CN110882378A - 一种蛋白在制备预防和治疗动脉粥样硬化及并发症药物的应用 - Google Patents
一种蛋白在制备预防和治疗动脉粥样硬化及并发症药物的应用 Download PDFInfo
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Abstract
本发明涉及一种蛋白在制备预防和治疗动脉粥样硬化及动脉粥样硬化相关的心脑血管疾病和外周血管疾病药物中的应用,其是将sDSS1蛋白用于制备预防和治疗这些疾病的药物。sDSS1蛋白能有效降低动脉粥样硬化模型动物血液中氧化型低密度脂蛋白水平,抑制血管内皮细胞和巨噬细胞对氧化型低密度脂蛋白的摄取,增加肝细胞对氧化型低密度脂蛋白的摄取,减少模型组动物中动脉粥样斑块面积,从而抑制动脉粥样硬化疾病进展或由动脉粥样硬化导致的心脑血管疾病及外周血管疾病的进展,极具临床应用价值。
Description
技术领域
本发明内容属于生物医药领域,涉及一种蛋白在制备预防和治疗动脉粥样硬化及动脉粥样硬化并发症的药物中的应用。
背景技术
动脉粥样硬化是指由于脂质在动脉血管管壁中积聚形成脂质条纹或粥样斑块导致动脉血管功能障碍。由于斑块形成导致的动脉血管舒缩功能下降、管腔狭窄甚至血栓,进而影响由该动脉所供血的组织器官的血供,导致组织器官的局部或全部的缺血。
自1955年至今的流行病学数据、临床前研究及临床试验数据表明,以低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-c)升高为特点的血脂异常是动脉粥样硬化性心血管疾病(atherosclerotic cardiovascular disease,ASCVD)重要的危险因素,与心血管疾病的发病率及死亡率呈正相关。ASCVD患者中LDL及氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)有明显升高,并且与心血管事件具有相关性[1]。降低LDL及oxLDL水平,可显著减少ASCVD的发病及死亡危险[2-7]。其他类型的血脂异常,如TG增高或高密度脂蛋白(high density lipoprotein,HDL)降低与ASCVD发病危险的升高也存在一定的关联[8,9]。
LDL在体内的活性氧(reactive oxygen species,ROS)作用下在动脉血管壁内皮层形成oxLDL。oxLDL可以通过参与血管内皮细胞的异常激活、巨噬细胞的吞噬及泡沫细胞的形成而促进动脉粥样硬化发生和发展[10-12]。血管内皮细胞在异常激活时,其表面所表达的整合素、粘附分子及其分泌的趋化因子等均明显增加。这些功能上的改变使循环系统中的单核细胞更容易粘附和迁移至血管内膜中,并进一步分化为巨噬细胞。巨噬细胞通过其表面的清道夫受体摄取oxLDL。随着脂质在巨噬细胞中的积聚,巨噬细胞逐渐转变为泡沫细胞并凋亡或坏死。巨噬细胞及泡沫细胞可以进一步释放更多的趋化因子招募更多的单核巨噬细胞及平滑肌细胞细胞向内膜迁移,最终形成动脉粥样斑块[13]。通过减少细胞对oxLDL的摄取,抑制血管内皮细胞的异常激活及巨噬细胞对oxLDL的摄取可以阻止或延缓动脉粥样硬化的进展[14-16]。
oxLDL来自于动脉血管壁中LDL的氧化修饰,其产生与机体内的氧化压力水平呈正相关。oxLDL/LDL比值可以作为反应患者的脂质氧化水平的评价指标[17-19]。OxLDL和LDL可分别通过肝细胞表面的清道夫受体I(scavenger receptor class B type I,SR-BI)和低密度脂蛋白受体(low density lipoprotein receptor,LDLR)进入肝细胞,进而被代谢清除[20,21]。因此,降低机体脂质氧化水平、增加oxLDL由肝细胞清除可以作为预防和治疗高脂血脂导致的心脑血管疾病的途径。
目前临床上对于动脉粥样硬化的治疗的首要策略是降血脂。已有的上市药物包括他汀类、贝特类、胆固醇吸收抑制剂、普罗考布、胆酸螯合剂、烟酸类及前蛋白转化酶枯草溶菌素9抑制剂等,都是以降低总胆固醇、LDL及甘油三酯为主要靶点。oxLDL是影响动脉粥样硬化进程的关键因素,但是受当前研究水平的限制,以oxLDL作为靶点的治疗方式大多处于基础研究层面。根据文献调研,已有3个oxLDL抗体申请了专利(专利号:US20040120893A1,US20070122419A1,CN101654481A),1个II期临床试验使用oxLDL单抗对动脉粥样硬化患者中的安全性进行评价(ClinicalTrials.gov临床试验编号:NCT01258907),但是尚无有效的oxLDL清除的药物。
Shfm1(split hand/split foot malformation type 1)基因是人蟹爪病中的关键基因之一,进化上高度保守,它所编码的蛋白DSS1参与到稳定基因组、同源基因重组、DNA损伤修复和细胞增殖等过程[22-26]。本专利发明人的研究结果显示DSS1蛋白作为标签可以通过耗能的酶促反应添加到氧化蛋白上,帮助细胞清除氧化蛋白[27]。这些结果显示DSS1蛋白在生物活动中的重要作用。
以上内容的引文如下:
1.Gao S,et al.Association between circulating oxidized LDL andatherosclerotic cardiovascular disease:a meta-analysis of observationalstudies.Can J Cardiol 2017.33(12):1624-1632.
2.Trialists C,et al.The effects of lowering LDL cholesterol withstatin therapy in people at low risk of vascular disease:meta-analysis ofindividual data from 27 randomised trials.Lancet2012.380(9841):581-90.
3.Califf R.M,et al.An update on the IMProved reduction of outcomes:Vytorin Efficacy International Trial(IMPROVE-IT)design.Am Heart J 2010.159(5):705-9.
4.National Cholesterol Education Program Expert Panel on Detection,E.and A.Treatment of High Blood Cholesterol in,Third Report of the NationalCholesterol Education Program(NCEP)Expert Panel on Detection,Evaluation,andTreatment of High Blood Cholesterol in Adults(Adult Treatment Panel III)finalreport.Circulation 2002.106(25):3143-421.
5.Law M.R,N.J Wald and S.G.Thompson.How much and how quickly doesreduction in serum cholesterol concentration lower risk of ischaemic heartdisease?BMJ 1994.308(6925):367-72.
6.Rossouw J.E,Lewis B and Rifkind B.M.The value of loweringcholesterol after myocardial infarction.N Engl J Med 1990.323(16):1112-9.
7.The Lipid Research Clinics Coronary Primary Prevention Trialresults I.Reduction in incidence of coronary heart disease.JAMA1984.251(3):351-64.
8.Ren J,et al.Long-term coronary heart disease risk associated withvery-low-density lipoprotein cholesterol in Chinese:the results of a 15-YearChinese Multi-Provincial Cohort Study(CMCS).Atherosclerosis 2010.211(1):327-32.
