CN108676071B - 一种抗Aβ蛋白聚集的七肽及其应用与编码该合成多肽的基因 - Google Patents
一种抗Aβ蛋白聚集的七肽及其应用与编码该合成多肽的基因 Download PDFInfo
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Abstract
本发明提供了一种抗Aβ42蛋白聚集的七肽及其应用与编码该合成多肽的基因。本发明的合成多肽具有显著抗Aβ42蛋白聚集的功效,进而具有改善记忆、延缓阿尔兹海默症发病进程的作用,可广泛应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品,进而能对包括AD疾病的神经退行疾病进行有效预防和治疗,改善神经退行疾病医疗状况,具有重大社会和经济效益。
Description
技术领域
本发明涉及多肽技术领域,特别涉及一种抗Aβ蛋白聚集的七肽及其应用与编码该合成多肽的基因。
背景技术
阿尔兹海默病(AD)又称老年痴呆症,是一种多因素诱发的中枢神经退行性疾病。AD临床表现为记忆力逐渐丧失,日常生活不能自理,病程后期大小便失禁,呈现缄默、肢体僵直症状,有强握、摸索和吮吸等原始反射,最终昏迷,因感染等原因死亡。AD病程大约十年,从发病到死亡呈现出一个完全无助的生存状态。AD的长期性,以及其对庇护自我认知能力的脆弱结构的攻击让病人、病人的家庭和整个社会承受了巨大的感情和财政负担。
目前,治疗AD的药物主要为乙酰胆碱酯酶抑制剂。乙酰胆碱酯酶抑制剂能够抑制乙酰胆碱酯酶的活性,减少脑内乙酰胆碱的分解,从而缓解AD症状。但AD的治疗始终缺乏特异性强、缓解且逆转病情,提高疾病治疗预后的有效药物。多肽类药物的出现为寻找高选择性、高效、低毒的AD治疗方法和药物研发提供了新方向。脑蛋白水解物为一种神经营养性多肽混合物,富含游离氨基酸、低分子多肽及镁、钾、磷、硒等多种元素,治疗阿尔兹海默症有一定疗效,多项研究显示脑蛋白水解物能显著改善患者的记忆、疲劳、眩晕、焦虑等症状。脑苷肌肽目前在临床上使用取得了较好的疗效。脑啡肽作为一种五肽,除抑制神经传递,起镇痛作用,同时因神经元保护作用成为了AD的潜力治疗药物。袁直等人在"一种用于治疗阿尔兹海默症的多肽"专利中公开了一种十三肽,可鳌合铜离子并有效抑制β-淀粉样肽的聚集。张奇志等发明了“一种用于治疗阿尔兹海默症的H1O2肽鼻腔溶液型喷雾剂”。四川百利药业有限责任公司研发“一种用于治疗阿尔兹海默症的多肽及基因疫苗”。已有的AD多肽类药物为多肽类药物的筛选提供理论支持,揭示了多肽作为AD药物的重大潜力。
AD的主要致病机制尚不清楚,已有的研究对AD发病机制提出多种假说,包括Aβ级联假说、微管相关蛋白-tau蛋白异常假说、中枢胆碱能损伤假说、基因突变或多型性学说、免疫功能突变假说、兴奋性氨基酸毒性学说等等。其中,Aβ级联假说是最有影响力的主流假说之一。Aβ级联假说提出β-淀粉样蛋白(Aβ)可导致老年斑形成以及神经纤维缠结(NFT)、神经细胞凋亡,对神经系统具有特异毒性,进一步诱发炎症变化,患者记忆力下降,导致AD的发生。已有研究证明,Aβ42是多种淀粉样蛋白中最具神经毒性的一种。Aβ42寡聚体与神经元和非神经元细胞膜上的多种成分,包括脂类、受体、离子通道等相结合引起一系列复杂的突触、神经元和神经元网络功能结构异常,导致学习、记忆等行为异常。本发明使用E22G-mCherry hek-293转基因细胞模型,通过胞内表达Aβ42-mCherry蛋白,良好的模拟了Aβ42在细胞内聚集、产生毒性的过程,模拟AD患者体内神经元细胞老年斑的病理发展过程,通过插入AD患者基因突变亚型E22G增强Aβ42的聚集性,并通过对Aβ42蛋白标记mCherry红色荧光蛋白追踪其的集聚,最终借助显微镜观察、拍摄等手段实现可视化快速筛选AD多肽类药物。
