CN113430230A - tau截断体蛋白在诱导tau病理聚集中的应用 - Google Patents
tau截断体蛋白在诱导tau病理聚集中的应用 Download PDFInfo
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Abstract
本发明公开了一种tau截断体蛋白在诱导tau病理聚集中的应用,首先构建了包括全长及11种tau截断体,其中tau151‑391猎获AD O‑tau的能力最强,因而将其在真核细胞中表达,提取蛋白超速离心处理后作为seeds来诱导tau的病理聚集。深入研究AD的发病机制以及针对以tau蛋白为靶标的AD治疗,获得有效的seeds尤为重要,由于获取AD患者死后的脑组织十分困难,无法提取脑组织中的AD O‑tau,因而寻找便捷有效的seeds替代AD O‑tau有重大意义。本发明不仅可以帮助我们构建AD模型,深入研究AD的发病机制、tau蛋白的传播特点,还可以为以tau为靶标的治疗提供AD模型动物。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种tau截断体蛋白在诱导tau病理聚集中的应用。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是多因素相关的疾病,老化是其最主要的病因。AD拥有病理特征:细胞外β-淀粉样蛋白(β-amyloid protein,Aβ)聚集形成的老年斑(senile plaque,SP)的沉积和细胞内大量的tau蛋白聚集形成神经原纤维缠结(neurofibrillary tangles,NFTs)和广泛神经元退变。
Tau蛋白是一种微管结合蛋白,在中枢神经系统的神经元中表达非常丰富,在身体其他细胞和中枢神经系统的星形胶质细胞和少突胶质细胞中表达相对较低。人类tau蛋白的基因位于17q21,含有16个外显子。在中枢神经系统,外显子2,3,10可发生可变剪接,产生六种不同的变异体(图1)。外显子10编码表达第二个微管结合片段,其编码与否决定了tau蛋白是含有3个还是4个微管结合重复序列,也就是我们简称的3R-tau或4R-tau。Tau蛋白对微管稳态有极其重要的作用,所以tau蛋白的可变剪接在发育过程中是被严格调控的。胎儿的脑中只表达3R-tau,正常成人脑中表达3R-tau和4R-tau,比例约为1。Tau蛋白结构上包含氨基端酸性区域、脯氨酸富集区、微管结合重复序列和羧基末端。其主要功能包括两个方面:促进微管的形成和维持微管结构的动态稳定性。
人tau基因(MAPT)位于17q21,含有16个外显子。在中枢神经系统,外显子2,3和10的可变剪接产生6种tau的变异体。黑色的小方块代表组成性外显子,灰色和白色方块代表可变剪接外显子。N1和N2代表有外显子1和1编码N末端氨基酸插入子1和2,PRD代表脯氨酸富集区,R1、R2、R3和R4分别代表4个微管结合重复序列。
Tau的截断是一种重要的翻译后修饰,在tau相关疾病中有重要病理作用。英国剑桥大学的Whischik’s小组在研究PHFs的结构时发现PHFs的核心区域中含有被截断的tau蛋白,tau蛋白病理性聚集与其被异常切割可能有关。Dr.Novak从AD患者脑中分离的tau151-391截断体,在大鼠脑中过表达,出现tau病理。我们最近的研究发现tau在AD患者脑中的截断明显增加,而且,我们观察到高分子量smear的tau基本不被N-末端的tau抗体识别,说明在AD患者脑中tau的N-末端的截断可能与tau的聚集或磷酸化也有重要关系。为了研究tau在N-末端和C-末端截断对tau病理改变的影响,我们目前构建了包括全长和11种tau截断体的真核表达质粒。
在AD患者脑中,tau蛋白在多个位点被Calpain、Caspase、AEP等蛋白酶截断。我们选择了最常见的AD相关截断位点:N-末端51、151、231的截断和C-末端391、421的截断,构建了全长和11种tau截断体的真核表达质粒pCI/tau1-441、pCI/tau1-421、pCI/tau1-391、pCI/tau51-441、pCI/tau51-421、pCI/tau51-391、pCI/tau151-441、pCI/tau151-421、pC/tau151-391、pCI/tau231-441、pCI/tau231-421和pCI/tau231-391(图2),在他们的N-末端都带有HA的标签。
