CN111499717B - 一种脑源肽及其应用 - Google Patents
一种脑源肽及其应用 Download PDFInfo
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- CN111499717B CN111499717B CN202010555836.3A CN202010555836A CN111499717B CN 111499717 B CN111499717 B CN 111499717B CN 202010555836 A CN202010555836 A CN 202010555836A CN 111499717 B CN111499717 B CN 111499717B
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种脑源肽HIBDAP,所述脑源肽的氨基酸序列为HSQFIGYPITLFVEKER。本发明还公开了一种融合肽TAT‑HIBDAP,所述融合肽TAT‑HIBDAP包括穿膜肽和所述的脑源肽HIBDAP。本发明还公开了所述的脑源肽HIBDAP或所述的融合肽TAT‑HIBDAP在制备预防或治疗新生儿缺氧缺血性脑损伤药物中的应用。本发明的多肽及其多肽类药物具有高选择性、高效性、耐受性好的优点,而相较于蛋白质药物,其生产更简单、成本更低。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种脑源肽及其应用。
背景技术
新生儿缺氧缺血性脑损伤(Hypoxic ischemic brain damage,HIBD)是新生儿死亡最常见的原因之一,可能留有永久性脑损害如脑瘫、癫痫、智力低下等后遗症。然而,迄今为止,除了亚低温治疗外,新生儿HIBD仍缺乏特效的治疗方法,并不能完全阻止神经系统后遗症的发生。因此,寻找有效干预新生儿HIBD的方法迫在眉睫。
近年来,多肽类药物因其诸多优点得到了广泛的关注,已广泛运用在代谢性疾病、肿瘤、心血管系统疾病、神经系统疾病的治疗中。
天然多肽具有化学和物理稳定性差、半衰期短的缺点,随着一系列新技术的发展,如通过替换特定氨基酸以减少多肽的降解、与白蛋白结合延长多肽的半衰期、通过化学修饰优化多肽的药代动力学等,可以克服天然多肽的内在缺陷,使得多肽类药物更易在临床中大范围推广。迄今,已有60多种多肽类药物投入市场用于病人,150多种多肽处于临床研发阶段,多肽类药物作为一种新的治疗途径有着巨大的潜力。
发明内容
发明目的:本发明通过多肽组学筛选技术发现一条来源于热休克蛋白90α(Hsp90α)210~226位氨基酸的多肽在HIBD患儿脑脊液中显著下调。在缺氧条件下,HSP90α与缺氧诱导因子1α(HIF-1α)结合激活HIF-1α而发挥重要作用。通过KEGG分析发现,Hsp90α参与调节NOD样受体(NLRs)信号通路中的NLRP3/Caspase-1轴,而该轴是激活细胞焦亡的经典途径之一。本发明通过研究发现,该条多肽HIBDAP与细胞穿膜肽合成的融合肽能在缺氧条件下通过抑制NLRP3/Caspase-1轴降低神经细胞焦亡,从而发挥神经保护作用。
本发明所要解决的技术问题是提供了一种新型脑源肽,该条肽的氨基酸序列:HSQFIGYPITLFVEKER,发明人将之命名为HIBDAP(Hypoxic ischemic brain damageassociated peptide)。考虑该条肽呈水溶性,且大于6个氨基酸的多肽一般不能穿过血脑屏障,本发明还要解决的技术问题是提供了一种融合肽,包括了穿膜肽(TAT)和HIBDAP,该融合肽的氨基酸序列为RKKRRQRRRAHSQFIGYPITLFVEKER,并证明该融合肽能穿透血脑屏障并穿透细胞膜进入神经细胞。
本发明还要解决的技术问题是提供了上述的融合多肽能降低焦亡通路中NLRP3、ASC、Caspase-1的表达,从而抑制神经细胞焦亡,为新生儿HIBD的治疗提供新的治疗方法。