发明内容
本发明的目的在于针对现有技术的不足,提供了一种抗Aβ蛋白聚集的七肽。
本发明的目的还在于提供编码所述的一种抗Aβ蛋白聚集的七肽的基因。
本发明的另一目的还在于提供所述的一种抗Aβ蛋白聚集的合成多肽的应用。
本发明的目的通过如下技术方案实现。
一种抗Aβ蛋白聚集的合成多肽即七肽,名称为WW-7,氨基酸序列为Trp-Asp-Gln-Trp-Cys-Ile-Trp,如序列表SEQ ID No:1所示;
其中,Trp为色氨酸的氨基酸相应残基,Asn为天冬酰胺的氨基酸相应残基,Gln为谷氨酸的氨基酸相应残基,Cys为半胱氨酸的氨基酸相应残基,Ile为异亮氨酸的氨基酸相应残基。
一种编码所述的抗Aβ蛋白聚集的合成多肽的基因,碱基序列为TGGGACCAATGGTGCATTTGG,如序列表SEQ ID No:2所示,基因长度为21个碱基;
其中,TGG为色氨酸的密码子,GAC为天冬氨酸的密码子,CAA为谷氨酸的密码子,TGC为半胱氨酸的密码子,ATT为异亮氨酸的密码子。
合成所述的一种抗Aβ蛋白聚集的合成多肽的方法,采用多肽固相合成法合成或基因工程技术,具体包括如下步骤:
其中,通过多肽固相合成法合成时,采用标准Fmoc方案,树脂选用2-ChlorotritylChloride Resin(2氯树脂);采用Fmoc保护氨基酸N端,各保护氨基酸为Fmoc-Trp(Boc)-OH、Fmoc-Asp(Tbu)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ile(Trt)-OH。树脂上的活性位点为卤素氯,多肽固相合成首先需要把树脂溶胀,再将第一个氨基酸的C端羧基与树脂上的活性位点氯反应,待首个氨基酸接在树脂上以后,进行脱水缩合接第二个氨基酸,缩合完成后再脱Fmoc保护。按照设计的氨基酸序列重复操作,依次接完其余的氨基酸并完成N端的乙酰化,最后用切割试剂把多肽从树脂上切割下来,获得粗品,粗品纯化后,获得具有抗Aβ42蛋白聚集功能的多肽。
通过基因工程技术合成时,将编码基因接入到载体中,再将载体转录到原核表达体系大肠杆菌中或真核表达体系酵母中进行表达,然后对目标多肽进行分离纯化,获得具有抗Aβ42蛋白聚集功能的多肽。
所述的一种抗Aβ蛋白聚集的合成多肽的应用,包括应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品。
优选的,所述药物的剂型包括膏剂、颗粒剂、丸剂、散剂、片剂、胶囊剂、口服液或糖浆剂。
优选的,所述食品为保健食品,剂型包括颗粒剂、胶囊剂、糖浆剂、片剂、粉剂、软糖、乳剂或口服液。
与现有技术相比,本发明具有如下优点和有益效果:
本发明的合成多肽Trp-Asp-Gln-Trp-Cys-Ile-Trp具有显著抗Aβ42蛋白聚集的功效,进而具有改善记忆、延缓阿尔兹海默症发病进程的作用,可广泛应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品,进而能对包括AD疾病的神经退行疾病进行有效预防和治疗,改善神经退行疾病医疗状况,具有重大社会和经济效益。
附图说明
图1a为实施例1合成的多肽WW-7的高效液相色谱图;
图1b为实施例1合成的多肽WW-7的液相色谱-质谱/质谱(LC-MS)图;
图2a为实施例2中阴性对照组(Control group)的IncuCyte Zoom长时间活细胞成像图;
图2b为实施例2中模型组(Model group)的IncuCyte Zoom长时间活细胞成像图;
图3a为实施例2中多肽WW-7浓度为0.05mM的多肽低剂量组的IncuCyteZoom长时间活细胞成像图;
图3b为实施例2中多肽WW-7浓度为0.5mM的多肽高剂量组的IncuCyteZoom长时间活细胞成像图;