9.Baigent C,et al.Efficacy and safety of cholesterol-loweringtreatment:prospective meta-analysis of data from 90,056participants in 14randomised trials of statins.Lancet 2005.366(9493):1267-78.
10.Gistera A.and G.K Hansson.The immunology of atherosclerosis.NatRev Nephrol 2017.13(6):368-380.
11.Libby P.Inflammation in atherosclerosis.Nature 2002.420(6917):868-74.
12.Libby P,Ridker P.M and Maseri A.Inflammation andatherosclerosis.Circulation 2002.105(9):1135-43.
13.Ross R.Atherosclerosis--an inflammatory disease.N Engl J Med1999.340(2):115-26.
14.Xu Y,et al.oxLDL/beta2GPI/anti-beta2GPI complex induced macrophagedifferentiation to foam cell involving TLR4/NF-kappa B signal transductionpathway.Thromb Res 2014.134(2):384-92.
15.Pirillo A,Norata G.D and Catapano A.L.LOX-1,OxLDL,andatherosclerosis.Mediators Inflamm 2013.2013:152786.
16.Singh K.K,et al.BRCA1 is a novel target to improve endothelialdysfunction and retard atherosclerosis.J Thorac Cardiovasc Surg 2013.146(4):949-960 e4.
17.Motamed M,et al.Oxidized low-Density lipoprotein(ox-LDL)to LDLratio(ox-LDL/LDL)and ox-LDL to high-Density lipoprotein ratio(ox-LDL/HDL).Clin Lab 2016.62(9):1609-1617.
18.Harmon M.E,et al.Associations of circulating cxidized LDL andconventional biomarkers of cardiovascular disease in a cross-sectional studyof the navajo population.PLoS One 2016.11(3):e0143102.
19.Pawlak K,Mysliwiec M and Pawlak D.Oxidized low-density lipoprotein(oxLDL)plasma levels and oxLDL to LDL ratio,are they real oxidative stressmarkers in dialyzed patients?Life Sci 2013.92(4-5):253-8.
20.Sun,B.,et al.,Distinct mechanisms for OxLDL uptake and cellulartrafficking by class B scavenger receptors CD36 and SR-BI.J Lipid Res 2007.48(12):2560-70.
21.Gillotte-Taylor,K.,et al.,Scavenger receptor class B type I as areceptor for oxidized low density lipoprotein.J Lipid Res 2001.42(9):1474-82.
22.Kragelund B.B,et al.DSS1/Sem1,a Multifunctional and IntrinsicallyDisordered Protein.Trends Biochem Sci 2016.41(5):446-459.
23.Li J,et al.DSS1 is required for the stability of BRCA2.Oncogene2006.25(8):1186-94.
24.Liu J,et al.Human BRCA2protein promotes RAD51filament formation onRPA-covered single-stranded DNA.Nat Struct Mol Biol 2010.17(10):1260-2.
25.van Silfhout A.T,et al.Split hand/foot malformation due tochromosome 7q aberrations(SHFM1):additional support for functionalhaploinsufficiency as the causative mechanism.Eur J Hum Genet 2009.17(11):1432-8.
26.Zhou Q,et al.Dss1interaction with Brh2as a regulatory mechanismfor recombinational repair.Mol Cell Biol 2007.27(7):2512-26.
27.Zhang Y,et al.DSSylation,a novel protein modification targetsproteins induced by oxidative stress,and facilitates their degradation incells.Protein Cell 2014.5(2):124-40.
发明内容
流行病学数据及大量临床研究表明,动脉粥样硬化是众多心脑血管疾病及外周血管疾病发病及进展的直接和关键因素之一。通过控制和延缓动脉粥样硬化的进展,可以降低心脑血管疾病及外周血管疾病的发病率及死亡率。在本发明中,我们(发明人)提供的sDSS1蛋白可以显著降低疾病模型动物中的氧化低密度脂蛋白水平,减少动脉粥样斑块形成,抑制动脉粥样硬化进展。因此,sDSS1蛋白具备预防和治疗动脉粥样硬化及动脉粥样硬化所导致的心脑血管疾病及外周血管疾病的潜力。
具体的技术方案如下:
一种蛋白在制备预防和治疗动脉粥样硬化或动脉粥样相关的心脑血管疾病及外周血管疾病药物中的应用,其特征在于,所述的应用是将sDSS1蛋白用于制备预防和治疗动脉粥样硬化或动脉粥样相关的心脑血管疾病和外周血管疾病药物。
优选地,所述的动脉粥样硬化是指动脉血管管壁中出现脂质积聚所致的脂质条纹或粥样斑块,导致动脉血管功能障碍。
优选地,所述的脂质是指血液中的胆固醇、胆固醇酯、甘油三酯、乳糜微粒(chylomicron,CM)、低密度脂蛋白(low density lipoprotein,LDL)、极低密度脂蛋白(very low density lipoprotein,VLDL)、中间密度脂蛋白(intermediate densitylipoprotein,IDL)、脂蛋白(a)(lipoprotein(a),Lp(a))、高密度脂蛋白(HDL)、氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)的一种或几种及其各自的代谢中间物。
优选地,所述的动脉血管功能障碍是指血管内径狭窄、血管外径扩张、血管收缩及扩增能力下降、血管弹性下降、血管脆性升高、血管壁钙化或钙质沉着、血栓导致的血管供血能力下降。
优选地,所述的动脉粥样相关的心血管疾病是指高血压、心绞痛、心肌梗塞、心肌供血不足、心律失常及猝死。
优选地,所述的动脉粥样相关的脑血管疾病是指脑供血不足、缺血性脑卒中及脑萎缩。
优选地,所述的动脉粥样相关的外周血管疾病是指肾功能不全、肾动脉狭窄、麻痹性肠梗阻或肢端缺血坏死。
优选地,所述的sDSS1蛋白包括人、黑猩猩、倭黑猩猩、大猩猩、红毛猩猩、白颊长臂猿、川金丝猴、恒河猴、滇金丝猴、东非狒狒、安哥拉疣猴、白顶白眉猴、鬼狒、豚尾猴的任一sDSS1蛋白序列形成的基础蛋白,其中人sDSS1的氨基酸序列如SEQ ID NO:1,黑猩猩sDSS1的氨基酸序列如SEQ ID NO:2,倭黑猩猩sDSS1的氨基酸序列如SEQ ID NO:3,大猩猩sDSS1的氨基酸序列如SEQ ID NO:4,红毛猩猩sDSS1的氨基酸序列如SEQ ID NO:5,白颊长臂猿sDSS1的氨基酸序列如SEQ ID NO:6,川金丝猴sDSS1的氨基酸序列如SEQ ID NO:7,恒河猴sDSS1的氨基酸序列如SEQ ID NO:8,滇金丝猴sDSS1的氨基酸序列如SEQ ID NO:9,东非狒狒sDSS1的氨基酸序列如SEQ ID NO:10,安哥拉疣猴sDSS1的氨基酸序列如SEQ ID NO:11,白顶白眉猴sDSS1的氨基酸序列如SEQ ID NO:12,鬼狒sDSS1的氨基酸序列如SEQ ID NO:13,豚尾猴sDSS1的氨基酸序列如SEQ ID NO:14。
优选地,所述的sDSS1蛋白是任一与以上方案所述的sDSS1蛋白相似度达到70%以上的第一种蛋白。
优选地,所述的sDSS1蛋白是任一以以上方案所述的sDSS1蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他多肽片段,用于融合的多肽片段的结构特征或氨基酸序列特征与以上方案所述的sDSS1蛋白碳端31个序列相同或相似的第二种蛋白。
优选地,所述的sDSS1蛋白是任一以以上方案所述的sDSS1蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他氨基酸片段,融合后的蛋白能实现跨膜转运功能的第三种蛋白。
优选地,所述的sDSS1蛋白是所述的基础蛋白、第一种蛋白、第二种蛋白或第三种蛋白与该蛋白自身、载体蛋白、抗体或其他任意长度氨基酸片段连接形成的融合蛋白。
优选地,所述的sDSS1蛋白是基于所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白进行的修饰产生的多肽/蛋白修饰物。
优选地,所述的多肽/蛋白修饰物是针对氨基酸侧链上的氨基、氨基酸侧链上的羰基、氮端末端氨基、碳端末端羰基、半胱氨酸、酪氨酸、丝氨酸、色氨酸进行的特异性或非特异性的1-20个位点的化学修饰。
优选地,所述多肽/蛋白修饰物的修饰方法包括糖基化修饰、脂肪酸修饰、酰基化修饰、Fc片段融合、白蛋白融合、聚乙二醇修饰、右旋糖苷修饰、肝素修饰、聚乙烯吡咯烷酮修饰、聚氨基酸修饰、多聚唾液酸修饰、壳聚糖及其衍生物修饰、凝集素修饰、海藻酸钠修饰、卡波姆修饰、聚乙烯吡咯烷酮修饰、羟丙基甲基纤维素修饰、羟丙基纤维素修饰、乙酰化修饰、甲酰化修饰、磷酸化修饰、甲基化修饰、磺酸化修饰以及其他医药上可用的多肽/蛋白药物修饰方法的一种或一种以上。
优选地,所述的sDSS1蛋白是利用基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白的氨基酸序列为基础进行的20种基本氨基酸以外的氨基酸进行的1-31个任意氨基酸位点替换的非天然氨基酸替代蛋白。
优选地,所述的非天然氨基酸替代蛋白的氨基酸替换包括羟脯氨酸、羟赖氨酸、硒代半胱氨酸、D-型氨基酸、人工合成的非天然氨基酸及其衍生物。
优选地,所述的sDSS1蛋白是把所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、多肽/蛋白修饰物或非天然氨基酸替代物与医药上可应用的药物载体形成的部分或全部复合体。
优选地,所述的复合体的药物载体包含肠溶衣制剂、胶囊、微球/囊、脂质体、微乳液、复乳液、纳米颗粒、磁颗粒、明胶和凝胶中的一种或一种以上。
优选地,所述的sDSS1蛋白是以个体自身sDSS1蛋白为靶点,通过外源药物影响个体自身sDSS1蛋白的水平。
优选地,所述的药物是以sDSS1蛋白、sDSS1蛋白的基因、sDSS1的基因的调控元件、sDSS1的基因的转录产物为药物作用靶点。
优选地,所述的药物是通过影响血液、脑脊液或淋巴液中蛋白酶/肽酶活性从而调节sDSS1蛋白在血液、脑脊液或淋巴液中的含量。
优选地,所述的药物是化学小分子药物、抗体、多肽/蛋白药物、核酸药物、纳米药物形成的第一种药物。
优选地,所述的sDSS1蛋白是以所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、多肽/蛋白修饰物、复合体、第一种药物中的任一一种成分的两种或多种的组合形成的第二种药物。
优选地,所述的sDSS1蛋白是以所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、多肽/蛋白修饰物、复合体、第一种药物中的任一一种成分的一种、两种或多种与医药上可用的赋形剂形成的第三种药物。
优选地,所述的sDSS1蛋白是通过表达体系把编码所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白的核苷酸序列导入体内并表达获得的第四种蛋白。
优选地,所述的表达体系是真核表达质粒载体、腺病毒、腺相关病毒、慢病毒、逆转录病毒、杆状病毒、疱疹病毒、伪狂犬病毒、ZFN基因编辑技术、TALEN基因编辑技术、CRISPR/Cas基因编辑技术及其他医疗上可用的基因编辑技术或病毒载体。