本发明最后要解决的技术问题是提供了所述融合肽TAT-HIBDAP在制备预防或治疗新生儿缺氧缺血性脑损伤药物中的应用。
为解决上述技术问题,本发明采用的技术方案如下:本发明提供了一种脑源肽HIBDAP,所述脑源肽的氨基酸序列为HSQFIGYPITLFVEKER。
本发明内容还包括一种融合肽TAT-HIBDAP,所述融合肽TAT-HIBDAP包括穿膜肽和所述的脑源肽HIBDAP。
其中,所述融合肽TAT-HIBDAP的氨基酸序列为RKKRRQRRRAHSQFIGYPITLFVEKER。
发明的多肽或融合肽可以委托公司合成,也可以自行合成。
本发明内容还包括所述的融合肽TAT-HIBDAP在制备预防或治疗新生儿缺氧缺血性脑损伤药物中的应用。
其中,所述应用通过检测融合肽TAT-HIBDAP对NLRP3、ASC和/或Caspase-1表达情况的影响。
其中,所述检测方法采用实时定量PCR和/或免疫印迹杂交方法。
其中,所述实时定量PCR中所采用的引物对序列分别为:
NLRP3:
F:5’-TGA AGA GTG TGA TCT GCG GAA AC-3’;
R:5’-GAA AGT CAT GTG GCT GAA GCT GT-3’;
ASC
F:5’-AGT TGA TGG TTT GCT GGA TGC T-3’;
R:5’-GGT CTG TCA CCA AGT AGG GCT G-3’;
Caspase-1
F:5’-AAC CTT GGG CTT GTC TTT-3’;
R:5’-CAG GAG GGA ATA TGT GGG-3’;
GAPDH
F:5’-AGA AGG CTG GGG CTC ATT TG-3’;
R:5’-AGG GGC CAT CCA CAG TCT TC-3’。
其中,所述免疫印迹杂交方法中所用到的抗体分别为NLRP3、ASC、Caspase-1或β-actin。
本发明内容还包括一种多肽类药物,所述多肽类药物包括所述的脑源肽HIBDAP或所述的融合肽TAT-HIBDAP。
其中,所述多肽类药物的剂型为为静脉注射剂、栓剂、灌肠剂、凝胶剂、泡沫剂、肠溶片、汤剂、合剂、糖浆剂、颗粒剂、丸剂、片剂、胶囊剂、冻干粉中的一种或几种。
有益效果:相对于现有技术,本发明具备以下优点:
与小分子药物相比,本发明多肽及其多肽类药物具有高选择性、高效性、耐受性好的优点,而相较于蛋白质药物,其生产更简单、成本更低。本发明通过实验证实,融合肽TAT-HIBDAP能够进入细胞,在缺氧缺血条件下增加细胞存活率、降低焦亡率,有脑保护作用。
附图说明
图1、高效液相色谱检测TAT-HIBDAP融合肽;
图2、多肽入胞图:荧光显微镜显示TAT-HIBDAP能够进入细胞膜;
图3、CCK8和流式图显示:TAT-HIBDAP能在缺氧缺血条件下增加细胞存活率、降低焦亡率,图中OGD代表的是氧糖剥夺组;
图4、PCR及Western blot结果显示在体外实验中,TAT-HIBDAP能在缺氧缺血条件下抑制NLRP3、ASC、Caspase-1的表达;NC:正常组;OGD:氧糖剥夺组;5μM+OGD:浓度为5μM多肽TAT-HIBDAP处理后氧糖剥夺;
图5、新生SD大鼠缺氧缺血后腹腔注射TAT-HIBDAP,提取缺氧缺血侧大脑皮层,PCR结果显示TAT-HIBDAP能在缺氧缺血条件下抑制NLRP3、ASC、Caspase-1的表达;NC:正常组;HI:缺氧缺血处理组;HI+NS:缺氧缺血+生理盐水注射组;HI+2859nmol/kg:缺氧缺血及浓度为2859nmol/kg的TAT-HIBDAP多肽处理组。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1获得多肽HIBDAP
1、样本选取南京医科大学附属儿童医院新生儿重症监护病房(NICU)4例HIBD患儿。对照组的脑脊液样本取自4例没有已知神经系统疾病的新生儿,他们需要进行腰椎穿刺诊断以进行常规脓毒症评估。加入蛋白酶抑制剂(Complete mini EDTA-free,Med ChemExpress,USA)后储存于液氮中;