图4为阴性对照组(Control group)、模型组(Model group)、0.05mM的多肽低剂量组和0.5mM的多肽高剂量组的Aβ42蛋白聚集率柱形图。
具体实施方式
以下结合具体实施例及附图对本发明的技术方案作进一步详细的描述,但本发明的保护范围及实施方式不限于此。
具体实施例中,本发明的一种抗Aβ蛋白聚集的合成多肽,名称为WW-7,氨基酸序列如氨基酸序列表SEQ ID NO:1所示,氨基酸序列为Trp-Asp-Gln-Trp-Cys-Ile-Trp;
其中,Trp为色氨酸的氨基酸相应残基,Asn为天冬酰胺的氨基酸相应残基,Gln为谷氨酸的氨基酸相应残基,Cys为半胱氨酸的氨基酸相应残基,Ile为异亮氨酸的氨基酸相应残基;
分子结构式如下所示:
编码上述的抗Aβ蛋白聚集的合成多肽的基因,碱基序列为TGGGACCAATGGTGCATTTGG,如序列表SEQ ID No:2所示,基因长度为21个碱基;
其中,TGG为色氨酸的密码子,GAC为天冬氨酸的密码子,CAA为谷氨酸的密码子,TGC为半胱氨酸的密码子,ATT为异亮氨酸的密码子。
本发明的抗Aβ42蛋白聚集的合成多肽WW-7可通过多肽固相合成法或基因工程技术合成;
其中,通过多肽固相合成法合成时,采用标准Fmoc方案,树脂选用2-chlorotritylchloride resin树脂;采用Fmoc保护氨基酸N端,各保护氨基酸为Fmoc-Trp(Boc)-OH、Fmoc-Asp(Tbu)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Ile(Trt)-OH。Fmoc合成的步骤如下:树脂上的活性位点为卤素氯,多肽固相合成首先需要把树脂溶胀,再将第一个氨基酸的C端羧基与树脂上的活性位点氯反应,待首个氨基酸接在树脂上以后,进行脱水缩合接第二个氨基酸,缩合完成后再脱Fmoc保护。按照设计的氨基酸序列重复操作,依次接完其余的氨基酸并完成N端的乙酰化,最后用切割试剂把多肽从树脂上切割下来,获得粗品,粗品纯化后,获得具有抗Aβ42蛋白聚集功能的多肽。
通过基因工程技术合成时,将编码基因接入到载体中,再将载体转录到原核表达体系大肠杆菌中或真核表达体系酵母中进行表达,然后对目标多肽进行分离纯化,获得抗Aβ42蛋白聚集的合成多肽。
具体实施例中,本发明基于Aβ级联假说,采用E22G-Aβ42-mCherry HEK-293转基因细胞模型对的获得的合成多肽WW-4进行AD老年斑体外研究。
E22G-Aβ42-mCherry HEK-293转基因细胞模型内能表达Aβ42-mCherry蛋白,良好地模拟了Aβ42在细胞内聚集、产生毒性的过程,模拟AD患者体内神经元细胞老年斑的病理发展过程,并通过对Aβ42蛋白标记mCherry红色荧光蛋白追踪其的集聚,最终借助显微镜观察、拍摄等手段实现可视化快速AD药物筛选。
实施例1
多肽固相合成法合成多肽WW-7
1、树脂选型
(1)采用标准Fomc方案,起始选用0.0125mmol 2-chlorotrityl chloride resin树脂(天津市南开合成科技有限公司),按照氨基酸序列Trp-Asp-Gln-Trp-Cys-Ile-Trp的C端到N端的序列特征,加入0.3mol第一个Fmoc保护氨基酸,将DCC和5%(质量分数)DMAP加入到反应器振荡反应,用甲基吡咯烷酮(NMP)冲洗树脂除去多余保护氨基酸。
(2)采用标准Fomc方案,起始选用0.0125mmol Wang树脂,按照氨基酸序列Trp-Asp-Gln-Trp-Cys-Ile-Trp的C端到N端的序列特征,加入0.3mol第一个Fmoc保护氨基酸,将DCC和5%(质量分数)DMAP加入到反应器振荡反应,用NMP冲洗树脂除去多余保护氨基酸。
2、合成过程
