CN107475260B - 抑制大脑中Aβ累积的BACE1亚型的功能研发及应用 - Google Patents
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Abstract
本发明属于生物工程中的DNA重组技术领域，涉及本发明属于生物工程技术领域，具体是基于BACE1选择性剪接机制的基因重组出一种对APP病理切割拮抗作用和Aβ形成抑制作用的47KD亚型的BACE1，通过ELISA实验检测47KD亚型BACE1对Aβ1‑40和Aβ1‑42生成的促进作用，并通过行为学实验验证其对AD鼠记忆损伤的校正作用，最后通过病理学实验验证其对脑内Aβ形成的校正作用。47KD亚型BACE1为AD分子靶点的深入研究及AD药物研发提供依据和线索。
Description
技术领域
本发明属于生物工程中的DNA重组技术领域，具体是基于BACE1选择性剪接机制的基因重组出一种对APP病理切割拮抗作用和Aβ形成抑制作用的BACE1亚型，并验证其对AD鼠记忆损伤和脑内Aβ形成的校正作用，以及其为AD分子靶点的深入研究及AD药物研发提供依据和线索。
背景技术
阿尔茨海默症(Alzheimer's disease AD)是老龄化社会的重要疾病，并成为威胁老年人群健康的四大杀手之一。淀粉样蛋白Aβ在大脑中不断生成和累积，引起显著的淀粉样斑块形成和继发性神经损伤，是AD重要的发病机制。Aβ是由APP(淀粉样蛋白前体)分子病理切割形成的，而β-分泌酶(BACE1)是Aβ生成的主要限速酶，也是AD药物研发和分子机制研究的核心靶点。
BACE1通常被认为是由501个氨基酸组成的55KD的蛋白酶，目前大部分的研究也将其作为靶点来进行AD的药物研发，然而多以失败告终，这提示了BACE1分子的复杂性。最新文献报道，BACE1的转录本通过选择性剪接机制可以产生分别编码含有501、476、457以及432个氨基酸的BACE1异构体，分别编码55KD，52KD，49KD和47KD的BACE1蛋白。B型I-476比起A型I-501在外显子4编码的区域缺少了25个氨基酸，C型I-457则在外显子3编码的区域内缺少了44个氨基酸，而D型I-432则同时缺失了B型I-476和C型I-457所缺失的所有氨基酸。在大脑内最为广泛表达的是A/型I-501，而其他三种形式在大脑中的表达量很低，主要在胰腺等组织中有所表达，所以研究者并没有关注其它三种形式对APP分子的切割功能。
大脑内最广泛表达的A型BACE1的蛋白结构域包括N端信号肽和前肽区域，中部的成熟蛋白催化域，以及由17个氨基酸组成的跨膜区域和24个氨基酸组成的C端延伸尾【图1】。BACE1的N端和C端各含有一个天冬氨酸活性催化区域，这两个天冬氨酸残基对于BACE1的活性至关重要，BACE1含有四个糖基化位点，糖基化是BACE1的成熟的必要条件，同时影响BACE1的活性。与A型BACE1相比，D型BACE1缺失146-214位氨基酸，缺失了Asn153，Asn172这两个糖基化位点，这一结构的改变是否会改变其功能，这值得我们深入研究。
发明内容
本发明是基于BACE1的选择性剪接和AD发生特点、治疗难点，通过生物工程发明一段特异的蛋白序列和核酸序列，证明其具有对APP病理切割拮抗作用和Aβ形成抑制作用，并通过动物实验验证其对AD鼠记忆损伤和脑内Aβ形成的校正作用。