2、将样品在液氮中研磨之后加入蛋白裂解液(8M Urea),吹打混匀,再加入终浓度1mM PMSF,2mM EDTA,10mM DTT,混匀之后冰上超声10min;4℃、12000r/min离心30min,取上清至新的离心管,Bradford法测定蛋白浓度,加入DTT至终浓度10mM,56℃水浴30min进行还原反应。随后,加入IAM至终浓度55mM,暗处室温放置30min进行烷基化反应。还原烷之后再进行Bradford法测定蛋白浓度,取等量蛋白(128μg)用10kD超滤管4℃、12000r/min离心30min进行超滤,收集穿透液,即为多肽样品;
3、将多肽样品用C18柱脱盐,真空冷冻干燥脱盐后的肽段;
4、用0.5M TEAB溶解所有多肽肽段,根据iTRAQ-8标试剂盒(AB SCIEX Inc.,Framingham,MA,USA)说明书进行标记,样本标记后混合,然后将混合后的肽段运用Ultimate 3000HPLC系统对肽段样品进行分级分离;色谱柱使用的是Durashell C18柱(5μm,4.6×250mm)。洗脱过程两个流动相是缓冲液A(10mM甲酸胺(PH=9.0),2%乙腈水溶液)和缓冲液B(10mM甲酸胺(PH=10.0)乙腈溶液),洗脱时间及缓冲液A、B比例(见表1),样品经分离柱穿流洗脱后,根据时间和峰型收集42份,每组样品再根据色谱图合并成12份,合并后的组分在Strata-X柱上脱盐并真空干燥。
表1液相方法
5、LC-MS/MS将真空干燥后的多肽样品溶解于2%乙腈/0.1%甲酸中得到多肽溶液,并使用与Eksigent nanoLC系统(SCIEX,USA)偶联的TripleTOF 5600plus质谱仪进行分析;将多肽溶液加到C18捕获柱(5μm,100μm×20mm)上,并以90min时间梯度,300nL/min的流速在C18分析柱(3μm,75μm×150mm)上进行梯度洗脱;两个流动相是缓冲液A(2%乙腈/0.1%甲酸/98%H2O)和缓冲液B(98%乙腈/0.1%甲酸/2%H2O);对于IDA(信息依赖性采集),以250ms的离子累积时间进行一级质谱图扫描,并以50ms的离子累积时间采集30个前体离子的二级质谱图;在350-1500m/z的范围内采集MS1光谱,并且在100-1500m/z的范围内采集MS2光谱,将前体离子动态排除时间设置为15s;
6、Proteinpilot软件鉴定蛋白及肽段;即可得到HIBDAP肽段,该HIBDAP肽段的氨基酸序列为HSQFIGYPITLFVEKER。该HIBDAP多肽可以根据氨基酸序列委托公司合成。
7、通过以上LC-MS/MS实验发现HIBDAP在HIBD患儿中显著下调,差异倍数为-3.1,其分子量为2671.5,具有稳定性强、半衰期长等特点(见表2),其前体蛋白参与NLR信号通路中NLRP3/Caspase-1轴的调节,这条多肽可能在HIBD中发挥了重要作用。
表2
实施例2构建融合肽TAT-HIBDAP
1、根据目标多肽的重量计算每种原料的重量;200mg成品预计需要4g的粗品;
2、将3gRINK树脂(天津南开大学树脂有限公司)放入150ml反应器中,并加入50mlDCM浸泡2小时;
3、用DMF洗涤树脂,然后抽干,如此重复4次,将RINK树脂抽干;
4、称取1mmol Fmoc-Arg(Pbf)-OH(RKKRRQRRRAHSQFIGYPITLFVEKER的C端第一个氨基酸)+20ml DCM+10ml DIEA加入到反应器中,然后将反应器置于30℃的摇床中反应2小时;
5、用甲醇溶液封闭(甲醇:DIEA:DCM=1:1:2)半小时,然后用DMF洗涤4次,抽干;
6、向反应器中加入50ml体积百分比20%哌啶溶液,脱去Fmoc保护基团,脱完保护后用DMF洗涤4次,然后抽干;
7、取少量RINK树脂用茚三酮法检测,RINK树脂有颜色,说明脱保护成功;