采用标准Fomc方案,选用偶合率较高的Wang树脂,按照氨基酸序列Trp-Asp-Gln-Trp-Cys-Ile-Trp的序列特征,使肽链从C端逐个向N端延伸,具体合成步骤如下:
加入20毫升20%哌啶/DMF溶液,5min后抽掉。再加入20毫升20%哌啶/DMF溶液,振荡15min,完成脱保护;抽掉哌啶溶液,取十几粒树脂,用乙醇洗三次,加入茚三酮,吡啶,苯酚各一滴,105℃-110℃加热5min,变深蓝色为阳性反应,可以继续接下一个氨基酸,如果不变色则为阴性,需要重新脱保护;依次用15毫升DMF,15毫升甲醇,15毫升DMF分别清洗两次;加入3倍树脂摩尔量的Fmoc-Ile-OH(异亮氨酸),3倍树脂摩尔量的HBTU,均用少量的DMF溶解,立刻加入10倍树脂摩尔量的DIEA,反应30min进行缩合;再次清洗,依次用15毫升DMF,15毫升甲醇,15毫升DMF分别清洗两次。按照以上方法重复操作,依次接完剩余的氨基酸完成肽链延伸。
合成后进行HPLC纯化:将粗品肽放入器皿中,用30-50ml浓度为50%的乙腈水溶液完全溶解,可以稍微超声2min;用0.45μm滤膜过滤溶解液;取3μl溶液用分析级HPLC分析粗品以便于后续制备。流动相是水和乙腈,时间30min,梯度洗脱,先将HPLC用起始梯度平衡5min然后进样,起始梯度为:水95%,乙腈5%,结束梯度为:水5%,乙腈95%;将溶解好的样品做进样准备。制备HPLC平衡10min,起始梯度为:水95%,乙腈5%,结束梯度为:水25%,乙腈75%,梯度时间40min。收集从检测器出来的样品。
合成的多肽经SHIMADZU高效液相色谱仪纯化,纯度达到99%以上,并采用液相色谱-质谱/质谱(LC-MS)进行定性分析,测定其氨基酸序列。
合成的多肽的高效液相色谱图和液相色谱-质谱/质谱(LC-MS)图分别如图1a和图1b所示,由图1a和图1b分析表明,合成的多肽的一级氨基酸序列是Trp-Asp-Gln-Trp-Cys-Ile-Trp,即得到目标多肽,合成获得抗Aβ42蛋白聚集的合成多肽。
实施例2
多肽WW-7体外抗Aβ42蛋白聚集的活性实验
1、实验方法
培养基的配制:高糖培养基(DMEM)、胎牛血清(FBS)、L-谷氨酰胺分别按照质量比8.75:1:0.25配制;同时加入1wt%的双抗(青霉素和链霉素)、0.1wt%的Hygromycin B和0.05wt%的Blasticidin S抗生素。
0.05mM和0.5mM的多肽(WW-7)溶液配制:称取10.4mg的多肽WW-7,用10mL的培养基溶解,过0.22μm的滤头后,母液浓度为1mM,再用培养基将母液稀释至实验所需浓度。
1mg/mL四环素溶液配制:称取20mg的四环素,用10mL的PBS缓冲液配制,过0.22μm的滤头后,-20℃避光保存备用。
细胞实验采用mCherry HEK-293细胞和E22G-Aβ42--mCherry HEK-293细胞进行培养和实验。实验分组:阴性对照组(Control group,mCherry HEK-293细胞,不表达Aβ42蛋白);模型组(Model group,E22G-Aβ42--mCherry HEK-293细胞,表达Aβ42蛋白);WW-7低剂量组(0.05mM的WW-7肽溶液,E22G-Aβ42-mCherry HEK-293细胞,表达Aβ42蛋白)和PW-5高剂量组(0.5mM的WW-7肽溶液,E22G-Aβ42-mCherry HEK-293细胞,表达Aβ42蛋白),每组设三个平行。
用24孔板进行细胞铺板,每孔的细胞个数是5000,贴壁24h后,按照实验分组分别加入培养基和多肽溶液。培养48h后,加入四环素(终浓度为20μg/mL)进行诱导,采用IncuCyte Zoom长时间活细胞成像仪进行实时跟踪拍照,观察四环素加入后,细胞中蛋白聚集情况变化,该过程持续72h后结束。仪器进行白光和荧光的双重拍照,拍照放大倍数为200倍,每间隔4h进行一次拍照,每孔拍9个视野,观察红色荧光聚集点个数,计算蛋白聚集率。