已有研究表明AD的病理改变中tau蛋白的异常聚集传播类似朊样传播,为模拟朊样传播,深入研究AD的发病机制以及针对以tau蛋白为靶标的AD治疗,获得有效的seeds尤为重要,由于获取AD患者死后的脑组织十分困难,因而无法从人脑组织中提取oligomer-tau(AD O-tau)作为seeds深入研究tau蛋白的朊样传播。在我们构建的11种tau截断体中,tau151-391猎获AD O-tau的能力最强,因而我们将其在真核细胞中表达,将获得的蛋白进一步处理后作为seeds来诱导细胞和小鼠产生AD样的tau病理聚集。这一发明,不仅可以帮助我们构建AD模型,深入研究AD的发病机制、tau蛋白的传播特点,还可以为以tau为靶标的治疗提供AD模型动物。
发明内容
为了研究AD发病机制以及筛选有效的抑制或逆转tau病理发生发展及传播的药物,构建有效的病理模型是必要条件,转基因动物成本高,繁殖率低,因此通过真核表达tau截断体蛋白,经过特殊处理后,作为seeds来诱导细胞或动物出现类似AD样的tau病理改变,成为有效的tau病理模型。
本发明采用以下技术方案:
一种tau截断体蛋白在诱导tau病理聚集中的应用,包括以下步骤:
1)真核细胞表达tau全长质粒pCI/neo-tau1-441和截断体质粒pCI/neo-tau151-391,得到真核表达的tau1-441和tau151-391截断体蛋白;
2)真核表达tau1-441和tau151-391截断体蛋白分别诱导表达HA-Tau151-391的HeLa细胞tau聚集;
3)小鼠海马注射真核表达tau1-441和tau151-391截断体蛋白;
4)组织固定与切片;
5)免疫组织化学。
进一步的,所述步骤1)具体包括以下步骤:
11)将冻存的HEK-293FT细胞从液氮中取出,37度水浴速溶,转至培养瓶中,加含有10%的FBS的基础培养基培养细胞;等HEK-293FT细胞长满至80%-90%时,可种至3个6cm的dish中,一共接种16个dish;
第二天开始准备转染质粒,转染试剂为lipo3000,其中8个dish转染pCI/neo-tau151-391,另外8个dish转染pCI/neo-tau1-441;A:250μl OPTI-MEM+5μg DNA+10μllipo3000,B:250μl OPTI-MEM+10μl lipo3000,A+B混匀静置10-15min,每个dish加500μlA+B混合液和4.5ml完全培养基,4-6h后每个dish换5ml培养基,培养基中含有OA20nM,36h后离心收集细胞;
12)分别收集的转染pCI/neo-tau151-391和pCI/neo-tau1-441的细胞,加入9ml浓度为50nM的细胞裂解液,将1ml ddH2O和5μl细胞裂解液混合超声1s停1s,直至细胞裂解液变澄清;13,000g离心5分钟,取上清4.5ml加500μl10%的sarkocyl溶液,室温静置1h,再235,000g,TLA-55,4℃离心,1h,分别收集上清和沉淀,沉淀即为真核细胞表达的作为seeds的蛋白质tau1-441和tau151-391,放-80℃保存。
进一步的,所述步骤2)具体包括以下步骤:
根据Fugene HD说明书,在HeLa细胞中转染pCI/HA-tau151-391质粒,4h后在细胞中加入Lipo3000处理的等量真核表达tau1-441或tau151-391截断体蛋白;
48h后,快速用PBS清洗三次,4%多聚甲醛固定15min,PBS清洗三次,0.3%Tritonin PBS室温处理15min,5%的封闭液封闭30min,细胞与在封闭液中的一抗4℃孵育过夜;PBS清洗三遍,最后滴加封片剂封片,使用激光共聚焦显微镜观察细胞内tau的聚集。
进一步的,所述步骤3)10月龄hTau小鼠腹腔注射2.5%的Avertin麻醉,海马注射真核表达tau1-441或者tau151-391截断体蛋白;注射的坐标如下:前囟向后2.5mm,向左/右旁开2.0mm,自硬膜向腹侧进针1.8mm;注射体积2.5μl,注射速度1.25μl/min,留针3min以防液体外溢。
进一步的,所述步骤4)具体包括以下步骤:
取小鼠,先经腹腔注射2.5%的2-Avertin麻醉剂,麻醉后,开胸暴露心脏,自心尖部将灌注针头经左心室插至升主动脉,灌注生理盐水后,改快速灌注含有4%多聚甲醛的0.1MPBS,待动物全身抽搐后,调慢灌注速度,约50滴/min,持续时间约30min后,取出大脑放入含有4%多聚甲醛的0.1MPBS中后固定4h;再先后转入含20%和30%蔗糖溶液中,待组织块下沉后,行冠状位冰冻连续切片,片厚40μm,漂于0.01M PBS中。
进一步的,所述步骤5)具体包括以下步骤:
51)挑取海马结构清晰的漂片,置于0.01M PBS缓冲液中漂洗3遍后,放入含有10%triton-100液体中20min,0.01M PBS缓冲液中漂洗3遍;放入含有10%山羊血清的封闭液中,在室温下振摇2h;
52)将封闭好的切片转入到1:500稀释的小鼠抗AT8单抗中,室温下轻轻振摇1h后放入4℃冰箱中过夜;