8、称取2mmol Fmoc-Glu(OtBu)-OH(RKKRRQRRRAHSQFIGYPITLFVEKER的C端第二个氨基酸)+40ml HOBT+20ml DIC加入到反应器中,然后将反应器置于30℃的摇床中反应1小时;
9、取少量树脂检测,用茚三酮法检测,若树脂有颜色,说明缩合不完全,继续反应;若树脂为无色,说明反应完全;待反应完全后,用DMF洗涤树脂4次,然后抽干;
10、向反应器中加入50ml体积百分比20%的哌啶溶液(哌啶/DMF=1:4),放在脱色摇床上摇晃20min,以此来脱去树脂上的Fmoc保护基团;脱完保护后用DMF洗涤4次,然后抽干检测保护是否脱去;
11、取少量树脂用茚三酮法检测,树脂有颜色,说明脱保护成功;
12、按照步骤8-11依次连接RKKRRQRRRAHSQFIGYPITLFVEKER的剩余氨基酸及乙酸;
13、用高浓度三氟乙酸将多肽保护基团全部切除,并从树脂上切割下来,送纯化;
14、通过高效液相色谱仪器(HPLC)将目标肽段与杂质分离,将目标肽段TAT-HIBDAP冻干成粉末,并送QC质检,检测HPLC和MS结果均符合要求。得到该融合肽TAT-HIBDAP的氨基酸序列为RKKRRQRRRAHSQFIGYPITLFVEKER。
从表3中的Area%可以得出本实施例的融合肽纯度大于95%。
表3
Peak Table
Detector A 214nm
Peak# | Ret.Time | Area | Height | Area% |
1 | 18.529 | 202070 | 19647 | 2.108 |
2 | 18.688 | 9133662 | 1063880 | 95.294 |
3 | 18.888 | 126833 | 24923 | 1.323 |
4 | 19.329 | 88757 | 8371 | 0.926 |
5 | 20.496 | 33371 | 5492 | 0.348 |
Total | 9584692 | 1122313 | 100.000 |
实施例3 TAT-HIBDAP在体外实验中的应用
1、将合成的TAT-HIBDAP和FITC标记的TAT-HIBDAP分别按浓度如1μM、5μM、10μM、20μM、40μM溶于无菌水中,将10μM FITC标记的TAT-HIBDAP加入PC12细胞(ATCC,Manassas,VA,USA)中30min,在荧光显微镜下观察多肽的入胞情况,结果显示多肽不仅能进入细胞膜,还能进入细胞核(图2);
2、PC12细胞用含或不含10μM TAT-HIBDAP的无糖培养基(无糖RPMI 1640培养基)处理1h后,置于2%02、5%CO2和93%N2的37℃环境中6h;
3、接种PC12细胞于96孔板,当细胞密度90%时,加入不同浓度(0、1μM、5μM、10μM、20μM、40μM)的多肽TAT-HIBDAP后,氧糖剥夺6h,避光条件下于每孔中加入10μL的CCK8溶液,后置于培养箱中孵育1h,采用酶标仪检测OD值,结果显示低浓度融合肽TAT-HIBDAP在缺氧缺血条件下能增强细胞活性(图3);
4、将步骤2处理的细胞用不含EDTA的胰酶消化,收集于流式管中,1500rpm离心5min后弃上清,1ml PBS清洗2次,轻柔吹打细胞后1500rpm离心5min,先后加入300μLBinding Buffer、2.5μL FITC和PI悬浮于灭菌的PBS缓冲液中,调节BD流式细胞仪参数,细胞计数为10000个,计算细胞平均荧光强度,结果显示融合肽TAT-HIBDAP能降低细胞焦亡率(图3);
