技术领域technical field
本发明属于生物化学与分子生物学领域，涉及一种抗人淀粉样β肽(amyloidβ,Aβ)的纳米抗体及其应用。The invention belongs to the field of biochemistry and molecular biology, and relates to a nanobody against human amyloid β peptide (amyloid β, Aβ) and application thereof.
背景技术Background technique
阿尔茨海默病(Alzheimer’s disease,AD)是进行性认知功能障碍和行为损害为特征的中枢神经系统变性疾病。2015年9月Alzheimer’s disease international,(ADI)的报告数据显示：目前约每3.2秒就有一人罹患老年痴呆，在老年痴呆患者中60-70％为阿尔茨海默病性痴呆，随着老龄人口基数和比例的持续增加，AD带来患者个人的痛苦和社会、家庭的压力将越来越严重。Alzheimer's disease (AD) is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment. In September 2015, the report data of Alzheimer's disease international, (ADI) showed that at present, about one person suffers from Alzheimer's disease every 3.2 seconds, and 60-70% of the patients with Alzheimer's disease are Alzheimer's disease dementia. With the aging population As the base and proportion continue to increase, AD will bring more and more serious personal pain to patients and social and family pressure.
淀粉样β肽(amyloidβ,Aβ)是存在于人脑的天然产物，其在神经细胞发育(Heo C,et al.J Neurochem.2007；102(2):493-500.)、睡眠调节(Kang JE,et al.2009；326(5955):1005-7)、抗菌(Soscia SJ,et al.PLoSOne 2010；5:e9505.)等方面发挥积极的调节作用。但遗传学分析发现Aβ前体蛋白(amyloid precursor protein,APP)(Goate A,etal.Nature.1991；349(6311):704-6；Jonsson T,et al.Nature.2012；488(7409):96-9)或与其代谢相关的基因如早老素发生突变与家族性AD的是否发生相关(Kamino K,etal.Neurosci Lett.1996；208(3):195-8；Sherrington R,et al.Nature.1995；375(6534):754-60.)，因此人们认为Aβ肯定与AD的发生相关。在AD发生过程中自由Aβ经历先增加后减少的过程(Maia LF,et al.EMBO Mol Med.2015；7(7):895-903.)，可能正是由于Aβ的代谢失衡，Aβ在某种未知条件的诱导下逐渐聚集形成可溶的Aβ聚集体以及不溶的Aβ纤维沉积。Amyloid β peptide (amyloidβ, Aβ) is a natural product present in the human brain. JE, et al.2009; 326(5955):1005-7), antibacterial (Soscia SJ, et al.PLoSOne 2010; 5:e9505.) and other aspects play a positive regulatory role. However, genetic analysis found that Aβ precursor protein (amyloid precursor protein, APP) (Goate A, et al. Nature.1991; 349(6311): 704-6; Jonsson T, et al. Nature.2012; 488 (7409): 96-9) or genes related to its metabolism, such as presenilin, are associated with the occurrence of familial AD (Kamino K, et al.Neurosci Lett.1996; 208(3):195-8; Sherrington R, et al. Nature .1995; 375(6534):754-60.), so it is believed that Aβ must be related to the occurrence of AD. In the process of AD, free Aβ experiences a process of first increasing and then decreasing (Maia LF, et al.EMBO Mol Med.2015; 7(7):895-903.), it may be due to the metabolic imbalance of Aβ, Aβ in a certain Under the induction of an unknown condition, they gradually aggregated to form soluble Aβ aggregates and insoluble Aβ fibril deposits.
动物模型研究和临床实验均发现普通抗体都可以在一定程度上干扰Aβ的代谢，但在缓解AD病情发展方面没有获得理想的结果。仔细分析治疗过程发现，其治疗机理主要是通过药物与血液内Aβ的结合打破血液与脑脊液Aβ的平衡，促使脑脊液内Aβ跨越血脑屏障进入血液；或者是通过抗体跨越血脑屏障起到直接治疗效果，但实际所用药物主要是IgG类抗体，血液内极低的Aβ水平和抗体难于跨越血脑屏障的缺陷，大大限制了其治疗效果(BardF,et al.Exp Neurol.2012；238(1):38-43.)。Both animal model studies and clinical experiments have found that common antibodies can interfere with the metabolism of Aβ to a certain extent, but have not achieved ideal results in alleviating the progression of AD. Careful analysis of the treatment process found that the therapeutic mechanism is mainly through the combination of drugs and blood Aβ to break the balance of blood and cerebrospinal fluid Aβ, and promote the Aβ in cerebrospinal fluid to cross the blood-brain barrier into the blood; However, the drugs actually used are mainly IgG antibodies. The extremely low Aβ level in the blood and the difficulty of antibodies crossing the blood-brain barrier greatly limit the therapeutic effect (BardF, et al. Exp Neurol.2012; 238(1) :38-43.).