优选地,所述的sDSS1蛋白是通过移植细胞在个体体内获得的所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白形成的第五种蛋白。
优选地,所述的细胞是任意一种人的干细胞、前体细胞或成体细胞。
优选地,所述的干细胞是胚胎干细胞、诱导多能干细胞、转分化得到的细胞,或者来源于原代培养的干细胞、由母细胞分化得到的多能或单能干细胞。
优选地,所述的sDSS1蛋白是通过血清、脑脊液、淋巴液或组织间液输注引入个体体内的第六种蛋白。
优选地,所述的sDSS1蛋白是通过移植组织或器官在个体体内获得的所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白形成的第七种蛋白。
优选地,所述的组织是脑、肝、肾、脾、胰岛的完整器官或部分组织块,或血液、脂肪、肌肉、骨髓、皮肤。
优选地,所述的预防药物是包含基础蛋白、第一种至第七种任一蛋白、融合蛋白、多肽/蛋白修饰物、非天然氨基酸替代蛋白、复合体、药物组合、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品、食品添加剂中的一种或多种。
优选地,所述的治疗药物是包含基础蛋白、第一种至第七种任一蛋白、融合蛋白、多肽/蛋白修饰物、非天然氨基酸替代蛋白、复合体、第一种药物、第二种药物、第三种药物、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品、食品添加剂中的一种或多种。
本发明的特点和/或有益效果有:
1.本发明提供的sDSS1蛋白在体外具有与oxLDL和LDL结合的能力;
2.本发明提供的sDSS1蛋白可以抑制人脐静脉内皮细胞(HUVEC)对oxLDL的摄取;
3.本发明提供的sDSS1蛋白可以抑制单核细胞THP-1分化的巨噬细胞对oxLDL的吞噬;
4.本发明提供的sDSS1蛋白可以增加肝细胞HepG2对oxLDL的摄取;
5.本发明提供的sDSS1蛋白可以降低模型动物中oxLDL/LDL比值水平;
6.本发明提供的sDSS1蛋白可以降低模型动物中动脉粥样斑块的面积,抑制动脉粥样硬化进展。
7.本发明提供的sDSS1蛋白是人和其他灵长类动物所具有的蛋白,分子量相对较小,免疫原性低,并且体内存在天然的蛋白降解机制,因此,临床应用不会引起明显的免疫反应或其他的毒副效应,安全可靠。
综上,本发明提供了一种用于预防和治疗动脉粥样硬化及动脉粥样硬化相关的心脑血管疾病和外周血管病的sDSS1蛋白药物,通过分子水平、细胞水平和动物水平的实验验证,sDSS1蛋白可以与LDL或oxLDL蛋白结合形成复合物,sDSS1蛋白添加到培养基中减少血管内皮细胞及单核巨噬细胞对oxLDL的摄取,增加肝细胞对oxLDL的摄取。在动物实验中,sDSS1蛋白可有效降低动脉粥样硬化模型小鼠的氧化型低密度脂蛋白水平,减小动脉粥样斑块面积,抑制动脉粥样硬化进展。sDSS1蛋白免疫原性较低,药效显著,具备用于临床上预防和治疗动脉粥样硬化及动脉粥样硬化相关的心脑血管疾病和外周血管疾病的潜力。
附图说明
下面结合附图,对本发明做进一步详细的阐述,以使本发明能够清楚、完整,但不是为了限制本发明的保护范围。
图1.分子实验显示sDSS1蛋白可以与oxLDL和LDL发生相互作用。
图1A.在醋酸钠/醋酸缓冲液(pH4.5)中,oxLDL或LDL与sDSS1蛋白孵育12小时,孵育产物经SDS-PAGE分离并用考马斯亮蓝法染色。结果显示,单独LDL蛋白(L1),oxLDL蛋白(L2)的条带分子量约70KD,单独sDSS1条带(L3,L6)分子量约15KD,LDL或oxLDL与sDSS1蛋白共孵育会发生相互作用(L4和L7对应LDL与sDSS1蛋白,L5和L8对应oxLDL与sDSS1蛋白)。
在SDS-PAGE胶上,与sDSS1蛋白反应后,反应体系中的oxLDL蛋白或LDL蛋白的条带(分子量约70KD)显著变浅;随着反应体系中sDSS1蛋白浓度增加,LDL或oxLDL形成的复合物更多,条带更深。
图1B.在磷酸盐缓冲液(pH7.2)中,oxLDL或LDL与sDSS1蛋白孵育12小时,孵育产物经SDS-PAGE分离并用考马斯亮蓝法染色。结果显示,单独LDL蛋白(L1),oxLDL蛋白(L2)的条带分子量约70KD,单独sDSS1条带(L3,L6)分子量约15KD,LDL或oxLDL与sDSS1蛋白共孵育会发生相互作用(L4和L7对应LDL与sDSS1蛋白,L5和L8对应oxLDL与sDSS1蛋白)。
在SDS-PAGE胶上,与sDSS1蛋白反应后,反应体系中的oxLDL蛋白或LDL蛋白的条带(分子量约70KD)变化不显著;随着反应体系中sDSS1蛋白浓度增加,LDL或oxLDL形成的复合物更多,条带更深。
图2.sDSS1蛋白可以抑制内皮细胞摄取oxLDL。
图2A.在人脐带静脉内皮细胞(HUVEC)的培养液中加入10μg/ml Dil-oxLDL,或加入Dil-oxLDL同时加入浓度为2μg/ml、5μg/ml、10μg/ml、20μg/ml的sDSS1蛋白。5小时后,Dil-oxLDL被HUVEC细胞摄取,在荧光显微镜下可以观察细胞内有数量不等的呈现红色荧光的吞噬小泡。加入sDSS1蛋白后,HUVEC细胞内的荧光减弱,且该效应呈现浓度依赖性。
图2B.Dil-oxLDL被HUVEC细胞摄取后,用流式细胞仪检测细胞内的荧光信号,发现随着加入培养液的sDSS1蛋白量增加,细胞的荧光信号逐渐减弱。
图2C.流式细胞仪检测结果统计发现,与对照细胞(10μg/ml Dil-oxLDL)相比,sDSS1蛋白可以显著抑制HUVEC细胞摄取oxLDL。培养液中加入2μg/ml sDSS1蛋白即可把oxLDL摄取降低一半,该效应呈现明显的剂量依赖性。
图3.sDSS1蛋白可以抑制巨噬细胞吞噬oxLDL。
图3A.THP-1细胞用100nM PMA诱导4天后分化为成熟的巨噬细胞。在培养液中加入10μg/ml Dil-oxLDL,或加入Dil-oxLDL同时加入浓度为2μg/ml、10μg/ml的sDSS1蛋白。5小时后观察,在对照细胞组,Dil-oxLDL被巨噬细胞吞噬,可以观察到细胞内明显的红色荧光吞噬小泡。加入sDSS1蛋白后,细胞内基本观察不到明显的荧光信号。
图3B.用流式细胞仪检测巨噬细胞的荧光信号,结果发现,对照组细胞(10μg/mlDil-oxLDL)可以检测到带荧光信号的细胞,而加入sDSS1蛋白的两个实验组细胞内基本没有带荧光信号的细胞出现。
图4.sDSS1蛋白可以提高人肝细胞细胞摄取oxLDL蛋白能力。
图4A.Dil-oxLDL加入Hep G2细胞9小时后,利用荧光显微镜检测细胞荧光。结果发现,在对照组和实验组细胞内可以观察到明显的荧光,这些荧光出现在数量不等的吞噬小泡中,说明Hep G2细胞摄取了Dil-oxLDL。