本发明利用生物工程技术，基因重组一段432个氨基酸多肽对应的DNA序列到pcDNA3.1真核表达载体。经序列分析证明重组成功后，将此真核表达重组多肽转染到人胚肾细胞中，免疫印迹证明多肽的蛋白表达，实现了多肽的重组。接着对多肽的拮抗APP病理切割功能进行了研究，细胞学实验表明此多肽具有APP病理切割拮抗作用和Aβ形成抑制作用。然后通过动物体内注射，行为学实验和病理学实验都证明此多肽具有AD鼠记忆损伤和脑内Aβ形成的校正作用
本发明的多肽重组表达质粒是将此多肽对应DNA序列连接进pcDNA3.1真核表达载体，多肽N端氨基酸序列为：
其对应的核苷酸序列为：
第一步，47KD亚型BACE1真核表达质粒构建及其表达检测
以pcDNA3.1-BACE1(55KD)为模板进行PCR。将PCR产物用DH5α感受态进行转化，并将转化出的菌落进行小量培养，然后将小量培养的菌液进行小量提取，最后将提取的质粒进行测序。
将正确连接的多肽真核表达载体转染293T细胞，48小时后搜集样品，免疫印迹结果证明了此多肽的表达。
第二步：47KD亚型BACE1的功能检测
1.细胞学实验
采用AD的细胞模型SHSY5Y的细胞上清液为样品，在共转染重组肽真核表达质粒和APP野生型质粒或APPSWE突变型质粒48h后，将细胞培养液离心，取1/100作为样品检测Aβ1-40和Aβ1-42的浓度。ELISA结果显示，47KD亚型的BACE1具有APP病理切割拮抗作用和Aβ形成抑制作用。
2.小鼠体内实验
选经过基因型鉴定的16月龄AD小鼠进行分组，相当于人类重度AD患者，每组分别腹腔注射高纯度的质粒，每周两次，每次每只20ug质粒，共2个月。在进行行为学检测后，将鼠脑进行固定包埋切片，通过免疫组化的方法检测脑内老年斑的形成及相关酶的变化。行为学和病理学结果显示47KD亚型的BACE1具有AD鼠记忆损伤和脑内Aβ形成的校正作用。
附图说明：
图1为BACE1蛋白结构图；
图2为构建BACE1突变质粒原理简图；
图3为47KD亚型BACE1测序结果比对；
图4为BACE1两种抗体原理图；
图5为BACE1突变质粒表达验证原理；
图6为BACE1突变质粒表达验证Westernblot分析；
图7为ELISA检测AD细胞模型SHSY5Y细胞培养液中Aβ1-40的浓度；
图8为ELISA检测AD细胞模型SHSY5Y细胞培养液中Aβ1-42的浓度；
图9为腹腔注射BACE1对AD小鼠行为认知作用的实验设计；
图10为质粒注射后AD小鼠Y迷宫入臂正确率统计图；
图11为AD小鼠定位航行典型轨迹图及6天潜伏期折线统计图；
图12为AD小鼠空间探索航行典型轨迹图及虚拟平台停留时间次数统计图；
图13为三种BACE1抗体检测腹腔注射BACE1突变质粒在AD小鼠脑内过表达情况；
图14为AD小鼠脑组织硫黄素染色及数理统计。
具体实施方式：
实施例1：47KD亚型BACE1真核表达质粒构建及其表达检测
1、47KD亚型BACE1真核表达载体构建
BACE1基因全长从Genebank得到，基因序列号为NM 001207048。D型I-432(47KD)BACE1与A型I-501(55KD)BACE1相比缺失146-214aa【图2】，以pcDNA3.1-BACE1(55KD)为模板，上游引物为5'-ACCTGCTTTGTGGTGCTGGCTTCCCCCTCA-3'，下游引物为5'-CAAAGCAGGTCGGTGCCCAGCTCCCCTTCC-3'，进行PCR。将PCR产物用DH5α感受态进行转化，并将转化出的菌落进行小量培养，然后将小量培养的菌液以碱裂解法进行小量提取，最后将提取的质粒进行测序，并进行比对，突变成功【图3】。
2、47KD亚型BACE1真核表达质粒的表达检测