Aβ42蛋白聚集率=视野中红色荧光点个数/细胞面积占比百分数。
2、实验结果
阴性对照组(Control group)是不表达目的蛋白Aβ42表达的细胞,模型组(Modelgroup)则是表达目的蛋白Aβ42的细胞,阴性对照组(Control group)和模型组(Modelgroup)的IncuCyte Zoom长时间活细胞成像图分别如图2a和图2b所示,多肽WW-7浓度为0.05mM的多肽低剂量组和0.5mM的多肽高剂量组的IncuCyte Zoom长时间活细胞成像图分别如图3a和图3b所示,而阴性对照组、模型组、0.05mM的多肽低剂量组和0.5mM的多肽高剂量组的Aβ42蛋白聚集率柱形图如图4所示,图中*代表p<0.05,**代表p<0.01;;
由图2a和图2b可知,阴性对照组没有红色荧光聚集点存在,而模型组细胞中Aβ42蛋白表达的红色荧光聚集点较阴性对照组显著增加;由图3a和图3b可知,多肽WW-7的低剂量组(0.05mM)和高剂量组(0.5mM)与模型组相比,细胞中的Aβ42蛋白红色荧光聚集点明显减少;而由图4可知,多肽WW-7的低剂量组(0.05mM)和高剂量组(0.5mM)与模型组相比,蛋白聚集率显著降低,而且高剂量组的效果较模型组变化更显著,呈一定浓度依赖性。
上述结果表明,多肽WW-7具有显著的抗Aβ42蛋白聚集功效,具有改善记忆、抑制阿尔兹海默症发展的作用,且呈一定浓度依赖性,可应用于制备抗Aβ42蛋白聚集的药物或食品,或者应用于制备预防或治疗阿尔兹海默症的药物或食品,进而能对包括AD疾病的神经退行疾病进行有效预防和治疗,改善神经退行疾病医疗状况。
以上实施例仅为本发明较优的实施方式,仅用于解释本发明,而非限制本发明,本领域技术人员在未脱离本发明精神实质与原理下所作的任何改变、替换、组合、简化、修饰等,均应为等效的置换方式,均应包含在本发明的保护范围内。
序列表
<110> 华南理工大学
<120> 一种抗Aβ蛋白聚集的七肽及其应用与编码该合成多肽的基因
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213> 七肽(Heptapeptide)
<400> 1
Trp Asp Gln Trp Cys Ile Trp
1 5
<210> 2
<211> 21
<212> DNA/RNA
<213> 七肽(Heptapeptide)
<400> 2
tgggaccaat ggtgcatttg g 21
Claims (4)
1.一种抗Aβ蛋白聚集的七肽,其特征在于,名称为WW-7,氨基酸序列为Trp-Asp-Gln-Trp-Cys-Ile-Trp,如序列表 SEQ ID No:1所示;
其中,Trp为色氨酸的氨基酸相应残基,Asp为天冬氨酸的氨基酸相应残基,Gln为谷氨酰胺的氨基酸相应残基,Cys为半胱氨酸的氨基酸相应残基,Ile为异亮氨酸的氨基酸相应残基。
2.一种编码权利要求1所述的抗Aβ蛋白聚集的七肽的基因,其特征在于,碱基序列为TGGGACCAATGGTGCATTTGG,如序列表 SEQ ID No:2所示,基因长度为21个碱基。
3.权利要求1所述的一种抗Aβ蛋白聚集的七肽的应用,其特征在于,应用于制备抗Aβ42蛋白聚集的药物,或者应用于制备预防或治疗阿尔兹海默症的药物。
4.根据权利要求3所述的应用,其特征在于,所述药物的剂型包括膏剂、颗粒剂、丸剂、散剂、片剂、胶囊剂、口服液或糖浆剂。
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