53)次日晨吸去一抗,用PBS清洗3遍后,加入1:500稀释的cy3荧光二抗,hochest(1:500)避光、室温下轻轻振摇2h;
54)0.01M PBS(pH 7.2)避光清洗3次,每次10min;
55)荧光封片液封片后激光共聚焦显微镜下观察。
有益效果
1、将质粒pCI/neo-tau151-391和pCI/neo-tau1-441分别转染到Hela细胞,可以发现tau的自聚集现象,如果在培养基中加入AD O-tau seeds,tau151-391可以大大增加异常tau的聚集。
2、将tau1-441和tau151-391截断体蛋白作为seeds分别加入转染了质粒tau151-391的Hela细胞培养基中,48h后可以检测到Hela细胞中有tau病理聚集,且tau151-391蛋白作为seeds的作用更强,说明截断体蛋白tau151-391可以替代AD O-tau作为seeds。
3、10月龄的hTau小鼠注射tau151-391截断体蛋白10w后,可以检测出小鼠神经元有tau病理聚集,并在脑区发生了朊样传播,证实截断体蛋白可以作为seeds诱导小鼠神经元出现AD样的tau病理改变,构建AD动物模型。
可以通过AD疾病模型的建立,可以深入研究AD发病的分子机制,tau蛋白的病理发展过程以及朊样传播特征;也可以使用该动物模型来研究某些因素,例如创伤性脑损伤、过表达或沉默磷酸酯酶来研究对tau病理发生发展的影响;更有意义的是,可以通过该模型的建立,有助于筛选出可以抑制或逆转tau病理发生发展及传播的药物。
附图说明
图1是tau的全长和11种tau的截断体示意图;
图2是分离不溶性tau蛋白聚集体的操作流程图;
图3是不溶性tau聚集体诱导细胞中tau的聚集的示意图;
图4是不溶性tau聚集体诱导Th/hTau小鼠tau的聚集的示意图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图及具体实施例对本发明的具体实施方式做详细的说明。
使用pCI/HA-tau作为模板,使用表1中列出的引物通过PCR扩增产生pCI/HA-Tau截断;在AD患者脑中,tau蛋白在多个位点被Calpain、Caspase、AEP等蛋白酶截断。我们选择了最常见的AD相关截断位点:N-末端51、151、231的截断和C-末端391、421的截断,构建了全长和11种tau截断体的真核表达质粒pCI/tau1-441、pCI/tau1-421、pCI/tau1-391、pCI/tau51-441、pCI/tau51-421、pCI/tau51-391、pCI/tau151-441、pCI/tau151-421、pC/tau151-391、pCI/tau231-441、pCI/tau231-421和pCI/tau231-391,如图1所示,在他们的N-末端都带有HA的标签。
在构建的11种tau截断体中,tau151-391猎获AD O-tau的能力最强,因而将其在真核细胞中表达,将获得的蛋白进一步处理后作为seeds来诱导细胞和小鼠产生AD样的tau病理聚集。这一发明,不仅可以有助于构建AD模型,深入研究AD的发病机制、tau蛋白的传播特点,还可以为以tau为靶标的治疗提供AD模型动物。
tau151-391蛋白氨基酸序列如SEQ ID NO:1,所示,DNA序列如SEQ ID NO:2所示;
表1引物的质粒信息
实施例1、一种tau截断体蛋白在诱导tau病理聚集中的应用,包括以下步骤:
1)真核细胞表达tau全长质粒pCI/neo-tau1-441和截断体质粒pCI/neo-tau151-391,得到真核表达的tau1-441和tau151-391截断体蛋白,具体步骤如图2所示;
11)将冻存的HEK-293FT细胞从液氮中取出,37度水浴速溶,转至培养瓶中,加含有10%的FBS的基础培养基培养细胞。等HEK-293FT细胞长满至80%-90%时,可种至3个6cm的dish中,一共接种16个dish。第二天开始准备转染质粒(转染试剂lipo3000),8个dish转染pCI/neo-tau151-391,另外8个dish转染pCI/neo-tau1-441。A:250μl OPTI-MEM+5μg DNA+10μllipo3000,B:250μl OPTI-MEM+10μl lipo3000,A+B混匀静置10-15min,每个dish加500μlA+B混合液和4.5ml完全培养基,4-6h后每个dish换5ml培养基,培养基中含有OA20nM,36h后离心收集细胞。