根据已有的两端特异性抗体【图4】，抗体BACE1190-214aa可以检测55KD、49KD这两种形式的BACE1，抗体BACE1146-189aa可以检测55KD、52KD这两种形式的BACE1；而已有的BACE1抗体可以检测55KD、52KD、49KD、47KD这四种亚型的BACE1。根据这三种抗体可以特异性识别的片段类型，设计了突变质粒表达验证实验【图5】。选择高转染效率的HEK293T细胞，在HEK293T细胞中过表达BACE1亚型的真核质粒，用上述三种抗体Western blot检测，结果显示突变质粒均构建成功且可以表达【图6】。
实施例2：47KD亚型BACE1的功能检测
1.ELISA检测AD细胞模型中47KD亚型BACE1对Aβ1-40和Aβ1-42的生成抑制
采用AD的细胞模型SHSY5Y的细胞上清液为样品，在共转染BACE1的突变质粒和APP野生型质粒或APPSWE突变型质粒48h后，将细胞培养液离心，取1/100作为样品检测47KD亚型BACE1对APP分子的切割作用，根据标准品浓度与吸光度曲线，计算样品浓度。与空白对照组相比，无论是过表达APP野生型还是过表达APPSWE突变型，只有47KD的BACE1能使Aβ1-40的浓度明显降低【图7】；对于Aβ1-42的生成检测实验中，也得到了相同的结果【图8】。
因此，可以确定D型BACE1(47KD)可以显著抑制Aβ1-40和Aβ1-42的生成，抑制率达到80％以上。这提示我们47KD的BACE1不同于传统的BACE1对APP病理切割和Aβ形成的促进作用，其具有APP病理切割的拮抗作用和Aβ形成的抑制作用。
2、Y迷宫探究47KD亚型BACE1对AD小鼠行为认知的作用
选择经过基因型鉴定的16月龄AD小鼠，相当于人类重度AD患者，正常饲养设置五组，每组6只实验鼠，包括对照组(空载体组)、A型BACE1(55KD)组、B型BACE1(52KD)组、C型BACE1(49KD)组和D型BACE1(47KD)组。每组分别腹腔注射高纯度的质粒，每周两次，每次每只20ug质粒，共2个月【图9】。
Y迷宫实验是一种用于检测小鼠空间认知能力和短期记忆能力的行为学手段，其操作简单，应用广泛。实验结果显示，空载体对照组和其他三种BACE1亚型在注射质粒后Y迷宫路径认知准确率下降，只有D型BACE1(47KD)过表达的转基因鼠的Y迷宫路径认知准确率略有上升【图10】，经过统计学分析具有统计学差异，并且该组小鼠的觅食和活动能力强于其它各组。实验结果说明只有D型BACE1(47KD)过表达可以增强转基因鼠APPswe/PS1dE9的学习短期记忆能力。
3、Morris水迷宫探究47KD亚型BACE1对AD小鼠行为认知的作用
Morris水迷宫是AD研究中关于脑学习记忆机制研究的一种非常普遍的实验手段。选择了16月龄的AD小鼠，正常饲养设置五组，每组6只实验鼠，包括空白组(空载体组)、A型BACE1(55KD)组、B型BACE1(52KD)组、C型BACE1(49KD)组和D型BACE1(47KD)组。每组分别腹腔注射高纯度的质粒，每周两次，每次每只20ug质粒；8周后通过Morris水迷宫实验检测每组模型鼠的学习记忆能力。
Morris水迷宫在直径为1.2米的圆形水池中进行，先将注射质粒8周的小鼠置于放有可见平台的水池中以确定游泳能力，并多次训练以加深小鼠对平台的位置记忆。随后进行定位航行试验，历时6天，每天分别从4个象限的固定入水点将小鼠面向池壁放入水中，并记录其寻找到水下隐蔽平台所用的时间，将该时间定义为潜伏期。根据各组小鼠在定位航行实验的典型轨迹图可知47KD亚型的BACE1组的小鼠找到隐匿于水下平台所走的路程较短，而其它三组则很难找到平台；随着测试天数的增加，各组小鼠找到隐匿平台所需的时间整体呈缩短趋势。总体趋势上，与空白对照组相比，d型BACE1(47KD)组小鼠找到隐匿平台所需的时间(潜伏期)大幅缩短【图11】。
