三天,收集培养上清液,将上清液置于AmiconUltra超滤管,4℃, 4000xg,离心20min,同时向每个培养皿加入1ml RIPA裂解液,冰上裂解 20min;
9)回收200μl超滤后的上清液于1.5ml EP管中,置于冰上待用;
10)收集细胞裂解液于1.5ml EP管中;
11)收集的细胞裂解液于4℃,15000xg,离心15min;
12)离心完毕,吸取200μl上层澄清的液体于另一1.5ml EP管;
13)分别加入50μl 5×SDS蛋白缓冲液于9)和12)步中的EP管,混匀;
14)100℃,金属浴,10min,到此截短型可溶性人ACE2蛋白提取完毕;
15)第四天,蛋白免疫印迹法检测截短分泌型ACE2和胞内ACE2的表达,电泳80V,2小时,转膜200mA,2小时,5%脱脂奶粉封闭1小时,一抗孵育过夜;
16)第五天,回收一抗,TBST洗涤蛋白条,10min/次,共3次,之后,二抗孵育1小时,TBST洗涤,10min/次,共3次,ECL发光液显色,迷你型化学发光成像分析系统(MiniChemi601)检测蛋白表达情况。
2.2AAVrh10编码的截短型可溶性人ACE2蛋白与SARS-CoV-2spike蛋白有效结合,具体包括:
1)第一天,将表达Spike蛋白的293T细胞接种于96孔板,2×104细胞 /100ul每孔;
2)第二天,将表达截短型可溶性人ACE2蛋白的细胞上清液与抗S蛋白抗体1:1混合;
3)将该混合物加入上述293T细胞中1小时;
4)PBS洗3遍;
5)加入Anti-human IgG二抗,4℃,孵育30min;
6)PBS洗涤3遍;
7)收集细胞于流式管中;
贝克曼流式细胞仪(CytoFLEX S B75442)检测结合了抗S蛋白抗体的细胞数量。
2.3AAVrh10编码的截短型可溶性人ACE2抑制SARS-CoV-2假病毒感染
1)第一天,BHK-ACE2细胞接种于96孔板,2×104细胞/100μl每孔;
2)第二天,分别转染1、10、100ng的AAV空载体、shACE2(含有上述人shACE2全长基因的载体,人shACE2全长基因为740个氨基酸)、shACE2PD (含有SEQ ID NO:1的载体)和shACE2(PD+Neck)(含有SEQ ID NO:2的载体) 于BHK-ACE2细胞,6小时后,更换培养基为含5%FBS和1%青霉素/链霉素的DMEM;
3)加入浓度为1×106PFU/ml SARS-CoV-2假病毒5μl/孔,培养24小时;
2.4AAVrh10编码的截短型可溶性人ACE2抑制SARS-CoV-2病毒样颗粒 (trVLP)感染,具体包括:
1)第一天,将CACO-2-N细胞接种到24孔板,1×105/500μl每孔;
2)第二天,分别以MOI=105和MOI=106剂量用AAVrh10-空载体、AAVrh10-shACE2(含有上述人shACE2全长基因的载体,人shACE2全长基因为740个氨基酸)、AAVrh10-shACE2(PD+Neck)(含有SEQ ID NO:2的载体) 感染细胞;
3)第三天,即24小时后,培养基更换为含5%FBS和1%青霉素/链霉素的DMEM,并加入1×106PFU/ml SARS-CoV-2病毒样颗粒(trVLP)20μl;
4)第四天,即24小时后,用RNA提取试剂盒提取细胞RNA,用Nano Drop 8000(Thermo,United States)测RNA的浓度,调整浓度使各个样本保持一致;
5)使用浓度一致的RNA作为模板,通过HifairTM III 1st Strand cDNASynthesis SuperMix forqPCR逆转录为cDNA;
6)以cDNA为模板做实时荧光定量PCR(qPCR),每个样本做三个复孔。设计引物如下:
正向引物:CGAAAGGTAAGATGGAGAGCC。
反向引物:TGTTGACGTGCCTCTGATAAG。
使用PerfectStart SYBR Green qPCR supermix和QuantStudio 7Flex (AppliedBiosystems,USA)定量实时PCR(qPCR)检测系统测量 SARS-CoV-2mRNA的水平。反应程序如下:
95.0℃,3min;95.0℃,10sec,56.0℃,30sec,循环44次;熔解曲线 65℃to 95℃,每次增长温度为0.5℃。GAPDH作为参照基因,使用2-ΔΔCt 方法计算基因的相对表达量。
2.5编码的截短型可溶性人ACE2 AAVrh10感染小鼠血清抑制 SARS-CoV-2假病毒感染,具体包括:
1)第一周,BALB/c小鼠(6-8周龄,雌性)随机分为PBS、AAVrh10-shACE2 (含有上述人shACE2全长基因的载体,人shACE2全长基因为740个氨基酸)、AAVrh10-shACE2(PD+Neck)(含有SEQ ID NO:2的载体)三组,每组各5 只,以浓度为1%的异氟烷,流速为400ml/min麻醉小鼠,小鼠进入深睡眠时,鼻内给药100μlAAV-shACE2、AAV-shACE2(PD+Neck)(2×1011GC,基因组拷贝)或PBS;