12)分别收集的转染pCI/neo-tau151-391和pCI/neo-tau1-441的细胞,加入9ml细胞裂解液(OA,50nM),1ml ddH2O和5μlOA(10μm)超声1s,停1s,直至细胞裂解液变澄清。分别13,000g离心5分钟,取上清4.5ml加500μl10%的sarkocyl溶液,室温静置1h,235,000g,TLA-55,4℃离心,1h,分别收集上清和沉淀,沉淀即为真核细胞表达的将作为seeds的蛋白质tau1-441和tau151-391,放-80℃保存。
将收集到的真核表达的蛋白tau1-441和tau151-391加入100μl生理盐水后分别进行超声,on1s,pause 3s,强度为400Hz,90min。然后将蛋白tau1-441,tau151-391稀释5倍、10倍、20倍、40倍、80倍,用dot blot方法,在硝酸纤维膜上每格(0.7x0.7cm)点5μl样,37℃烘干一小时。将膜用封闭液(5%的脱脂奶粉溶于TBS)封闭30min,HA一抗过夜,TBST洗三遍,GAM二抗孵育2h,TBST洗三遍,将膜置于配置好的ECL(ThermoFisher Scientific)显色液中(临用前A、B液等体积混匀),室温2min,压片(注意膜正反不要放错)、曝光、显影。根据显影结果用生理盐水稀释蛋白,使两种蛋白质的tau浓度含量相等,为后续小鼠海马注射蛋白量提供依据。
2)真核表达tau1-441和tau151-391截断体蛋白分别诱导表达HA-Tau151-391的HeLa细胞tau聚集;
根据Fugene HD说明书,在HeLa细胞中转染pCI/HA-tau151-391质粒,4h后在细胞中加入Lipo3000处理的等量tau1-441或tau151-391蛋白(8孔chamber中每孔1μl tau1-441或tau151-391蛋白与0.667μl Lipofectamin2000混合于Opti-MEM中,共20μl体积,室温20min,培养液终体积250μl),48h后,快速用PBS清洗三次,4%多聚甲醛固定15min,PBS清洗三次,0.3%Triton in PBS室温处理15min,5%的封闭液(5%newborn goat serum,0.1%TritonX-100,and 0.05%Tween20 in PBS)封闭30min,细胞与在封闭液中的一抗(Rabbitanti-HA)4℃孵育过夜。PBS清洗三遍,最后滴加封片剂ProLongTMGold antifadereagent(ThermoFisher Scientific),封片,使用激光共聚焦显微镜观察细胞内tau的聚集。如图3所示,细胞免疫荧光显示,在加入了tau151-391蛋白seeds后,出现tau聚集的细胞数大于加入tau全长蛋白seeds组,提示截断体蛋白tau151-391有类似AD O-tau seeds朊样传播的作用,可以诱导细胞内发生tau病理性聚集。
3)小鼠海马注射真核表达tau1-441和tau151-391截断体蛋白;
10月龄hTau小鼠腹腔注射2.5%的Avertin麻醉,海马注射真核表达tau1-441或者tau151-391截断体蛋白;注射的坐标如下:前囟向后2.5mm,向左/右旁开2.0mm,自硬膜向腹侧进针1.8mm;注射体积2.5μl,注射速度1.25μl/min,留针3min以防液体外溢。如图4所示,10w后免疫荧光实验证实,tau151-391截断体蛋白可以引起小鼠脑内神经元发生AD样的tau病理聚集和朊样传播。
4)组织固定与切片;
取小鼠,先经腹腔注射2.5%的2-Avertin麻醉剂,麻醉后,开胸暴露心脏,自心尖部将灌注针头经左心室插至升主动脉,灌注生理盐水后,改快速灌注含有4%多聚甲醛的0.1MPBS(pH7.2),待动物全身抽搐后,调慢灌注速度,约50滴/min,持续时间约30min后,取出大脑放入含有4%多聚甲醛的0.1MPBS(pH7.2)中后固定4h。再先后转入含20%和30%蔗糖溶液中,待组织块下沉后,行冠状位冰冻连续切片,片厚40μm,漂于0.01M PBS中。
5)免疫组织化学;
51)挑取海马结构清晰的漂片,置于0.01M PBS缓冲液(pH7.2)中漂洗3遍后,放入含有10%triton-100液体中20min,0.01M PBS缓冲液(pH7.2)中漂洗3遍。放入含有10%山羊血清的封闭液中,在室温下振摇2h。
52)将封闭好的切片转入到1:500稀释的小鼠抗AT8单抗中,室温下轻轻振摇1h后放入4℃冰箱中过夜;
53)次日晨吸去一抗,用PBS清洗3遍后,加入1:500稀释的cy3荧光二抗,hochest(1:500)避光、室温下轻轻振摇2h;
54)0.01M PBS(pH 7.2)避光清洗3次,每次10min;
55)荧光封片液封片后激光共聚焦显微镜下观察。
实施例2、tau病理模型的应用举例
(1)筛选可抑制tau病理的基因。