为了提高抗体跨越血脑屏障的能力，目前通常对抗体进行包埋修饰或将靶标抗体与具有跨血脑屏障能力的蛋白(脑血管内皮细胞相关抗原抗体或具有特殊转胞吞作用的蛋白)构建成嵌合体。无论哪一种策略，都进一步增加了抗体的制备成本。而分子量较小(12-15kD)等电点较高(pI>9.2)的来自驼科动物重链抗体可变区的纳米抗体在不进行任何修饰的条件下可以非常高效的跨越血脑屏障(Li T,et al.FASEB J.2012；26(10):3969-79.)，因此纳米抗体在脑疾病治疗中具有很好的应用前景；另外，获得的纳米抗体和普通抗体一样可以用于以Aβ为靶标的诊断、检测与治疗。In order to improve the ability of antibodies to cross the blood-brain barrier, antibodies are usually modified by embedding or the target antibody is constructed with a protein capable of crossing the blood-brain barrier (cerebrovascular endothelial cell-associated antigen antibody or a protein with special transcytosis) into a chimera. No matter which strategy, it further increases the cost of antibody preparation. Nanobodies from camelid heavy chain antibody variable regions with smaller molecular weight (12-15kD) and higher isoelectric point (pI>9.2) can cross the blood-brain barrier very efficiently without any modification ( Li T, et al.FASEB J.2012; 26(10):3969-79.), so nanobodies have good application prospects in the treatment of brain diseases; in addition, the obtained nanobodies can be used in the same way as ordinary antibodies Diagnosis, detection and treatment targeting Aβ.
发明内容Contents of the invention
本发明的目的之一是提供一种抗淀粉样β肽的纳米抗体。One of the objectives of the present invention is to provide a nanobody against amyloid β peptide.
一种抗淀粉样β肽的纳米抗体，所述纳米抗体氨基酸序列包括框架区FR和互补决定区CDR，其特征在于，所述的框架区FR由下组的FR的氨基酸序列组成：如SEQ ID NO.1所示的FR1，如SEQ ID NO.2所示的FR2，如SEQ ID NO.3所示的FR3，如SEQ ID NO.4所示的FR4；所述的互补决定区CDR由下组的CDR的氨基酸序列组成：如SEQ ID NO.5所示的CDR1，如SEQ IDNO.6所示的CDR2，如SEQ ID NO.7所示的CDR3。A nanobody against amyloid beta peptide, the amino acid sequence of the nanobody includes a framework region FR and a complementarity determining region CDR, characterized in that the framework region FR is composed of the amino acid sequence of the FR of the following group: such as SEQ ID FR1 shown in NO.1, FR2 shown in SEQ ID NO.2, FR3 shown in SEQ ID NO.3, FR4 shown in SEQ ID NO.4; the complementarity determining region CDR is composed of the following The amino acid sequence composition of the CDRs of the group: CDR1 as shown in SEQ ID NO.5, CDR2 as shown in SEQ ID NO.6, and CDR3 as shown in SEQ ID NO.7.
进一步地，所述的抗淀粉样β肽的纳米抗体，其具有SEQ ID NO.8所示的氨基酸序列。Further, the anti-amyloid β peptide nanobody has the amino acid sequence shown in SEQ ID NO.8.
本发明目的之二是提供一种上述所述的纳米抗体在制备治疗淀粉样β肽代谢失衡引起的疾病的药物中的应用。优选地，所述的淀粉样β肽代谢失衡引起的疾病为阿尔茨海默病。The second object of the present invention is to provide the application of the nanobody described above in the preparation of medicines for treating diseases caused by the imbalance of amyloid beta peptide metabolism. Preferably, the disease caused by the imbalance of amyloid β peptide metabolism is Alzheimer's disease.
本发明目的之三是提供一种上述所述的纳米抗体在制备检测淀粉样β肽的检测试剂中的应用。The third object of the present invention is to provide an application of the nanobody described above in the preparation of a detection reagent for detecting amyloid β peptide.
本发明还提供一种DNA分子，其编码本发明所述的抗淀粉样β肽的纳米抗体。The present invention also provides a DNA molecule encoding the nanobody against amyloid beta peptide of the present invention.
本发明的有益效果：Beneficial effects of the present invention:
本发明提供了一种特异性的抗Aβ的纳米抗体，该纳米抗体对Aβ具有与已发表抗体不同的氨基酸序列，可以将其对应的基因转入到原核的或真核表达体系中进行表达，获得的抗体可以应用在各种以Aβ为靶标的疾病的治疗药物的制备以及Aβ的检测试剂的制备中。The present invention provides a specific anti-Aβ nanobody. The nanobody has an amino acid sequence different from that of published antibodies against Aβ. The corresponding gene can be transferred into a prokaryotic or eukaryotic expression system for expression. The obtained antibody can be used in the preparation of therapeutic drugs for various diseases targeting Aβ and the preparation of detection reagents for Aβ.
附图说明Description of drawings
图1为本发明纳米抗体的框架区、互补决定区的位置以及氨基酸序列示意图。Fig. 1 is a schematic diagram showing the positions of the framework region, complementarity determining region and amino acid sequence of the Nanobody of the present invention.
