CN115043927B - 针对抗α-突触核蛋白K80Hcy抗体的抗原肽及其应用 - Google Patents
针对抗α-突触核蛋白K80Hcy抗体的抗原肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种针对抗α‑突触核蛋白K80Hcy抗体的抗原肽及其应用，属于生物技术领域。本发明发现同型半胱氨酸硫内酯(HTL)可以修饰α‑突触核蛋白(α‑synuclein)80位的赖氨酸残基，PD病人脑中有更多的α‑synuclein K80Hcy。以本发明的抗原肽AC‑TAVAQK(Hcy)TVEG‑COOH为抗原制备得到抗体能够特异性识别α‑synuclein K80Hcy，而不识别没有修饰的α‑synuclein，其可以用于检测α‑synuclein K80Hcy程度、用于PD发病机制的研究及PD的早期诊断。
Description
技术领域
本发明属于生物技术领域，涉及一种针对抗α-突触核蛋白K80Hcy抗体的抗原肽及其应用。
背景技术
帕金森氏病(Parkinson’s disease,PD)是一种慢性、进行性神经退行性疾病，它是世界上第二大常见神经退行性疾病1,2。60岁以上人群的PD患病率约为1％，其发病率随着年龄的增长而升高。随着人口老龄化，到2030年，全球PD患病人数预计将达到8.7-9.3百万3。迄今为止，PD发病的原因尚不清楚，因此也缺乏针对发病机制、延缓病程的治疗措施。
流行病学证据表明，人体内过多的同型半胱氨酸(Hcy)与帕金森病风险的增加有关4-7。然而，其分子机制尚不清楚。有研究发现，同型半胱氨酸硫内酯(HTL)8，一种同型半胱氨酸的高反应型硫酯，可以共价修饰蛋白质的赖氨酸残基。
参考文献：
1 Bloem,B.R.,Okun,M.S.&Klein,C.Parkinson's disease.Lancet 397,2284-2303,
doi:10.1016/S0140-6736(21)00218-X(2021).
2 Tolosa,E.,Garrido,A.,Scholz,S.W.&Poewe,W.Challenges in thediagnosis of Parkinson's disease.Lancet Neurol 20,385-397,doi:10.1016/S1474-4422(21)00030-2(2021).
3 Tysnes,O.B.&Storstein,A.Epidemiology of Parkinson's disease.JNeural Transm(Vienna)124,901-905,doi:10.1007/s00702-017-1686-y(2017).
4 Mattson,M.P.,Kruman,II&Duan,W.Folic acid and homocysteine in age-related disease.Ageing Res Rev 1,95-111,doi:10.1016/s0047-6374(01)00365-7(2002).
5 Licking,N.et al.Homocysteine and cognitive function in Parkinson'sdisease.Parkinsonism Relat Disord 44,1-5,doi:10.1016/j.parkreldis.2017.08.005(2017).
6 Jimenez-Jimenez,F.J.,Alonso-Navarro,H.,Garcia-Martin,E.&Agundez,J.A.G.Cerebrospinal and blood levels of amino acids as potential biomarkersfor Parkinson's disease:review and meta-analysis.Eur J Neurol 27,2336-2347,doi:10.1111/ene.14470(2020).
7 Boelens Keun,J.T.,Arnoldussen,I.A.,Vriend,C.&van de Rest,O.DietaryApproaches to Improve Efficacy and Control Side Effects of Levodopa Therapyin Parkinson's Disease:A Systematic Review.Adv Nutr 12,2265-2287,doi:10.1093/advances/nmab060(2021).