图4B.用流式细胞仪检测细胞荧光值,可以看到在对照组Hep G2细胞内能检测到明显的Dil荧光信号,培养液加入5μg/ml sDSS1没有显著影响细胞内荧光水平,而50μg/mlsDSS1蛋白可以显著提高细胞内Dil信号强度
图4C.统计流式细胞仪检测结果显示,与对照组相比,50μg/ml sDSS1蛋白加入到培养液后Hep G2细胞吸收oxLDL能力提高了约27%,与对照组有显著性提高(**p<0.01)。
图5.sDSS1蛋白对人肝细胞细胞摄取LDL蛋白能力没有显著影响。
图5A.Dil-LDL加入Hep G2细胞10小时后,利用荧光显微镜检测细胞荧光。结果发现,在对照组和实验组细胞内可以观察到明显的荧光,这些荧光出现在数量不等的吞噬小泡中,说明Hep G2细胞摄取了Dil-LDL。检查带荧光细胞数量,没有出现明显的差异。
图5B.用流式细胞仪检测细胞荧光值,可以看到在对照组Hep G2细胞内能检测到明显的Dil荧光信号,培养液加入5μg/ml sDSS1或50μg/ml sDSS1蛋白并不会导致细胞内Dil荧光信号强度的明显变化。
图6.sDSS1蛋白降低小鼠血浆oxLDL水平及血浆oxLDL/LDL比值,LDL水平没有显著差异。
ApoE-/-小鼠分为两组,对照组及sDSS1蛋白给药组,对照组给予200μl生理盐水每天腹腔注射一次,给药组按照10mg/kg体重(10mpk)或30mg/kg体重(30mpk)每天腹腔注射sDSS1蛋白一次,每天按照40mg/kg体重(40mpk)腹腔注射阿托伐他汀(Atorvastatin,ATV)用作阳性对照药,连续给药7天。给药结束后检测小鼠血浆oxLDL及LDL水平。
图6A.检测血浆LDL水平发现,与对照组小鼠相比,10mpk或30mpk注射sDSS1蛋白没有导致小鼠血浆LDL水平出现明显变化。
图6B.检测血浆oxLDL水平,结果发现sDSS1蛋白给药后,低剂量注射sDSS1蛋白没有显著影响小鼠血浆oxLDL水平,而高剂量给药明显降低小鼠的oxLDL水平,显示与40mpkATV相同的药效。
图6C.分析血浆中oxLDL/LDL比值,结果显示高剂量给药sDSS1蛋白组小鼠的血浆oxLDL/LDL比值明显低于对照组,与ATV显示相似的药效。(*p值<0.05)。
图7.sDSS1蛋白抑制ApoE-/-小鼠动脉粥样硬化斑块形成。16周龄ApoE-/-小鼠连续按照3mpk注射sDSS1或40mpk注射ATV,连续注射13周,结束后解剖主动脉进行油红O染色并统计斑块面积。
图7A.ApoE-/-小鼠主动脉正面(en face)染色显示动脉粥样斑块。动脉粥样斑块被油红O染色呈现橘红色,无斑块处不着色为透明。结果显示,阳性药物组小鼠动脉粥样斑块面积较模型组明显减少;sDSS1蛋白组小鼠动脉粥样斑块面积较模型组同样明显减少,某些区域效应甚至优于阳性药物。
图7B.测量油红O染色面积与全主动脉面积并计算斑块面积比值。阳性药物组及sDSS1蛋白组斑块面积比明显小于模型组(**p<0.01),阳性药物组及sDSS1蛋白组斑块面积比无明显差异。
具体实施方式
以下内容将结合实例对本发明中的优选方案进行说明和验证,不是对本发明的范围进行限定。本发明的所有范围限定以权利要求书中的限定为准。
下述实施案例中所用的实验方法如无特殊说明,均为常规实验方法。
下述实施案例中所用的sDSS1蛋白为本公司自行生产并进行质量控制,经检测蛋白纯度大于95%,内毒素(内毒素含量小于3EU/mg蛋白)和其他杂质残留符合标准,可用于动物实验而不引起明显的动物毒性反应。
下述实施案例中材料和试剂,除了sDSS1蛋白其他均可以通过商业途径获取。
实施例1.sDSS1蛋白可以与oxLDL或LDL发生相互作用。
1.1实验材料与方法
实验材料:sDSS1蛋白,氧化型低密度脂蛋白(oxLDL)(索莱宝,货号:H7980);低密度脂蛋白(LDL)(索莱宝,货号:H7960)。
实验方法:分别在20mM醋酸钠/醋酸缓冲液(pH4.5)或20mM磷酸盐缓冲液(pH7.2)条件下,将2μg的oxLDL或LDL与6μg的sDSS1蛋白或10μg的sDSS1蛋白加入1.5ml的EP管中混合,在37℃孵育12小时。孵育产物添加上样缓冲液混匀,100℃变性处理10分钟,制成上样样品。制备的样品用聚丙烯酰胺凝胶电泳(SDS-PAGE)进行电泳分离,分离完成后将SDS-PAGE胶进行考马斯亮蓝染色显示蛋白条带。
1.2实验结果
在醋酸盐缓冲液体系(pH4.5)中,染色后的SDS-PAGE胶上可见LDL蛋白和oxLDL蛋白位于约70KD(L1,L2),sDSS1蛋白位于约15KD(L3、L6)。当将LDL或oxLDL与sDSS1蛋白混合孵育后,LDL或oxLDL与sDSS1蛋白会发生相互作用(L4和L7对应LDL与sDSS1蛋白,L5和L8对应oxLDL与sDSS1蛋白)。在SDS-PAGE胶上,LDL或oxLDL与sDSS1蛋白反应后,反应体系中的oxLDL蛋白或LDL蛋白的条带变浅;随着反应体系中sDSS1蛋白浓度增加,LDL或oxLDL与sDSS1蛋白形成的复合物更多,弥散条带浓度更深(L4vs.L7;L5vs.L8)(图1A)。
在磷酸盐缓冲液体系(pH7.2)中,染色后的SDS-PAGE胶上可见LDL蛋白和oxLDL蛋白位于约70KD(L1,L2),sDSS1蛋白位于约15KD(L3、L6)。当将LDL或oxLDL与sDSS1蛋白混合孵育后,LDL或oxLDL与sDSS1蛋白会发生相互作用(L4和L7对应LDL与sDSS1蛋白,L5和L8对应oxLDL与sDSS1蛋白)。在SDS-PAGE胶上,LDL或oxLDL与sDSS1蛋白反应后,反应体系中的oxLDL蛋白或LDL蛋白的条带显著变浅;随着反应体系中sDSS1蛋白浓度增加,LDL或oxLDL与sDSS1蛋白形成的复合物更多,弥散条带浓度更深(L4vs.L7;L5vs.L8)(图1B)。
这些结果说明,sDSS1蛋白可以与oxLDL或LDL发生相互作用并形成复合物,这些复合物不能被SDS-PAGE分开。
实施例2.sDSS1蛋白可以降低血管内皮细胞摄取oxLDL。
2.1实验材料与方法
实验材料:sDSS1蛋白,Dil-oxLDL(赛默飞世尔科技,货号:L34358),人脐静脉内皮细胞(HUVEC)(PromoCell,货号:C-12200)
实验方法:HUVEC细胞按照细胞数30万每孔接种到6孔板中,贴壁24小时后,加入1.5ml 10μg/ml的Dil-oxLDL,或加入1.5ml10μg/ml的Dil-oxLDL同时加入浓度为2μg/ml、5μg/ml、10μg/ml、20μg/ml的sDSS1蛋白。继续于培养箱中孵育5小时后,使用荧光显微镜观察细胞内荧光信号,用胰酶消化细胞成单细胞用于流式细胞仪检测荧光强度。
2.2实验结果
