CN115947818A - 一种血管生成素1突变体的设计及其制备方法和应用 - Google Patents
一种血管生成素1突变体的设计及其制备方法和应用 Download PDFInfo
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Abstract
本发明提出了一种增强与Tie2结合的血管生成素1突变体Ang1A451D的设计及其制备方法和应用。本发明通过序列同源性分析、晶体结构分析和分子动力学计算等最终确定蛋白突变位点:在Ang1的与受体Tie2结合区上丙氨酸突变成天冬氨酸,利用点突变PCR技术获得突变基因Ang1A451D。本发明还公开了表达载体的构建以及转染哺乳动物细胞高效重组表达的方法,制备的血管生成素1突变体增强与Tie2的结合力(约50倍),并可以提高Tie2的胞内磷酸化水平,促进内皮细胞的稳态。因此,可将其作为缓解脓毒症炎症风暴的候选药物,其在生物工程制药业及基因工程、生物化学、分子生物学等研究领域具有重要应用价值。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种血管生成素1突变体的设计及其制备方法和应用。
背景技术
脓毒症(Sepsis)是由细菌等病原微生物侵入机体,引起器官功能失调导致全身炎症,具有极高致死率的综合征,据全球最新统计,每年大约有4900万人感染此病,其中近1100万人死亡,占全球死亡人数的20%(Rudd KE,Lancet,2020,395(10219):200-11)。2017年,世界卫生组织(WHO)将脓毒症列为需极高度优先使用医疗资源的疾病(Reinhart K,
N
Engl J Med,2017,377(5):414-7)。其中严重的脓毒症常并发一种独特且致命的综合征:弥散性血管内凝血症(Disseminated Intravascular Coagulation,DIC)。DIC一般表现为血管中有很多(微)血块,并且出血情况较严重,致病因素不同,其对应的病理生理机制及临床特点也存在很大差异。目前,对DIC的治疗基本上都是依赖于抗生素治疗、血流动力学稳定性以及对衰竭器官的维持(Van wyngene L,
EMBO Mol Med,2018,10(8)),加深DIC潜在机制的了解有助于对该综合征的诊断和预防。
Ang-Tie轴包含血管生成素(Ang1、Ang2和Ang4,Ang3是小鼠中Ang4的同源物)和具有免疫球蛋白和表皮生长因子同源性结构域的酪氨酸蛋白激酶(Tie1和Tie2)。尽管Ang1和Ang2以相似的亲和力与Tie2结合,但它们对内皮细胞具有不同的调控作用(Teichert M,
Nat Commun,2017,8:16106)。Ang1有三个结构域,其中一个负责受体结合的C端纤维蛋白原样结构域(Receptor Binding Domain,RBD)、一个寡聚化纤维蛋白原样结构域的中央卷曲螺旋结构域(Colied Coil Domain,CCD)和一个短N末端结构域组成(Super ClusteringDomain,SCD)(Procopio WN,
J Biol Chem,1999,274(42):30196-201;Davis S,
Nat Struct
Biol,2003,10(1):38-44;Davis S,
Cell,1996,87(7):1161-9)。Ang1的中央卷曲结构和N末端超结构域导致蛋白质聚集,可溶性低,天然状态的Ang1蛋白会同时存在二聚、四聚或多聚的状态。Tie2是一种酪氨酸激酶样受体(Teichert M,
Nat Commun,2017,8:16106),是血液和淋巴管发育和病理过程的关键调节因子,包括肿瘤血管生成、动脉粥样硬化和血管渗漏,例如脓毒症。
Ang1是Tie2的重要激动剂。Ang1非常紧密地结合Tie2,其亲和力在纳摩尔级别,使Tie2簇在内皮细胞-内皮细胞(EC-EC)连接处诱导形成,进而增加血管稳定性(尤其是在血管生成过程后),抑制组织纤维化,并在抗血管生成治疗期间调节血管正常化。在正常生理条件下,Ang2水平较低,但炎症和缺氧刺激会增加Ang2的表达,降低血管稳定性,促进内皮细胞活化、血管生成和重塑(Eklund L,