2)第七周,尾静脉采血100μl,4℃静置3小时;
3)4℃,1000xg,离心30min,收集上层淡黄色血清;
4)上述血清按1:30和1:90倍稀释,按2.3步骤检测血清假病毒中和能力。
3.实验结果
如图13A所示,AAV-shACE2(含有上述人shACE2全长基因的载体,人 shACE2全长基因为740个氨基酸)、AAV-shACE2PD(含有SEQ ID NO:1的载体)和AAV-ACE2(PD+Neck)(含有SEQID NO:2的载体)在细胞上清液中均表达,而仅AAV-shACE2在细胞中有表达;如图13B所示,AAV-ACE2(PD+Neck)显示出与AAV-shACE2相当的结合S蛋白的能力,而AAV-shACE2PD结合S 蛋白能力相对较弱;如图13C所示,AAV-ACE2(PD+Neck)显示出与AAV-shACE2 相当的抑制SARS-CoV-2假病毒感染的能力,而AAV-shACE2PD的抑制能力相对较弱;如图13D所示,AAVrh10-ACE2(PD+Neck)抑制SARS-CoV-2病毒样颗粒 (trVLP)感染的能力最强,AAVrh10-shACE2次之,AAVrh10-shACE2PD的抑制能力最弱;如图13E所示,AAVrh10-shACE2和AAVrh10-ACE2(PD+Neck)感染组小鼠血清显示强大的抑制SARS-CoV-2假病毒感染的能力,说明本申请提供的SEQ IDNO:1~2具有广阔的应用前景。
实施例5
本申请实施例提供了AAVrh10递送的不同物种的截短型可溶性ACE2抑制SARS-CoV-2病毒感染细胞试验,具体包括:
2.1AAVrh10编码的截短型可溶性人ACE2抑制SARS-CoV-2病毒样颗粒 (trVLP)感染,具体包括:
1)第一天,将CACO-2-N细胞接种到24孔板,1×105/500μl每孔;
2)第二天,转染400ngAAV空载体、AAV-shACE2(含有上述人shACE2 全长基因的载体,人shACE2全长基因为740个氨基酸)、AAV-hACE224-83 (含有SEQ ID NO:6)、AAV-cACE224-83(含有SEQ ID NO:4的载体)、 AAV-mACE224-83(含有SEQ ID NO:5的载体)和AAV-dACE224-83(含有SEQ ID NO:3的载体)于CACO-2-N细胞;
3)6小时后,培养基更换为含5%FBS和1%青霉素/链霉素的DMEM,并加入1×106PFU SARS-CoV-2病毒样颗粒(trVLP);
4)第四天,即48小时后,用RNA提取试剂盒提取细胞RNA,用Nano Drop 8000(Thermo,United States)测RNA的浓度,调整浓度使各个样本保持一致;
5)使用浓度一致的RNA作为模板,通过HifairTM III 1st Strand cDNASynthesis SuperMix forqPCR逆转录为cDNA;
6)以cDNA为模板做Q-PCR,每个样本做三个复孔。设计引物如下:
正向引物:CGAAAGGTAAGATGGAGAGCC。
反向引物:TGTTGACGTGCCTCTGATAAG。
使用PerfectStart SYBR Green qPCR supermix和QuantStudio 7Flex(AppliedBiosystems,USA)定量实时PCR(qPCR)检测系统测量SARS-CoV-2 mRNA的水平。反应程序如下:
95.0℃,3min;95.0℃,10sec,56.0℃,30sec,循环44次;熔解曲线65℃ to 95℃,每次增长温度为0.5℃。GAPDH作为参照基因,使用2-ΔΔCt方法计算基因的相对表达量。
2.2AAVrh10编码的截短型可溶性人ACE2抑制SARS-CoV-2假病毒感染:
1)第一天,BHK-ACE2细胞接种于96孔板,2×104细胞/100μl每孔;
2)第二天,分别用MOI=106的AAV空载体、AAV-shACE2(含有上述人 shACE2全长基因)、AAV-shACE2(PD+Neck)(含有SEQ ID NO:2的载体)、 AAV-hACE224-83(含有SEQ ID NO:6的载体)和AAV-dACE224-83(含有SEQ ID NO:3的载体)病毒感染BHK-ACE2细胞;
3)第三天,加入浓度为1×106PFU/ml SARS-CoV-2假病毒5μl/孔,培养 24小时;
3.实验结果
如图14A所示,狗、猫、鼠、人四个物种的ACE2多肽序列都显示出对 SARS-CoV-2trVLP的抑制作用,其中物种狗的ACE2多肽效果最好,这与上述计算分析结果一致,提示狗ACE2多肽在抑制SARS-CoV-2感染方面的有益作用。如图14B所示,狗的ACE2(SEQ ID NO:3)多肽显示强大的 SARS-CoV-2假病毒抑制能力,甚至优于shACE2(人shACE2全长基因)和shACE2(PD+Neck)(SEQ ID NO:2)。
实施例6
AAVrh10递送截短型可溶性ACE2抑制SARS-CoV-2S蛋白介导细胞和细胞融合,具体包括:
1)第一天,将HEK-293T细胞和Hela细胞分别接种到6孔板中,5×105/2ml 每孔;
2)第二天,将3μg pcDNA3.1-SARS-CoV-2和2μg PVAX-GFP共转染 HEK-293T细胞(效应细胞);同时以MOI=105的AAVrh10-shACE2(含有上述人shACE2全长基因的载体,人shACE2全长基因为740个氨基酸)、 AAVrh10-shACE2(PD+Neck)(含有SEQ ID NO:2的载体)、AAVrh10-hACE224-83 (含有SEQ ID NO:6的载体)、AAVrh10-dACE224-83(含有SEQ ID NO:3的载体)病毒感染Hela细胞;
3)第四天,即48小时后,弃去HEK-293T细胞培养液,收集Hela细胞上清液各2ml,加入HEK-293T细胞中,37℃培养;
4)6小时后,以细胞表面表达ACE2的VeroE6细胞作为靶细胞,将上述效应细胞和靶细胞按1:1混合,在DMEM中共培养24小时;
5)第五天,在荧光显微镜(EVOS数字倒置显微镜,Invitrogen)下观察效应细胞和靶细胞的融合情况。
3.实验结果
如图15所示,四个截短型ACE2片段均能够有效抑制SARS-CoV-2S蛋白介导的细胞融合,其中,AAVrh10-shACE2(PD+Neck)(SEQ ID NO:2)比 AAVrh10-dACE224-83(SEQ ID NO:3)更有效地抑制细胞融合。 AAVrh10-shACE2(PD+Neck)(SEQ ID NO:2),可以保证ACE2的二聚体结构,从而对SARS-CoV-2具有很强的中和作用,而AAVrh10-dACE224-83(SEQ ID NO:3)对SARS-CoV-2突变株具有广泛的中和作用,可以有效阻断 SARS-CoV-2感染,是具有巨大潜在应用价值的ACE2片段。
综上所述,本申请中AAVrh10血清型自恒河猴体内分离,人类感染机会较少,故使用AAVrh10载体可减少发生宿主血清学免疫反应的机会,同时使其携带的目的基因在特定组织长效表达,简化蛋白制备流程,经济,有助于大规模应用;其次,本申请采用AAVrh10编码不同物种的截短型可溶性ACE2 功能域肽段,截取ACE2有效与S蛋白结合的部分肽段,减小蛋白的大小,使之既可以结合S蛋白中和病毒,又可以避免过敏反应的发生,也不会干扰常规的生理反馈系统。针对不同物种ACE2的分析有助于寻找与SARS-CoV-2 具有高效亲和力的最优片段,提高抑制病毒进入能力。
可见,本申请提供的SEQ ID NO:1~SEQ ID NO:6不仅对SARS-CoV-2 RBD变体保持高亲和力,而且显示出良好的安全性,可以用作治疗病毒感染的潜在治疗剂。综合考虑结合亲和力、致敏性和免疫原性,狗ACE2肽表现最好,因此是最有价值的诱饵肽。
以上所述仅是本申请的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本申请原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本申请的保护范围。
序列表
<110> 中山大学·深圳,中山大学
<120> ACE2功能结构域肽段及其在制备预防和/或治疗新型冠状病毒药物中的应用
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Claims (10)
1.ACE2功能结构域肽段,其特征在于,ACE2功能结构域肽段具有SEQ ID NO:1~6任一项所示的氨基酸序列,或者具有与SEQ ID NO:1~6任一项所示的氨基酸序列存在至少80%同源性的序列。
2.ACE2功能结构域肽段在制备预防和/或治疗新型冠状病毒药物中的应用;所述ACE2功能结构域肽段具有SEQ ID NO:1~6任一项所示的氨基酸序列,或者具有与SEQ ID NO:1~6任一项所示的氨基酸序列存在至少80%同源性的序列。
3.ACE2功能结构域核酸,其特征在于,其编码权利要求1所述的ACE2功能结构域肽段的基因片段。
4.一种表达载体,包含权利要求3所述的ACE2功能结构域核酸。
5.根据权利要求4所述的表达载体,其特征在于,所述表达载体包括ACE2功能结构域核酸和十号血清型腺相关病毒AAVrh10载体;
所述ACE2功能结构域核酸位于所述AAVrh10载体的启动子和所述AAVrh10载体的polyA尾巴之间。
6.根据权利要求4所述的表达载体,其特征在于,所述AAVrh10载体的两端具有反向终端重复序列ITR。
7.根据权利要求4所述的表达载体,其特征在于,所述AAVrh10载体的启动子为人巨细胞病毒启动子CMV。
8.根据权利要求4所述的表达载体,其特征在于,所述AAVrh10载体的polyA尾巴为SV40。
9.一种宿主细胞,其包含权利要求4所述的表达载体。
10.一种用于治疗或预防新型冠状病毒感染的药物组合物,其包括权利要求4~8任意一项所述的表达载体。
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