HUVEC细胞培养液中加入Dil-oxLDL并继续孵育5小时后,荧光显微镜下可以观察到HUVEC细胞内明显的荧光,荧光基中出现在吞噬小泡中。并且,加入sDSS1s蛋白后,可以观察到细胞内的荧光强度降低,细胞内带荧光的吞噬小泡数量下降。该现象呈现剂量依赖性,在高浓度组,细胞内荧光微弱(图6A)。用流式细胞仪检测细胞荧光强度,结果显示添加浓度为2μg/ml或10μg/ml sDSS1蛋白后,HUVEC细胞内的荧光明显变弱(图6B)。统计学结果也显示,sDSS1蛋白可以显著降低HUVEC细胞内荧光值,且该效应有明显的剂量依赖效应,提示sDSS1蛋白降低那片细胞摄取oxLDL。培养液加入2μg/ml后,细胞摄取oxLDL减少一半。这些结果表明,sDSS1蛋白可以减少HUVEC细胞摄取oxLDL蛋白。
实施例3.sDSS1蛋白抑制巨噬细胞吞噬oxLDL。
3.1实验材料与方法
实验材料:sDSS1蛋白,Dil-oxLDL,佛波酯(PMA,西格玛奥德里奇公司,货号:P1585),人单核细胞THP-1(中国科学院典型培养物保藏委员会细胞库,目录号:SCSP-567)。
实验方法:THP-1细胞按照细胞数25万每孔接种到6孔板中,培养液中包含100ng/mL PMA。细胞被PMA激活48小时后,换成新鲜培养基培养四天,帮助细胞贴壁完成。弃去旧培养基,加入1.5ml10μg/ml的Dil-oxLDL,或加入1.5ml 10μg/ml的Dil-oxLDL同时加入浓度为2μg/ml、5μg/ml、10μg/ml、20μg/ml的sDSS1蛋白。继续于培养箱中孵育5小时后,用荧光显微镜观察细胞内荧光信号,胰酶消化细胞成单细胞用于流式细胞仪检测荧光强度。
3.2实验结果
Dil-oxLDL加入巨噬细胞5小时后,检测细胞荧光。结果发现,在对照组巨噬细胞内可以观察到明显的荧光,这些荧光出现在数量不等的吞噬小泡中,说明巨噬细胞摄取了oxLDL。但是,培养液中加入sDSS1蛋白后,细胞内没有观察到明显的荧光,或者荧光十分微弱(图3A)。用流式细胞仪检测细胞荧光值,可以看到在对照组细胞内能检测到带荧光的细胞,而加入2μg/ml或10μg/ml的sDSS1蛋白后,没有检测到带荧光的细胞(图3B)。这些结果说明,sDSS1蛋白可以抑制巨噬细胞摄取oxLDL蛋白。
实施例4.sDSS1蛋白促进肝细胞吞噬oxLDL,不影响吞噬LDL。
4.1实验材料与方法
实验材料:sDSS1蛋白,Dil-oxLDL,Dil-LDL(ThermoFisher,L3482),人肝癌细胞Hep G2(中国科学院典型培养物保藏委员会细胞库,目录号:SCSP-510)。
实验方法:①.检测Hep G2细胞吞噬oxLDL时,Hep G2细胞按照细胞数25万每孔接种到6孔板中,12小时后,所有细胞贴壁完成。弃去旧培养基,加入1.5ml 10μg/ml的Dil-oxLDL,或加入1.5ml10μg/ml的Dil-oxLDL同时加入浓度为5μg/ml和50μg/ml的sDSS1蛋白。继续于培养箱中孵育9小时后,用荧光显微镜观察细胞内荧光信号,胰酶消化细胞成单细胞用于流式细胞仪检测荧光强度。②.检测Hep G2细胞吞噬LDL时,Hep G2细胞按照细胞数25万每孔接种到6孔板中,12小时后,所有细胞贴壁完成。弃去旧培养基,加入1.5ml 10μg/ml的Dil-LDL,或加入1.5ml 10μg/ml的Dil-LDL同时加入浓度为5μg/ml和50μg/ml的sDSS1蛋白。继续于培养箱中孵育10时后,用荧光显微镜观察细胞内荧光信号,胰酶消化细胞成单细胞用于流式细胞仪检测荧光强度。
4.2实验结果
Dil-oxLDL加入Hep G2细胞9小时后,利用荧光显微镜检测细胞荧光。结果发现,在对照组和实验组细胞内可以观察到明显的荧光,这些荧光出现在数量不等的吞噬小泡中,说明Hep G2细胞摄取了oxLDL(图4A)。用流式细胞仪检测细胞荧光值,可以看到在对照组Hep G2细胞内能检测到明显的Dil荧光信号,培养液加入5μg/ml sDSS1没有显著影响细胞内荧光水平,而50μg/ml sDSS1蛋白可以显著提高细胞内Dil信号强度(图4B)。统计流式细胞仪检测结果显示,与对照组相比,50μg/ml sDSS1蛋白加入到培养液后Hep G2细胞吸收oxLDL能力提高了约27%(图4C)。这些结果说明,sDSS1蛋白可以提高人肝细胞细胞摄取oxLDL蛋白能力。
在Hep G2细胞培养物中加入Dil-LDL10小时后,可以观察到荧光染料出现在细胞内,分布在囊泡中。与单独添加Dil-LDL组相比,添加5μg/ml或50μg/ml sDSS1蛋白后,摄取Dil-LDL的Hep G2细胞数量没有出现明显的变化(图5A)。用流式细胞仪分析细胞结果显示,添加不同浓度sDSS1蛋白后,细胞内荧光强度基本一致(图5B),提示sDSS1蛋白对人肝细胞摄取LDL没有明显影响。
这些结果说明,sDSS1蛋白可以提高人肝细胞摄取oxLDL水平,但是没有显著影响肝细胞摄取LDL。
实施例5.sDSS1蛋白给药降低ApoE-/-小鼠血浆oxLDL水平和oxLDL/LDL比率。
5.1实验材料与方法
实验动物:ApoE-/-小鼠,8-10周龄,雄性,购于南京大学-南京生物医药研究院,实验前动物置于动物房适应一周。
实验材料:sDSS1蛋白,oxLDL ELISA试剂盒(武汉伊莱瑞特生物科技股份有限公司,货号:E-EL-M0066c),LDL ELISA试剂盒(武汉伊莱瑞特生物科技股份有限公司,货号:E-EL-M1363c)
实验方法:ApoE-/-小鼠随机分为两组,对照组和sDSS1蛋白组。对照组给予200μl生理盐水每天腹腔注射一次,sDSS1蛋白组按照10mg/kg每天腹腔注射sDSS1蛋白一次,连续7天。给药7天后采集小鼠血液,以EDTA-Na为抗凝剂,分离血浆,使用oxLDL ELISA试剂盒测定血浆oxLDL水平,使用LDL ELISA试剂盒测定血浆LDL水平。
在各孔中加入oxLDL标准品或样品各100μl,37℃孵育90分钟;倒去孔内液体,加入100μl生物素化oxLDL抗体工作液,37℃孵育60分钟;洗涤3次;加入100μl酶结合工作液,37℃孵育30分钟;洗涤5次;加入90μl底物溶液,37℃孵育15分钟;加入50μl终止液,立即在450nm波长处测量OD值;使用标准品建立标准曲线,依照标准曲线计算各样品的oxLDL水平。
在各孔中加入LDL标准品或样品各100μl,37℃孵育90分钟;倒去孔内液体,加入100μl生物素化LDL抗体工作液,37℃孵育60分钟;洗涤3次;加入100μl酶结合工作液,37℃孵育30分钟;洗涤5次;加入90μl底物溶液,37℃孵育15分钟;加入50μl终止液,立即在450nm波长处测量OD值;使用标准品建立标准曲线,依照标准曲线计算各样品的LDL水平。
oxLDL/LDL比值=oxLDL浓度(ng/ml)÷LDL浓度(ng/ml)
5.2实验结果
ApoE-/-小鼠按照10mg/kg体重腹腔注射sDSS1蛋白,连续7天后,分别检测血浆LDL和oxLDL水平。结果显示,sDSS1蛋白给药后,小鼠血浆LDL水平保持在正常水平,与对照组小鼠没有明显的差异(图6A)。但是,sDSS1蛋白注射可以显著降低小鼠血浆oxLDL含量,抑制效果与阳性对照药ATV相似(图6B)。经过进一步分析,sDSS1蛋白注射后的小鼠血浆oxLDL/LDL比值也显著低于对照组小鼠,与对照药ATV的效应基本一致(图6C)。综合这些结果,sDSS1蛋白可以降低小鼠血浆oxLDL水平,但是不影响血浆LDL水平,最终导致血浆oxLDL/LDL比率下降。