Clin Sci (Lond),2017,131(1):87-103)。这促使一些研究机构和制药公司以Ang1/Tie2轴为靶点开展与血管疾病相关的基础和临床研究。例如:COMP-Ang1,可以克服Ang1天然的局限性,且可激活Ang-Tie轴,此蛋白可以促进血管生成、抗炎、减少血管疾病的血管渗漏,已被用作增强靶向癌症治疗的联合治疗剂(WallaceRG,
Vascul Pharmacol,2021,141:106919)。也有研究者用源自血清补给C4结合蛋白α(C4BPα)去替代天然Ang1中央卷曲结构域和寡聚结构域,即C4BP-Ang1。该蛋白在小鼠体内可改善DIC引起的血管渗漏(Liu P,
Biotechnol Bioeng,2021,118(1):423-32)。DIC发病时,降低Tie2激活程度会对Ang-Tie轴造成严重干扰,包括Ang1和Tie2的表达量减小、产生可溶性Tie受体和影响Ang2拮抗活性。已有DIC小鼠模型实验表明,脂多糖(Lipopolysaccharide,LPS)给药后磷酸化Tie2与总Tie2的比例显著下降(Mofarrahi M,
Am J Physiol Lung Cell
Mol Physiol,2008,294(5):L955-63);LPS会诱导协同受体Tie1胞外域裂解,不但减小Tie2的激活,还能促进Ang2的拮抗活性,进而抑制Ang-Tie轴的信号转导(Kim M,
J Clin
Invest,2016,126(9):3511-25;Korhonen EA,
J Clin Invest,2016,126(9):3495-510)。因此,研究Ang-Tie轴与DIC的关系具有非常重要的意义,靶向Ang-Tie药物的研发也逐渐吸引了科研者的注意,结合了本实验室研究基础,我们也探索了多种方法来进一步研究Ang-Tie轴在DIC发生发展中的潜在机制。
发明内容
本发明的目的在于针对上述DIC现状,提供一种增强与Tie2结合的血管生成素1的突变体Ang1A451D及其应用,该突变体Ang1A451D具有显著增强与Tie2结合的能力,可促进Ang-Tie轴正反馈以及有效预防DIC炎症风暴。
为实现上述目的,本发明采用如下技术方案:
本发明首先提供了一种血管生成素1的突变体Ang1A451D,所述突变体Ang1A451D的氨基酸序列如SEQ ID NO.1所示。
进一步的,所述突变体Ang1A451D的突变点位于血管生成素1 Ang1的受体结合区Ang1-RBD。
进一步的,所述突变体Ang1A451D突变体为血管生成素1 Ang1第451位丙氨酸的一个位点突变。
进一步的,所述突变体Ang1A451D突变体是将血管生成素1 Ang1氨基酸序列第451位点上的丙氨酸突变成天冬氨酸。
本发明还提供了上述一种血管生成素1的突变体Ang1A451D在增强sTie2的结合力以及促进Ang1-Tie2轴正反馈的应用。
本发明还提供了上述一种血管生成素1的突变体Ang1A451D在制备预防脓毒症炎症风暴的药物中的应用。
本发明的优点在于:
本发明公开了一种增强与Tie2结合的血管生成素突变体Ang1A451D,该突变体是一个全新的突变体,在此之前从未有人报道过。我们通过获得实验证实,突变体Ang1A451D增强了与Tie2的结合力(约50倍),可增强对Ang-Tie轴在维持内皮细胞的稳定性,该突变体可以应用于缓解DIC炎症风暴等症状,也包括其他涉及内皮细胞稳定性相关的炎症疾病,适用于生物工程制药业及基因工程、生物化学、分子生物学等研究领域。
附图说明
图1:Ang1-Tie2结构图以及预测的突变位点。橙色为Ang1-RBD晶体结构,蓝色为Tie2晶体结构;基于晶体复合物,MD模拟计算出降低Ang1与Tie2结合自由能的突变点。
图2:pCMV3-SP-N-FLAG-Ang1的质粒图谱。