在第7天撤去水中隐匿平台，小鼠在水中寻找原平台的位置进行空间探索实验，在搜索时间相同和路程接近的情况下，47KD亚型的BACE1组的小鼠能够对原平台位置有准确的记忆和识别，不同于其他组的盲目寻找【图12】。分析小鼠的长期记忆能力和空间记忆能力的结果发现，47KD亚型BACE1组的小鼠经过原平台位置的次数明显高于其他各组，说明47KD亚型的BACE1能够加深小鼠对平台位置的长期记忆，即对阿尔茨海默症的记忆认知损伤的恢复作用显著。各组小鼠在原平台所在的象限的活动时间同样是47KD亚型的BACE1组的小鼠明显高于其他组，说明47KD亚型的BACE1能够加深小鼠对平台的空间方向的记忆，即对空间认知和位置记忆有显著的改善作用，且对AD患病小鼠空间认知和位置记忆能力的恢复作用明显。
4、免疫组化验证腹腔注射不同亚型的BACE1在AD小鼠大脑中的过表达情况
将16月龄起连续腹腔注射BACE1突变质粒8周的AD小鼠处死，固定包埋其鼠脑组织并用三种BACE1抗体免疫组化。在同一时间同一条件下进行染色和图片拍摄，结果显示，与对照组相比，四组过表达BACE1不同亚型质粒的小鼠大脑海马区染色加深；可以检测55KD、49KD两种亚型BACE1的自制抗体BACE1190-214aa结果显示，海马区49KD亚型的BACE1组和55KD亚型的BACE1组的染色明显深于其余各组；可以检测55KD、52KD两种亚型的BACE1的自制抗体BACE1146-189aa结果显示，海马区52KD亚型的BACE1组和55KD亚型的BACE1组的染色明显深于其余各组，大脑皮层区BACE1的表达与海马区的结果一致【图13】。以上结果证明腹腔注射BACE1不同亚型质粒可以在小鼠大脑组织中正常表达.。
5、硫黄素S染色探究47KD亚型BACE1对AD小鼠大脑组织β-淀粉样斑块的影响
大脑的边缘***包括杏仁核和海马等结构，对记忆起着关键性作用，其中大脑海马区与特定的学习和记忆相关，是存储短期记忆关键部位，同时也是人类学习记忆声光味等事件的大脑区域，即长时记忆区域。硫磺素S染色主要用于显示细胞外淀粉样物质，不可溶性的Aβ蛋白在脑组织中沉积所形成β-淀粉样斑块具有与硫磺素S结合的特性，因而选择这种方法进行组织切片染色的方法分析了16月龄起连续腹腔注射BACE1突变质粒2个月的AD小鼠脑组织内β-淀粉样斑块沉积的改变，并分区域进行定性观察和定量分析，结果发现47KD亚型的BACE1具有显著地减低AD小鼠大脑海马区β-淀粉样斑块的沉积作用，对海马周围皮质区和远海马端皮质区同样具有明显的干预作用【图14】。将转基因小鼠大脑内β-淀粉样斑块总体数量进行量化分析和数理统计，结果证明只有注射47KD亚型BACE1的转基因小鼠大脑内β-淀粉样斑块在斑块个数上都较空白载体组有明显降低，说明47KD亚型的BACE1,具有显著减少和纠正AD鼠脑内β-淀粉样斑块的形成和沉积的作用。
Claims (1)
1.一种47KD亚型的BACE1在制备降低阿尔茨海默症(AD)鼠脑内β-淀粉样斑块的形成和沉积，使其记忆损伤矫正的药物中的用途，包括在AD鼠中过表达47KD的BACE1，其中47KD亚型BACE1与55KD亚型BACE1相比缺失146-214aa，该47KD亚型的BACE1的核苷酸序列如下：
氨基酸序列如下：
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