实施例6.sDSS1蛋白抑制ApoE-/-小鼠动脉粥样硬化斑块形成。
6.1实验材料与方法
实验动物:ApoE-/-小鼠,8周龄,雄性,购于南京大学-南京生物医药研究院,实验前动物置于动物房适应一周。
实验材料:sDSS1蛋白,阿托伐他汀(Atorvastatin,ATV)。
实验方法:ApoE-/-小鼠自9周龄开始给予含0.25%胆固醇的高脂饲料饲养,自高脂饲料饲养第10周开始,根据总胆固醇水平随机分为3组,每组10只。模型组给予生理盐水200μL腹腔注射每天1次,阳性药物组给予ATV 40mg/kg体重经口给药每天1次,sDSS1蛋白组给予sDSS1蛋白3mg/kg腹腔注射每天1次。于高脂饲料饲养第22周,即给药第13周,开始处死动物,解剖并分离全主动脉。将分离的全主动脉行en face油红O染色,评价动脉粥样硬化斑块形成情况。
6.2实验结果:
en face染色结果显示,模型组小鼠全主动脉发展出了明显的动脉粥样斑块,被油红O染为红色,无斑块处不着色为透明。阳性药物组小鼠动脉粥样斑块面积较模型组明显减少,sDSS1蛋白组小鼠动脉粥样斑块面积较模型组明显减少(图7A)。使用Image Pro plus测量油红O染色面积与全主动脉面积并计算比值。ANOVA分析显示,阳性药物组及sDSS1蛋白组斑块面积比明显小于模型组(**p<0.01),阳性药物组及sDSS1蛋白组斑块面积比无明显差异。这些结果说明,sDSS1蛋白可以明显抑制ApoE-/-小鼠中动脉粥样硬化斑块的形成,减缓动脉粥样硬化的进展。
序列表
<110> 上海清流生物医药科技有限公司
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Claims (35)
1.一种蛋白在制备预防和治疗动脉粥样硬化或动脉粥样相关的心脑血管疾病及外周血管疾病药物中的应用,其特征在于,所述的应用是将sDSS1蛋白用于制备预防和治疗动脉粥样硬化或动脉粥样相关的心脑血管疾病和外周血管疾病药物。
2.根据权利要求1所述的应用,其特征在于,所述的动脉粥样硬化是指动脉血管管壁中出现脂质积聚所致的脂质条纹或粥样斑块,导致动脉血管功能障碍。
3.根据权利要求2所述的应用,其特征在于,所述的脂质是指血液中的胆固醇、胆固醇酯、甘油三酯、乳糜微粒(chylomicron,CM)、低密度脂蛋白(low density lipoprotein,LDL)、极低密度脂蛋白(very low density lipoprotein,VLDL)、中间密度脂蛋白(intermediate density lipoprotein,IDL)、脂蛋白(a)(lipoprotein(a),Lp(a))、氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)的一种或几种及其各自的代谢中间物。
4.根据权利要求2所述的应用,其特征在于,所述的动脉血管功能障碍是指血管内径狭窄、血管外径扩张、血管收缩及扩增能力下降、血管弹性下降、血管脆性升高、血管壁钙化或钙质沉着、血栓导致的血管供血能力下降。
5.根据权利要求1所述的应用,其特征在于,所述的动脉粥样相关的心血管疾病是指高血压、心绞痛、心肌梗塞、心肌供血不足、心律失常及猝死。
6.根据权利要求1所述的应用,其特征在于,所述的动脉粥样相关的脑血管疾病是指脑供血不足、缺血性脑卒中及脑萎缩。
7.根据权利要求1所述的应用,其特征在于,所述的动脉粥样相关的外周血管疾病是指肾功能不全、肾动脉狭窄、麻痹性肠梗阻或肢端缺血坏死。
8.根据权利要1所述的应用,其特征在于,所述的sDSS1蛋白包括人、黑猩猩、倭黑猩猩、大猩猩、红毛猩猩、白颊长臂猿、川金丝猴、恒河猴、滇金丝猴、东非狒狒、安哥拉疣猴、白顶白眉猴、鬼狒、豚尾猴的任一sDSS1蛋白序列形成的基础蛋白,其中人sDSS1的氨基酸序列如SEQ ID NO:1,黑猩猩sDSS1的氨基酸序列如SEQ ID NO:2,倭黑猩猩sDSS1的氨基酸序列如SEQ ID NO:3,大猩猩sDSS1的氨基酸序列如SEQ ID NO:4,红毛猩猩sDSS1的氨基酸序列如SEQ ID NO:5,白颊长臂猿sDSS1的氨基酸序列如SEQ ID NO:6,川金丝猴sDSS1的氨基酸序列如SEQ ID NO:7,恒河猴sDSS1的氨基酸序列如SEQ ID NO:8,滇金丝猴sDSS1的氨基酸序列如SEQ ID NO:9,东非狒狒sDSS1的氨基酸序列如SEQ ID NO:10,安哥拉疣猴sDSS1的氨基酸序列如SEQ ID NO:11,白顶白眉猴sDSS1的氨基酸序列如SEQ ID NO:12,鬼狒sDSS1的氨基酸序列如SEQ ID NO:13,豚尾猴sDSS1的氨基酸序列如SEQ ID NO:14。
9.根据权利要求8所述的应用,其特征在于,所述的sDSS1蛋白是任一与基础蛋白相似度达到70%以上的第一种蛋白。
10.根据权利要求8所述的应用,其特征在于,所述的sDSS1蛋白是以所述的基础蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他多肽片段,用于融合的多肽片段的结构特征或氨基酸序列特征与所述的基础蛋白碳端31个序列相同或相似的第二种蛋白。
11.根据权利要求8所述的应用,其特征在于,所述的sDSS1蛋白是以所述基础蛋白氮端58个氨基酸为基础,在氮端或碳端融合其他氨基酸片段,融合后的蛋白能实现跨膜转运功能的第三种蛋白。
12.根据权利要求8-11任一所述的应用,其特征在于,所述的sDSS1蛋白是所述的基础蛋白、第一种蛋白、第二种蛋白或第三种蛋白与该蛋白自身、载体蛋白、抗体或其他任意长度氨基酸片段连接形成的融合蛋白。
13.根据权利要求8-12任一所述的应用,其特征在于,所述的sDSS1蛋白是基于所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白进行的修饰产生的多肽/蛋白修饰物。
14.根据权利要求13所述的应用,其特征在于,所述的多肽/蛋白修饰物是针对氨基酸侧链上的氨基、氨基酸侧链上的羰基、氮端末端氨基、碳端末端羰基、半胱氨酸、酪氨酸、丝氨酸、色氨酸进行的特异性或非特异性的1-20个位点的化学修饰。
15.根据权利要求13所述的应用,其特征在于,所述多肽/蛋白修饰物的修饰方法包括糖基化修饰、脂肪酸修饰、酰基化修饰、Fc片段融合、白蛋白融合、聚乙二醇修饰、右旋糖苷修饰、肝素修饰、聚乙烯吡咯烷酮修饰、聚氨基酸修饰、多聚唾液酸修饰、壳聚糖及其衍生物修饰、凝集素修饰、海藻酸钠修饰、卡波姆修饰、聚乙烯吡咯烷酮修饰、羟丙基甲基纤维素修饰、羟丙基纤维素修饰、乙酰化修饰、甲酰化修饰、磷酸化修饰、甲基化修饰、磺酸化修饰以及其他医药上可用的多肽/蛋白药物修饰方法的一种或一种以上。