5、采用实时定量PCR及免疫印迹杂交(Western Blot)方法检测NLRP3、ASC及Caspase-1的表达情况,序列为:NLRP3(F:5’-TGAAGAGTG TGATCT GCG GAAAC-3’;R:5’-GAAAGT CAT GTG GCT GAA GCT GT-3’);ASC(F:5’-AGT TGA TGG TTT GCT GGATGCT-3’;R:5’-GGT CTG TCACCAAGTAGG GCT G-3’);Caspase-1(F:5’-AAC CTT GGG CTT GTC TTT-3’;R:5’-CAG GAG GGA ATA TGT GGG-3’);GAPDH(F:5’-AGAAGG CTG GGG CTCATT TG-3’;R:5’-AGG GGC CAT CCACAG TCT TC-3’);抗体:NLRP3(Cat:19771-1-AP;Proteintech,Chicago,USA);ASC(Cat:sc-514414;Santa Cruz,CA,USA);Caspase-1(Cat:sc-514;Santa Cruz,CA,USA)、β-actin(Cat:8457S;Cell Signaling Technology,MA,USA);
实时定量PCR反应体系和反应条件:
逆转录:
本实验通过Hiscript II Q RT SuperMix for qPCR(R222-01)进行逆转录。
按以下体系配置逆转录试剂:(10μL/孔)
涡旋混匀后离心,分别加入八联排管中,开始反应。
反应结束后,可将得到的cDNA样本置于冰上,或-20℃保存。
2.6.2实时定量PCR反应体系
通过AceQ qPCR SYBER Green Master Mix(R131-03,Vazyme,Nanjing,China)进行PCR。取GAPDH为内参。
按以下体系配置PCR试剂:(10μL/孔)
涡旋混匀后离心,将样本分别加入96孔板中,离心后开始反应。
Western blot步骤:加入蛋白裂解液提取蛋白,使用BCA试剂盒(Pierce,Rockford,IL,USA)测定蛋白浓度,加入上样缓冲液(Abcam,Cambridge,MA,UK),在100℃煮5min。样品在10%SDS-PAGE上分离,转移PVDF膜上(Millipore,MA,USA),用脱脂奶粉在室温下封闭,之后孵育一抗NLRP3(Cat:19771-1-AP;Proteintech,Chicago,USA)、ASC(Cat:sc-514414;Santa Cruz,CA,USA),Caspase-1(Cat:sc-514;Santa Cruz,CA,USA)、β-actin(Cat:8457S;Cell Signaling Technology,MA,USA)在4℃过夜。用含有0.1%Tween 20的TBST洗涤5次,每次10分钟,然后在室温下用入耦联辣根过氧化物酶的二抗处理1h,洗涤5次,每次10分钟。最后用显影仪进行测定,用图像分析系统(image J)定量。
PCR和Western blot结果显示氧糖剥夺时,NLRP3、ASC和Caspase-1表达均是上升的,加入TAT-HIBDAP预处理时,其表达是下降的,可以看出TAT-HIBDAP能在缺氧缺血条件下抑制焦亡通路中NLRP3、ASC和Caspase-1的表达(图4),具有潜在的脑保护作用。
实施例4动物实验
1、构建新生大鼠缺血缺氧(HI)模型
取7日龄SD清洁级大鼠,雌雄对半,体重12-18g,假手术组大鼠5%水合氯醛(1mL/100g)麻醉后仅切开颈部皮肤和肌肉,暴露左颈总动脉,但不结扎动脉,不做缺氧处理,HI模型组大鼠水合氯醛麻醉后,分离出左颈总动脉并永久结扎,再将大鼠放入含8%氧气和92%氮气的低氧箱2h,温度和湿度分别保持在37℃和50-80%,之后与母鼠合笼,HI且腹腔注射盐水的实验组大鼠处理同上,与母鼠合笼前腹腔注射生理盐水0.005ml/g,HI且腹腔注射多肽的实验组大鼠处理同上,与母鼠合笼前腹腔注射浓度为2359nmol/kg的TAT-HIBDAP多肽溶液0.005ml/g。于术后48h断头处死新生大鼠。每组留取6-8只取结扎侧脑组织,分离皮层置于液氮中保存备用。
2、采用实时定量PCR方法检测皮层中NLRP3、ASC及Caspase-1的表达情况。