图3:相关蛋白的表达与纯化。(A)Ang1-RBD经Superdex 200 Increase 10/300 GL纯化色谱图;(B)Ang1-RBD色谱图上洗脱洗脱16.78 mL处样品12% SDS-PAGE鉴定结果;(C)Ang1-RBDA451D经Superdex 75 Increase 10/300 GL纯化色谱图;(D)Ang1-RBDA451D色谱图上洗脱洗脱13.03 mL处样品12% SDS-PAGE鉴定;(E)Ang1经Superdex 200 Increase 10/300GL纯化色谱图;(F)Ang1洗脱样品WB鉴定;(G)Ang1A451D经Superdex 200 Increase 10/300GL纯化色谱图;(H)Ang1A451D洗脱样品免疫印迹法(Western Blot,WB)鉴定。
图4:表面等离子体共振测定Ang1-RBD与sTie2结合。(A)sTie2与Ang1-RBD结合动力学曲线;(B)sTie2与Ang1-RBD结合亲和力曲线。(C)sTie2与Ang1-RBDA451D结合动力学曲线;(D)sTie2与Ang1-RBDA451D结合亲和力曲线。
图5:Ang1A451D对胞内Tie2磷酸化水平的检测。(A)加入LPS、Ang1和Ang1A451D后对内皮细胞
p-Tie2捕获的WB图像;(B)Image J软件对WB图像灰度值定量分析。其中“+”代表加入,“-”代表未加。在WB实验中,用1 μM LPS、Ang1和Ang1A451D各15 nM分别去刺激内皮细胞1h,加入裂解液制样,分别使用anti-
p-Tie2抗体、anti-Tie2抗体和内参anti-GAPDH抗体去识别内源物。
图6:Ang1A451D对Ang1-Tie2轴下游FOXO1(Forkhead Transcription Factor 1)的影响。(A)光学图像,红色箭头代表入核,橙色箭头代表出核,比例尺=30 μm;(B)FOXO1入核率,数据代表平均值±SEM,n=3。
图7:Ang1A451D对小鼠DIC模型炎症因子的影响。(A)LPS诱导1.5 h时血浆内TNF-α情况,值为平均值±SEM,ns代表没有显著性差异,nd代表未检测到信号;(B)LPS诱导1.5 h和4h时血浆内IL-6情况,值为平均值±SEM。
具体实施方式
下面将结合附图和实施例对本发明的方法和其优点作进一步说明,但这些实施例并不构成对本发明权利要求范围的限制。在不脱离本发明主要特征的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
实施例一:Ang1突变点的选择和筛选
基于Ang1-Tie2复合物晶体结构(PDB code:4K0V)分析人血管生成素1 Ang1和酪氨酸蛋白激酶受体Tie2之间的相互作用位点(图1),其中Ang1的RBD结构域介导了与Tie2的相互作用。我们逐一分析相互作用面上的Ang1侧的氨基酸,并提出了可能会增强Ang1对Tie2亲和力的5个氨基酸突变位点(图1),接着通过分子动力学(Molecular Dyanimcs,MD)计算了预测的5个点突变对Ang1和Tie2结合自由能的影响(表1),亲和力大小与自由能成负相关,亲和力越强其自由能就会越低,以期提高与Tie2结合力并增强Tie2的激活从而进一步增强Ang-Tie轴的信号。根据结果,我们最终确定了A451D作为突变实验的位点。
表1. 利用MD法计算Ang1点突变对其与Tie2结合自由能的影响(单位为kcal/mol)
注:加粗为自由能绝对值较大的突变点。
实施例二:Ang1与sTie2的构建、表达和纯化
天然的Ang1含有多个结构域,且稳定性较差,做为治疗剂应用的可能性较低,且该分子具有高度复杂的调节模式,且涉及多个结构相似分子,实验纯化难度较大,在分析Ang1-Tie2复合物晶体结构(图1)时,我们发现与受体Tie2结合的是配体Ang1的C端RBD结构域,因此我们对蛋白Ang1进行了截短,获得蛋白Ang1-RBD,其氨基酸序列如SEQ ID NO.2所示。
(1)Ang1及其突变体的表达载体的构建。
以含有Ang1全长DNA的质粒(pCMV3-SP-N-FLAG-Ang1,质粒图谱如图2所示)为模板,使用引物:5’-TTTAGAGACTGTGCAGATGTATATCAAGCTGG-3’(上游引物),5’-GCTACCGCCTCCACCCTTATCG-3’(下游引物),通过PCR的方法扩增出Ang1-RBD基因片段(核苷酸序列如SEQ ID NO.3所示),用限制性内切酶KpnI和XhoI切割质粒pCMV3-SP-N-FLAG-Ang1,使用核酸外切酶Ⅲ将Ang1-RBD片段连接到pCMV3-SP-N-FLAG质粒中。将酶连产物经过42℃热激发转化至大肠杆菌DH5α,涂平板,挑单菌落,进行基因测序,将含有正确Ang1-RBD序列的