16.根据权利要求8-11任一所述的应用,其特征在于,所述的sDSS1蛋白是利用所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白的氨基酸序列为基础进行的20种基本氨基酸以外的氨基酸进行的1-31个任意氨基酸位点替换的非天然氨基酸替代蛋白。
17.根据权利要求16所述的应用,其特征在于,所述的非天然氨基酸替代蛋白的氨基酸替换包括羟脯氨酸、羟赖氨酸、硒代半胱氨酸、D-型氨基酸或者人工合成的非天然氨基酸及其衍生物。
18.根据权利要求8-17任一所述的应用,其特征在于,所述的sDSS1蛋白是把所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、多肽/蛋白修饰物或非天然氨基酸替代物与医药上可应用的药物载体形成的部分或全部复合体。
19.根据权利要求18所述的应用,其特征在于,所述的复合体的药物载体包含肠溶衣制剂、胶囊、微球/囊、脂质体、微乳液、复乳液、纳米颗粒、磁颗粒、明胶和凝胶中的一种或一种以上。
20.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是以个体自身sDSS1蛋白为靶点,通过外源药物影响个体自身sDSS1蛋白的水平。
21.根据权利要求20所述的应用,其特征在于,所述的药物是以sDSS1蛋白、sDSS1蛋白的基因、sDSS1的基因的调控元件、sDSS1的基因的转录产物为药物作用靶点。
22.根据权利要求20所述的应用,其特征在于,所述的药物是通过影响血液中蛋白酶/肽酶活性从而调节sDSS1蛋白在血液、脑脊液、淋巴液中的含量。
23.根据权利要求20-22任一所述的应用,其特征在于,所述的药物是化学小分子药物、抗体、多肽/蛋白药物、核酸药物、纳米药物形成的第一种药物。
24.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是以所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、多肽/蛋白修饰物、复合体、第一种药物中的任一一种成分的两种或多种的药物组合形成的第二种药物。
25.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是以所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白、多肽/蛋白修饰物、复合体、第一种药物中的任一一种成分的一种、两种或多种与医药上可用的赋形剂形成的第三种药物。
26.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是通过表达体系把编码所述的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白、融合蛋白的核苷酸序列导入体内并表达获得的第四种蛋白。
27.根据权利要求26所述的应用,其特征在于,所述的表达体系是真核表达质粒载体、腺病毒、腺相关病毒、慢病毒、逆转录病毒、杆状病毒、疱疹病毒、伪狂犬病毒、ZFN基因编辑技术、TALEN基因编辑技术、CRISPR/Cas基因编辑技术及其他医疗上可用的基因编辑技术或病毒载体。
28.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是通过移植细胞在个体体内获得的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白形成的第五种蛋白。
29.根据权利要求28所述的应用,其特征在于,所述的细胞是任意一种人的干细胞、前体细胞或成体细胞。
30.根据权利要求29所述的应用,其特征在于,所述的干细胞是诱导多能干细胞、转分化得到的细胞,或者来源于原代培养的干细胞、由母细胞分化得到的多能或单能干细胞。
31.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是通过血清、组织间液输注引入个体体内的第六种蛋白。
32.根据权利要求1所述的应用,其特征在于,所述的sDSS1蛋白是通过移植组织或器官在个体体内获得的基础蛋白、第一种蛋白、第二种蛋白、第三种蛋白或融合蛋白形成的第七种蛋白。
33.根据权利要求32所述的应用,其特征在于,所述的组织是脑、肝、肾、脾、胰岛的完整器官或部分组织块,或血液、脂肪、肌肉、骨髓、皮肤。
34.根据权利要求1所述的应用,其特征在于,所述的预防药物是包含基础蛋白、第一种至第七种任一蛋白、融合蛋白、多肽/蛋白修饰物、非天然氨基酸替代蛋白、复合体、药物组合、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品、食品添加剂中的一种或多种。
35.根据权利要求1所述的应用,其特征在于,所述的治疗药物是包含基础蛋白、第一种至第七种任一蛋白、融合蛋白、多肽/蛋白修饰物、非天然氨基酸替代蛋白、复合体、第一种药物、第二种药物、第三种药物、表达体系、细胞、组织、器官、体液、组织液的蛋白药物、多肽药物、核酸药物、化学小分子药物、细胞产品、商业化移植组织、注射液、冻干粉、保健品、食品添加剂中的一种或多种。
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CN115287276A (zh) * | 2022-03-07 | 2022-11-04 | 兰州大学 | Sem1蛋白、表达sem1蛋白工程化益生菌在制备治疗和/或预防心脏病药物中的应用 |
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SG11202102442WA (en) | 2021-04-29 |
EP3854804A4 (en) | 2022-06-29 |
KR20210075087A (ko) | 2021-06-22 |
US20220040257A1 (en) | 2022-02-10 |
WO2020052570A1 (zh) | 2020-03-19 |
EP3854804A1 (en) | 2021-07-28 |
AU2019339037A1 (en) | 2021-05-13 |
CN112839953A (zh) | 2021-05-25 |
CA3112416A1 (en) | 2020-03-19 |
CN112839953B (zh) | 2024-06-11 |
MX2021005010A (es) | 2022-11-30 |
JP2022500491A (ja) | 2022-01-04 |
IL281394A (en) | 2021-04-29 |
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