构建靶基因腺相关病毒载体,注射入4月龄C57BL/6小鼠海马区,使靶基因高表达。2周后,同一位置注射tau151-391seeds,诱导tau病理产生。14月龄时,取鼠脑进行免疫荧光,比较注射靶基因病毒和对照病毒的鼠脑tau病理强度(tau病理特异性抗体mouse-AT8阳性细胞的数目),判断靶基因是否影响tau病理产生,从而筛选出可有效抑制tau病理的基因。
(2)筛选可抑制tau病理的药物
4月龄C57BL/6小鼠海马区注射tau151-391seeds,诱导tau病理产生。注射后第二天,通过腹腔注射或灌胃给小鼠施加相应浓度的药物,持续10个月。取鼠脑进行免疫荧光,比较给药组和对照组之间tau病理强度(tau病理特异性抗体mouse-AT8阳性细胞的数目),判断药物是否影响tau病理传播,从而筛选出可有效抑制tau病理的药物。
序列表
<110> 南通大学
<120> tau截断体蛋白在诱导tau病理聚集中的应用
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195 200 205
Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His
210 215 220
Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala
225 230 235 240
Glu
<210> 2
<211> 723
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atcgccacac cgcggggagc agcccctcca ggccagaagg gccaggccaa cgccaccagg 60
attccagcaa aaaccccgcc cgctccaaag acaccaccca gctctggtga acctccaaaa 120
tcaggggatc gcagcggcta cagcagcccc ggctccccag gcactcccgg cagccgctcc 180
cgcaccccgt cccttccaac cccacccacc cgggagccca agaaggtggc agtggtccgt 240
actccaccca agtcgccgtc ttccgccaag agccgcctgc agacagcccc cgtgcccatg 300
ccagacctga agaatgtcaa gtccaagatc ggctccactg agaacctgaa gcaccagccg 360
ggaggcggga aggtgcagat aattaataag aagctggatc ttagcaacgt ccagtccaag 420
tgtggctcaa aggataatat caaacacgtc ccgggaggcg gcagtgtgca aatagtctac 480
aaaccagttg acctgagcaa ggtgacctcc aagtgtggct cattaggcaa catccatcat 540
aaaccaggag gtggccaggt ggaagtaaaa tctgagaagc ttgacttcaa ggacagagtc 600
cagtcgaaga ttgggtccct ggacaatatc acccacgtcc ctggcggagg aaataaaaag 660
attgaaaccc acaagctgac cttccgcgag aacgccaaag ccaagacaga ccacggggcg 720
gag 723
<210> 3
<211> 65
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
acgcgtcgac atgtacccat acgatgttcc agattacgct atggctgagc cccgccagga 60
gttcg 65
<210> 4
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ataagaatgc ggccgctcac aaaccctgct tggccaggga gg 42
<210> 5
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acgcgtcgac atgtacccat acgatgttcc agattacgct atcgccacac cgcggggagc 60
ag 62
<210> 6
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ataagaatgc ggccgctcag tctaccatgt cgatgctgcc gg 42
Claims (6)
1.一种tau截断体蛋白在诱导tau病理聚集中的应用,其特征在于,包括以下步骤:
1)真核细胞表达tau全长质粒pCI/neo-tau1-441和截断体质粒pCI/neo-tau151-391,得到真核表达的tau1-441和tau151-391截断体蛋白;
2)真核表达tau1-441和tau151-391截断体蛋白分别诱导表达HA-Tau151-391的HeLa细胞tau聚集;
3)小鼠海马注射真核表达tau1-441和tau151-391截断体蛋白;
4)组织固定与切片;
5)免疫组织化学。
2.根据权利要求1所述一种tau截断体蛋白在诱导tau病理聚集中的应用,其特征在于,所述步骤1)具体包括以下步骤:
11)将冻存的HEK-293FT细胞从液氮中取出,37度水浴速溶,转至培养瓶中,加含有10%的FBS的基础培养基培养细胞;等HEK-293FT细胞长满至80%-90%时,可种至3个6cm的dish中,一共接种16个dish;
第二天开始准备转染质粒,转染试剂为lipo3000,其中8个dish转染pCI/neo-tau151-391,另外8个dish转染pCI/neo-tau1-441;A:250μl OPTI-MEM+5μg DNA+10μl lipo3000,B:250μlOPTI-MEM+10μl lipo3000,A+B混匀静置10-15min,每个dish加500μlA+B混合液和4.5ml完全培养基,4-6h后每个dish换5ml培养基,培养基中含有OA20nM,36h后离心收集细胞;
12)分别收集的转染pCI/neo-tau151-391和pCI/neo-tau1-441的细胞,加入9ml浓度为50nM的细胞裂解液,将1ml ddH2O和5μl细胞裂解液混合超声1s停1s,直至细胞裂解液变澄清;13,000g离心5分钟,取上清4.5ml加500μl10%的sarkocyl溶液,室温静置1h,再235,000g,TLA-55,4℃离心,1h,分别收集上清和沉淀,沉淀即为真核细胞表达的作为seeds的蛋白质tau1-441和tau151-391,放-80℃保存。
3.根据权利要求1所述一种tau截断体蛋白在诱导tau病理聚集中的应用,其特征在于,所述步骤2)具体包括以下步骤:
根据Fugene HD说明书,在HeLa细胞中转染pCI/HA-tau151-391质粒,4h后在细胞中加入Lipo3000处理的等量真核表达tau1-441或tau151-391截断体蛋白;
48h后,快速用PBS清洗三次,4%多聚甲醛固定15min,PBS清洗三次,0.3%Triton inPBS室温处理15min,5%的封闭液封闭30min,细胞与在封闭液中的一抗4℃孵育过夜;PBS清洗三遍,最后滴加封片剂封片,使用激光共聚焦显微镜观察细胞内tau的聚集。
4.根据权利要求1所述一种tau截断体蛋白在诱导tau病理聚集中的应用,其特征在于,所述步骤3)10月龄hTau小鼠腹腔注射2.5%的Avertin麻醉,海马注射真核表达tau1-441或者tau151-391截断体蛋白;注射的坐标如下:前囟向后2.5mm,向左/右旁开2.0mm,自硬膜向腹侧进针1.8mm;注射体积2.5μl,注射速度1.25μl/min,留针3min以防液体外溢。
5.根据权利要求1所述一种tau截断体蛋白在诱导tau病理聚集中的应用,其特征在于,所述步骤4)具体包括以下步骤:
取小鼠,先经腹腔注射2.5%的2-Avertin麻醉剂,麻醉后,开胸暴露心脏,自心尖部将灌注针头经左心室插至升主动脉,灌注生理盐水后,改快速灌注含有4%多聚甲醛的0.1MPBS,待动物全身抽搐后,调慢灌注速度,约50滴/min,持续时间约30min后,取出大脑放入含有4%多聚甲醛的0.1MPBS中后固定4h;再先后转入含20%和30%蔗糖溶液中,待组织块下沉后,行冠状位冰冻连续切片,片厚40μm,漂于0.01M PBS中。
6.根据权利要求1所述一种tau截断体蛋白在诱导tau病理聚集中的应用,其特征在于,所述步骤5)具体包括以下步骤:
51)挑取海马结构清晰的漂片,置于0.01M PBS缓冲液中漂洗3遍后,放入含有10%triton-100液体中20min,0.01M PBS缓冲液中漂洗3遍;放入含有10%山羊血清的封闭液中,在室温下振摇2h;
52)将封闭好的切片转入到1:500稀释的小鼠抗AT8单抗中,室温下轻轻振摇1h后放入4℃冰箱中过夜;
53)次日晨吸去一抗,用PBS清洗3遍后,加入1:500稀释的cy3荧光二抗,hochest(1:500)避光、室温下轻轻振摇2h;
54)0.01M PBS(pH 7.2)避光清洗3次,每次10min;
55)荧光封片液封片后激光共聚焦显微镜下观察。
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