DH5α菌种,进行扩大培养,采用氯化铯法抽提pCMV3-SP-N-FLAG-Ang1-RBD质粒,以备下面实验使用。
通过PCR技术,使用定点突变引物:5’-GGTGGTTTGATGACTGTGGCCCCTC-3’(上游引物),5’-ATCCTCCTGTTAACATGAGGGCACATTTGC-3’(下游引物),分别以含有Ang1-RBD基因片段及Ang1全长DNA的质粒为模板,扩增得到Ang1-RBDA451D(氨基酸序列如SEQ ID NO.4所示)和Ang1A451D(氨基酸序列如SEQ ID NO.1所示)的突变质粒,PCR体系及程序设置如下:
分别加入1µL DpnI(Takara)至上述PCR产物中,37℃水浴锅消化3h。采用EZNA胶回收试剂盒(OMEGA)对PCR产物进行胶回收。42℃热激发转化至大肠杆菌
DH5α,涂平板,挑单克隆测序,将含有正确突变的菌种进行25%甘油菌保存于-80℃冰箱待用。
(2)蛋白的表达与纯化。
将质粒Ang1-RBD、Ang1-RBDA451D、Ang1和Ang1A451D通过真核表达系统HEK293F细胞来表达完成,对于每个蛋白的表达,以20 mL培养体系(100 mL培养瓶)举例如下:转染前一天,取样计数细胞密度,计算细胞活率;以2×106个/mL的密度将细胞密度接种到新鲜SMM293-TII(货号:M293TII)培养基中,置于37℃,5% CO2,150-175 rpm的恒温摇床中培养;转染当天,取样计数细胞密度和活率。细胞密度应该在3-5×106个/mL,活率高于90%,调整细胞密度至3×106个/mL,每瓶细胞液体积为20 mL,接着配制转染液:用新鲜SMM 293-TII培养基稀释20 µg质粒DNA至总体积为2 mL,温和混匀,接着在此管中按照5:1 w/w PEI/DNA的比率逐滴加入聚乙烯亚胺PEI(Polyethylenimine,PEI,23966-2,Polysciences,Inc.)剧烈混匀,DNA/PEI复合物在室温静置20 min,作为转染液;将转染液逐滴加入到预先准备好的细胞培养液中,滴加的同时轻轻摇动培养瓶,摇匀后放回摇床继续培养,在转染后第24 h加入0.7 mL SMS 293-SUPl加料液,此后每隔48 h添加一次SMS 293-SUPl加料液(每次0.7mL),转染后6天低速获得培养基并高速离心去除细胞碎片。通过多次挂柱的方法,将细胞上清的目的蛋白挂在层析柱上(FLAG标签亲和层析介质),用100 mM Glycine,10 mM NaCl,pH=3.0的洗脱缓冲液将目的蛋白洗脱至收集管中(预先装有按照中和缓冲液:洗脱缓冲液=1:10 v/v的1 mL 1 M Tris-HCl,pH 8-9),将洗脱蛋白Ang1-RBD和Ang1-RBDA451D进行12% SDS-PAGE鉴定,捕获的Ang1和Ang1A451D进行WB鉴定,获得结果(图3),剩下样品浓缩置于-80℃冰箱储存。
实施例三:表面等离子体共振测定Ang1-RBD及其突变体与sTie2的结合常数
所有表面等离子体共振(SPR)测定均在Biacore T200仪器(GE Health SciencesInc.)上进行,运行缓冲液含有10 mM HEPES、150 mM NaCl、3 mM EDTA和0.05%(v/v)Surfactant P20,所有溶液均通过0.22 μm孔径过滤器过滤并在室温下脱气。CM5芯片放入仪器后,首先用运行缓冲液冲洗一遍管路,然后使用N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)以20 μL/min的流速激活7 min。接着固定sTie2,将40μM的sTie2蛋白在10 mM乙酸钠(pH=5.0)中的溶液偶联到芯片上,直到达到>10000(RU)。使用1 M乙醇胺将芯片上活化的游离结合位点再封闭7 min。所有实验均在25℃下以30 μL/min的流速和120 s的接触时间进行,然后在pH 1.5甘氨酸解离600 s以及60 s再生。使用BIACORE评价软件,采用1:1动力学拟合,计算动力学参数(