8 Jakubowski,H.Quality control in tRNA charging--editing ofhomocysteine.Acta Biochim Pol 58,149-163(2011).
发明内容
本发明发现同型半胱氨酸硫内酯(HTL)可以修饰α-突触核蛋白(α-synuclein)80位的赖氨酸残基，将其命名为α-synuclein K80Hcy(α-synuclein蛋白第80位赖氨酸发生同型半胱氨酸化修饰)。本发明还发现A53T转基因小鼠脑组织中α-synuclein K80Hcy水平呈年龄依赖性增加，并且PD病人脑中有更多的α-synuclein K80Hcy。检测实验动物标本及人体液中的α-synuclein K80Hcy对PD的诊断与发病机制的研究具有重要的作用。
本发明的目的在于针对现有技术无法特异性检测α-synuclein K80Hcy的问题，提供一种针对α-synuclein K80Hcy的抗原肽及其抗体，用于PD的发病机制研究和疾病早期诊断。
本发明的目的通过以下技术方案实现：
一种针对抗α-synuclein K80Hcy抗体的抗原肽，其氨基酸序列为：AC-TAVAQK(Hcy)TVEG-COOH(SEQ ID NO.1)，其中K(Hcy)表示同型半胱氨酸化修饰的赖氨酸。
上述抗原肽在制备抗α-synuclein K80Hcy抗体中的应用。
一种抗α-synuclein K80Hcy的抗体，以上述抗原肽为抗原制备得到。进一步地，所述的抗体以上述抗原肽和载体蛋白交联后得到的交联多肽为抗原制备得到，所述的载体蛋白优选为KLH。所述的抗体为兔多克隆抗体，其制备方法包括以下步骤：以上述抗原肽与载体蛋白KLH交联得到的物质为免疫原免疫家兔，收集家兔血液制备抗血清。
本发明的抗原肽具有很好的免疫原性，以其为抗原制备的α-synuclein K80Hcy多克隆抗体特异性好，效价高，其可以用于检测α-synuclein K80Hcy程度，用于PD发病机制的研究及PD的早期诊断，用于制备检测α-synuclein K80Hcy程度的试剂，以及用于制备PD早期诊断的试剂。
一种检测α-synuclein K80Hcy程度的试剂，包含上述抗体。
一种PD早期诊断试剂，包含上述抗体。
本发明相对于现有技术具有如下的优点及效果：目前尚无特异性识别α-synuclein K80Hcy的抗体，本发明的特异性抗α-synuclein K80Hcy的抗体解决了这一问题。
附图说明
图1为酶联免疫吸附实验检测抗体的滴度。
图2为质谱鉴定到α-synuclein K80Hcy。
图3为HTL修饰α-synuclein形成α-synuclein K80Hcy，抗α-synuclein K80Hcy抗体能够识别α-synuclein K80Hcy，而不识别没有修饰的α-synuclein。
图4为使用抗α-synuclein K80Hcy抗体进行免疫组织化学实验，检测到PD病人脑内的α-synuclein K80Hcy。
具体实施方式
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
实施例1兔多克隆抗体(抗α-synuclein K80Hcy抗体)的制备
本实施例所用抗原肽(AC-TAVAQK(Hcy)TVEG-COOH)由人工化学合成得到(上海艾比玛特生物医药有限公司)。合成的抗原肽进行质谱鉴定，鉴定正确后利用HPLC进行纯化和纯度鉴定。
将上述抗原肽偶联到载体蛋白KLH上得到抗原肽-KLH偶联物，以其作为免疫原免疫新西兰兔制备多克隆抗体。具体步骤如下：
(1)实验前采集动物血液，并制备免疫前血清。采集的血液离心后收集上清即得到血清。
(2)初次免疫：每只兔子皮下多点注射乳化的1mL抗原肽-KLH偶联物和弗氏完全佐剂的混合液(体积比1:1)，其中，抗原肽含量为0.5mg。
(3)初次免疫一周后，按照初次免疫的方法进行第二次加强免疫。
(4)第二次加强免疫一周后，按照初次免疫的方法进行第三次加强免疫。
(5)直到第五次加强免疫一周后，采集血液制备抗血清。
实施例2检测抗α-synuclein K80Hcy抗体滴度
(1)将修饰抗原肽与未修饰抗原肽分别按10ng包被ELISA板，依次倍比稀释递减包被量。修饰抗原肽：AC-TAVAQK(Hcy)TVEG-COOH；未修饰抗原肽：AC-TAVAQKTVEG-COOH。
(2)次日，倒空液体并拍干残留液体，洗涤液每隔5分钟加入，冲洗三次。
(3)每孔加入150μL封闭液，37℃孵育1h。
(4)倒空液体并拍干残留液体，洗涤液冲洗三次。
(5)每孔加入按梯度稀释的抗α-synuclein K80Hcy抗体100μL，37℃孵育1h。
(6)倒空液体并拍干残留液体，洗涤液冲洗三次。
(7)每孔加入按1:10000稀释的二抗，37℃孵育1h。
(8)倒空液体并拍干残留液体，洗涤液冲洗三到四次。
(9)拍干孔中残留液体，每孔加入100μL混合的显色液A、B，37℃避光显色30min。
(10)每孔加入50～100μL 2M H2SO4终止显色，并立即读取450nm OD值，检测结果见图1，抗α-synuclein K80Hcy抗体可以较好的区别修饰抗原肽与未修饰抗原肽。
实施例3抗α-synuclein K80Hcy抗体特异性的细胞学检测
(1)细胞转染：在HEK293细胞内转染表达带GST标签的α-synuclein(GST-α-synuclein，GST-α-syn)质粒。