PCR结果显示,HI处理后,NLRP3、ASC及Caspase-1显著上调,HI后加入TAT-HIBDAP处理时,NLRP3、ASC及Caspase-1表达下降,可以看出TAT-HIBDAP能在HI后抑制焦亡通路中NLRP3、ASC和Caspase-1的表达(图5),具有脑保护作用。
序列表
<110> 南京市儿童医院
<120> 一种脑源肽及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> PRT
<213> 脑源肽HIBDAP(Artificial Sequence)
<400> 1
His Ser Gln Phe Ile Gly Tyr Pro Ile Thr Leu Phe Val Glu Lys Glu
1 5 10 15
Arg
<210> 2
<211> 27
<212> PRT
<213> TAT-HIBDAP(Artificial Sequence)
<400> 2
Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala His Ser Gln Phe Ile Gly
1 5 10 15
Tyr Pro Ile Thr Leu Phe Val Glu Lys Glu Arg
20 25
<210> 3
<211> 23
<212> DNA
<213> NLRP3 F(Artificial Sequence)
<400> 3
tgaagagtgt gatctgcgga aac 23
<210> 4
<211> 23
<212> DNA
<213> NLRP3 R(Artificial Sequence)
<400> 4
gaaagtcatg tggctgaagc tgt 23
<210> 5
<211> 22
<212> DNA
<213> ASC F(Artificial Sequence)
<400> 5
ggtctgtcac caagtagggc tg 22
<210> 6
<211> 22
<212> DNA
<213> ASC R(Artificial Sequence)
<400> 6
ggtctgtcac caagtagggc tg 22
<210> 7
<211> 18
<212> DNA
<213> Caspase-1 F(Artificial Sequence)
<400> 7
aaccttgggc ttgtcttt 18
<210> 8
<211> 18
<212> DNA
<213> Caspase-1 R(Artificial Sequence)
<400> 8
caggagggaa tatgtggg 18
<210> 9
<211> 20
<212> DNA
<213> GAPDH F(Artificial Sequence)
<400> 9
agaaggctgg ggctcatttg 20
<210> 10
<211> 20
<212> DNA
<213> GAPDH R(Artificial Sequence)
<400> 10
aggggccatc cacagtcttc 20
Claims (4)
1.一种脑源肽HIBDAP,其特征在于,所述脑源肽的氨基酸序列为HSQFIGYPITLFVEKER。
2.一种融合肽TAT-HIBDAP,其特征在于,所述融合肽TAT-HIBDAP由穿膜肽和权利要求1所述的脑源肽HIBDAP组成,所述融合肽TAT-HIBDAP的氨基酸序列为RKKRRQRRRAHSQFIGYPITLFVEKER。
3.一种多肽类药物,其特征在于,所述多肽类药物包括权利要求1所述的脑源肽HIBDAP或权利要求2所述的融合肽TAT-HIBDAP。
4.根据权利要求3所述的多肽类药物,其特征在于,所述多肽类药物的剂型为静脉注射剂、栓剂、灌肠剂、凝胶剂、泡沫剂、汤剂、合剂、糖浆剂、颗粒剂、丸剂、片剂、胶囊剂、冻干粉中的一种或几种。
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