K on 和
K off ),
K D值通过
K off /
K on 计算得到。得到结果(表2,图4)表明,Ang1-RBD与sTie2结合的
K D值为7.723×10-6 M,而Ang1-RBDA451D与sTie2结合的
K D值为1.696×10-7 M,增强了约50倍。
表2. 表面等离子体共振测定Ang1-RBD与sTie2结合常数
实施例四:Ang1A451D调控胞内Tie2磷酸化水平的检测
根据Ang-Tie轴相关调节情况,我们用将LPS、Ang1和Ang1A451D加入到HUVEC细胞混合孵育1 h后通过免疫印迹实验(WB)对内皮细胞内源
p-Tie2定量,根据条带深浅来评估突变体在体外对
p-Tie2的影响。具体操作如下:
将HUVEC细胞消化后铺在12孔板中,2×106个/孔细胞,将铺好后的孔板放入37ºC,5% CO2,饱和湿度培养箱过夜培养,次日将药物以15 nM终浓度加入培养液中,加入药物刺激1 h后,吸弃培养液,每孔加入100 μL的2×SDS(含有PMSF的RIPA裂解液,碧云天,货号:P0013B),把细胞全部收集到1.5 mL离心管中金属浴15 min,10% SDS-PAGE凝胶80 V、30min;120 V、90 min;恒流转膜0.15 A、90 min,5% BSA封闭1 h,一抗(GAPDH、
p-Tie2和Total-Tie2(胞外重组多克隆抗体))4℃冰箱过夜孵育;用1×TBST将膜洗三遍;二抗孵育1h,然后用1×TBST洗5遍将二抗洗干净;最后高敏ECL(Enhanced Chemiluminescent)化学发光试剂,用于检测HRP(辣根过氧化物酶)标记的目的条带,检测灵敏度最高可达皮克(pg)级,曝光后立即用成像仪捕获结果(图5A),然后使用Image J软件对其灰度值进行定量分析(图5B)。
结果显示与未处理细胞组相比,加入LPS组HUVEC内源的
p-Tie2明显减少,加入Ang1A451D组捕获细胞内源的
p-Tie2明显增加,该实验可表明:加入Ang1A451D后,Ang-Tie轴中Tie2磷酸化水平增加,具有促进该轴正反馈调节作用。
实施例五:Ang1A451D对Ang1-Tie2轴下游FOXO1的影响
Ang-Tie轴正常情况下,Ang2表达很低,Ang1决定Ang2的表达水平,Ang1结合并激活Tie2,Tie2被磷酸化后,会激活下游PI3K/AKT信号通路,磷酸化的AKT使核外排的FOXO1失活,进一步抑制Ang2的表达;炎症情况下,Ang2上调与Ang1竞争结合Tie2,协同受体Tie1的胞外域被切割,从而抑制Tie2的磷酸化和PI3K/AKT信号通路失活。因此,有活性的FOXO1增加Ang2的表达,从而抑制Tie2信号通路的正反馈,从而使血管不稳定或者渗漏,本实验要验证与sTie2结合更强的突变体是否能够调节此通路,用核定位的方法来研究突变体Ang1A451D对FOXO1进出核位置的影响。具体操作如下:
将EA.hy926细胞消化后铺在96孔板中,每孔5000个细胞,将铺好后的孔板放入37ºC,5% CO2,饱和湿度培养箱过夜培养,次日将重组蛋白以15 nM和50 nM两个不同浓度加入培养液中,每个重组蛋白浓度设置两个复孔,加入重组蛋白刺激1 h后,每孔用200 μL预热1×PBS洗涤一下漂浮的细胞,然后每孔加入100 μL 4%PFA组织固定液置于37ºC,5% CO2,饱和湿度培养箱固定10 min,用泵吸干之后,每孔加入100 μL 4%多聚甲醛在4℃冰箱透膜10min;每孔用200 μL预热1×PBS洗涤洗涤一下孔里的杂质,共三遍,每孔加入100 μL 5% BSA封闭1 h;每孔加入50 μL Anti-FOXO1一抗4℃冰箱过夜孵育;次日每孔用200 μL预热1×PBS洗涤三遍,将一抗洗干净,接着每孔加入100 μL预先配制的二抗,室温孵育40 min,每孔用200 μL预热1×PBS洗涤三遍,后在高内涵成像仪上捕捉FOXO1位置变化。