(2)制样：细胞转染后，向细胞培养基中加入HTL(0.1、0.5、1mM)，处理24h后，收集细胞，PBS洗一遍，加裂解液(50mM柠檬酸钠，5mM DTT，0.1％CHAPS，0.5％TritonX-100，pH＝6.0)冰上裂解30min，取上清至新的EP管中，加入5×SDS loading buffer，混匀，100℃沸水煮10min。
为了鉴定α-synuclein上的修饰位点，选择0.1mM HTL处理的细胞，收集裂解细胞，纯化GST-α-synuclein，跑胶后，进行质谱鉴定，结果见图2，GST-α-synuclein第80位赖氨酸发生同型半胱氨酸化修饰。
(3)跑胶：将制好的样品按照图3的顺序进行点样，80V恒压跑胶20min后，120V恒压跑胶到Marker分开至合适的位置。
(4)转膜：提前配好转膜液，4℃预冷，按照负极-海绵-滤纸-胶-NC膜-滤纸-海绵-正极的顺序将胶和膜用转膜夹固定好，220mA恒流转膜75min。
(5)封闭：转膜完成后，将膜用TTBS溶液洗涤一次后，置于5％奶粉中，放置于摇床上室温封闭1h。
(6)孵育一抗：封闭完成后，孵育一抗，一抗采用3％的奶粉进行稀释(GST抗体(anti-GST)：1:10000，抗α-synuclein K80Hcy抗体(anti-K80Hcy)：1:1000，GAPDH抗体(anti-GAPDH)：1:10000)，4℃摇床孵育过夜。
(7)洗膜：室温采用TTBS溶液洗膜40min，每10min换一次洗膜液。
(8)孵育二抗：采用3％的奶粉将二抗按照1:10000比例进行稀释，室温孵育1h。
(9)洗膜：室温采用TTBS溶液洗膜1h，每10min换一次洗膜液。
(10)显影：使用ECL显影液对膜进行曝光显影。
结果见图3，抗α-synuclein K80Hcy抗体能够识别细胞中α-synuclein K80Hcy，而不识别未修饰的α-synuclein K80Hcy，并且修饰含量随着HTL的浓度增加而增加。
实施例4抗α-synuclein K80Hcy抗体特异性的人脑组织切片检测
(1)脑片：PD患者和非PD对照的脑组织石蜡切片。
(2)脱蜡，水化脑片：将脑组织石蜡切片放入二甲苯中，进行脱蜡，将蜡脱干净后，脑片依次放入95％、85％、75％的无水乙醇中，分别放置5min，然后放入ddH2O中。
(3)抗原修复：将脑片放入100mM柠檬酸钠(pH＝6.0)抗原修复液中，92℃抗原修复20min，待自然冷却后，PBS洗三遍，每次5min。
(4)3％双氧水孵育10min，阻断内源性过氧化物酶，以减少非特异性背景染色，PBS洗三遍，每次5min。
(5)封闭：3％BSA封闭30min。
(6)孵一抗：孵育抗α-synuclein K80Hcy抗体，抗体采用3％BSA溶液按照1:500的比例稀释，4℃孵育过夜。
(7)洗一抗：PBS洗三遍，每次5min。
后面步骤采用中杉金桥公司的免疫组化试剂盒进行。
(8)加入100μL一抗放大剂(试剂盒中的C液)，孵育10min，PBS洗三遍，每次5min。
(9)加入100μL二抗(试剂盒中的D液)，避光，孵育10min，PBS洗三遍，每次5min。
(10)配制新鲜显色液：将试剂盒中的E液和F液按照3:100的比例进行配制，混匀，待用。
(11)显色：在脑片上加入100μL新鲜显色液进行孵育，并在显微镜下观察，脑片被染上色后用去离子水进行冲洗，终止显色反应。
(12)苏木素染核。
(13)透明，将组织片子依次放入75％无水乙醇-85％无水乙醇-95％无水乙醇-二甲苯，分别放置5min。
(14)封片：中性树胶封片。
(15)显微镜观察，拍照。
结果见图4，使用抗α-synuclein K80Hcy抗体可以检测到PD患者脑内α-synucleinK80Hcy。
序列表
<110> 武汉大学
<120> 针对抗α-突触核蛋白K80Hcy抗体的抗原肽及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Thr Ala Val Ala Gln Lys Thr Val Glu Gly
1 5 10
Claims (8)
1. 一种针对抗α-synuclein K80Hcy抗体的抗原肽，其特征在于：其氨基酸序列为：AC-TAVAQK(Hcy)TVEG-COOH，其中K(Hcy)表示同型半胱氨酸化修饰的赖氨酸；所述的α-synuclein K80Hcy表示α-synuclein蛋白第80位赖氨酸发生同型半胱氨酸化修饰。
2. 权利要求1所述的抗原肽在制备抗α-synuclein K80Hcy抗体中的应用，其特征在于：所述的抗α-synuclein K80Hcy抗体为多克隆抗体，其以权利要求1所述的抗原肽和载体蛋白KLH交联后得到的交联多肽为抗原制备得到。
3. 一种抗α-synuclein K80Hcy的抗体，其特征在于：以权利要求1所述的抗原肽和载体蛋白KLH交联后得到的交联多肽为抗原制备得到；所述的抗α-synuclein K80Hcy抗体为多克隆抗体。
4. 根据权利要求3所述的抗α-synuclein K80Hcy的抗体，其特征在于：所述的抗体为兔多克隆抗体，其制备方法包括以下步骤：以权利要求1所述的抗原肽与载体蛋白KLH交联得到的物质为免疫原免疫家兔，收集家兔血液制备抗血清。
5. 权利要求3或4所述的抗体在制备检测α-synuclein K80Hcy程度的试剂中的应用。
6.权利要求3或4所述的抗体在制备PD早期诊断的试剂中的应用。
7. 一种检测α-synuclein K80Hcy程度的试剂，其特征在于：包含权利要求3或4所述的抗体。
8.一种PD早期诊断试剂，其特征在于：包含权利要求3或4所述的抗体。
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