获得结果(图6)显示:体外加入Ang1和Ang1A451D均具有影响Ang-Tie轴下游信号通路的功能,且Ang1A451D的效果更强。由此推测Ang1A451D可以促进Ang-Tie轴的正反馈。
实施例六:Ang1A451D对小鼠DIC模型炎症因子的影响
动物实验上,检测了Ang1A451D对DIC模型小鼠的炎症因子的作用,脂多糖(Lipopolysaccharide,LPS)是DIC的重要介质,也是脓毒症发生的重要始动因素。其原理是LPS给药后磷酸化Tie2与总Tie2的比例显著下降;先在体内加入一定浓度的Ang1和Ang1A451D,30 min后再加入LPS诱导DIC模型,通过酶联免疫吸附实验(Enzyme LinkedImmunosorbent Assay,ELISA)的方法检测检测炎症因子TNF-α和白细胞介素(Interleukin,IL-6)的变化。
(a)材料
8-12周龄雄性C57BL/6J小鼠,购买自福州吴氏实验动物(福州,中国)。
Ang1和Ang1A451D经实施例一获得。
E. coli LPS 055:B5和PBS(Lot No.RNBJ1164)均购买于美国Sigma公司。
(b)步骤
1. 将8-12周龄雄性C57BL/6J小鼠随机分为4组,每组4只;按照分组1×PBS,16mg/kg LPS,16 mg/kg LPS+10 mg/kg Ang1,16 mg/kg LPS+10 mg/kg Ang1A451D;
2. 通过尾静脉注射预先准备好的蛋白;
3. 注射药物30 min后腹腔注射LPS,分别在1.5 h和4 h进行颌下取血,至少200 μL;
4. 3000 rpm,5 min取血浆进行Elisa实验,Elisa所有标准品和样品一式三份,每次测定都需要做一条标准曲线;
5. 包被:稀释包被中的捕获抗体缓冲液,96孔板每孔100 μL,密封板在4°C冰箱孵育过夜;
6. 洗板:次日,将高吸附板用PBST洗涤三次,并将板倒置在吸水纸上用力敲击残留缓冲液;
7. 封闭:每孔加入100 μL 3% BSA(PBS溶解)的封闭液,密封置于室温摇床封闭1h;
8. 加血浆样品:将血浆用3% BSA(PBST溶解)适量倍数稀释,100 μL/孔,密封置于室温摇床封闭2 h;
9. 洗板:操作如上;二抗:加入HRP-抗体缓冲液,100 μL/孔,密封置于室温摇床封闭40 min-1 h;
10. 显色:洗板后100 μL/孔TMB显色液,密封置于室温摇床封闭10-30 min,等到标曲第四个显色即可;
11. 终止:每孔加入100 μL终止液,分别在450 nm和570 nm处测吸收。
结果(图7)显示,与加入LPS的模型组相比,加入Ang1A451D的突变体后,1.5 h时TNF-α组和4 h时IL-6组炎症因子显著降低,具有缓解DIC炎症风暴的效果。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (6)
1.一种血管生成素1的突变体Ang1A451D,其特征在于:所述突变体Ang1A451D的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的血管生成素1的突变体Ang1A451D,其特征在于:所述突变体Ang1A451D的突变点位于血管生成素1 Ang1的受体结合区Ang1-RBD。
3.根据权利要求2所述的血管生成素1的突变体Ang1A451D,其特征在于:所述突变体Ang1A451D突变体为血管生成素1 Ang1第451位丙氨酸的一个位点突变。
4.根据权利要求3所述的血管生成素1的突变体Ang1A451D,其特征在于:所述突变体Ang1A451D突变体是将血管生成素1 Ang1氨基酸序列第451位点上的丙氨酸突变成天冬氨酸。
5.一种如权利要求1所述的突变体Ang1A451D在增强sTie2的结合力以及促进Ang1-Tie2轴正反馈中的应用。
6.一种如权利要求1所述的突变体Ang1A451D在制备预防脓毒症炎症风暴的药物中的应用。
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