图2为SDS-PAGE电泳检测纳米抗体在IPTG诱导条件下的表达情况。M为蛋白质分子量标准；泳道3为未加IPTG诱导的全菌蛋白；泳道1与2为IPTG诱导后的全菌蛋白。Figure 2 is the SDS-PAGE electrophoresis detection of the expression of nanobodies under IPTG-induced conditions. M is the protein molecular weight standard; Lane 3 is the whole bacteria protein induced without IPTG; Lane 1 and 2 are the whole bacteria protein induced by IPTG.
图3为SDS-PAGE电泳检测金属螯合层析分离纯化纳米抗体情况。M为蛋白质分子量标准，泳道1为流穿液；泳道2-4为50mM咪唑洗脱；泳道5-11为150mM咪唑洗脱；泳道12-13为500mM咪唑洗脱。Fig. 3 is the situation of SDS-PAGE electrophoresis detection of separation and purification of nanobodies by metal chelation chromatography. M is protein molecular weight standard, lane 1 is flow-through; lanes 2-4 are 50mM imidazole elution; swimming lanes 5-11 are 150mM imidazole elution; swimming lanes 12-13 are 500mM imidazole elution.
图4为ELISA检测纳米抗体对不同序列(Aβ12-35、Aβ40、Aβ42)与状态(单体、可溶聚集体和纤维)Aβ的结合能力。对照组与实验组的区别是前者包被抗原未加入纳米抗体，后者加入了纳米抗体。Fig. 4 is an ELISA detection of the binding ability of Nanobodies to different sequences (Aβ12-35 , Aβ40 , Aβ42 ) and states (monomer, soluble aggregate and fiber) Aβ. The difference between the control group and the experimental group is that the former is coated with antigen without adding nanobody, while the latter is added with nanobody.
图5为MTT检测纳米抗体对Aβ处理细胞SH-SY5Y的保护情况。Figure 5 shows the protection of the nanobody on Aβ-treated cells SH-SY5Y detected by MTT.
具体实施方式Detailed ways
下述非限制性实施例可以使本领域技术人员更全面理解本发明，本申请公开的DNA序列和氨基酸序列可以按照本领域常用的其他方法获得。而且可以将本申请获得的新的氨基酸序列对应的基因构建到任何合适的载体，用任何合适的表达体系进行表达。下述内容仅仅是对本申请要求保护的范围的示例性说明，一些根据所公开的内容做出的改变和修饰，也应当属于本申请要求保护的范围。本文实施例中所使用的药品及试剂若无特殊说明均按常规途径获得。The following non-limiting examples can enable those skilled in the art to understand the present invention more comprehensively. The DNA sequence and amino acid sequence disclosed in this application can be obtained by other methods commonly used in the art. Moreover, the gene corresponding to the novel amino acid sequence obtained in this application can be constructed into any suitable vector and expressed with any suitable expression system. The following content is only an exemplary description of the scope of protection claimed in this application, and some changes and modifications made according to the disclosed content should also fall within the scope of protection claimed in this application. The medicines and reagents used in the examples herein were obtained by conventional means unless otherwise specified.
不同序列的Aβ(Aβ12-35、Aβ40、Aβ42)的单体、可溶聚集体和纤维购自杭州中肽生化有限公司。Monomers, soluble aggregates and fibers of Aβ with different sequences (Aβ12-35 , Aβ40 , Aβ42 ) were purchased from Hangzhou Zhongpei Biochemical Co., Ltd.
实施例1纳米抗体库的建立Example 1 Establishment of Nanobody Library
本发明使用的噬菌体展示库是以T7噬菌体为载体的非免疫库，建立步骤如下：The phage display library used in the present invention is a non-immune library with T7 phage as the carrier, and the establishment steps are as follows:
(1)提取2岁雄性羊驼外周血淋巴细胞的总RNA(PuerLinkTM RNA MiniKit，Life Technologies：12183018A)；以UP primer 1为引物将提取的总RNA反转录为cDNA，并采用两轮巢式PCR扩增VHH基因；其中第一轮PCR为以转录的cDNA为模版，以UPprimer 1和DOWN primer 1为引物，PCR扩增后回收大小为650～750bp的条带，再以此为模版，进行第二轮PCR扩增，上游和下游引物分别为UP primer 2和DOWN primer 2，回收得到450～500bp的PCR产物；(1) Total RNA was extracted from peripheral blood lymphocytes of 2-year-old male alpaca ( PuerLinkTM RNA MiniKit, Life Technologies: 12183018A); the extracted total RNA was reverse-transcribed into cDNA using UP primer 1 as a primer, and the VHH gene was amplified by two rounds of nested PCR; the first round of PCR was based on the transcribed cDNA Template, use UPprimer 1 and DOWN primer 1 as primers, recover a band with a size of 650-750bp after PCR amplification, and then use this as a template to perform the second round of PCR amplification. The upstream and downstream primers are UP primer 2 and DOWN primer 2, recover the PCR product of 450-500bp;
UP primer 1: 5’-GGTACGTGCT GTTGAACTGT TCC-3’UP primer 1: 5’-GGTACGTGCT GTTGAACTGT TCC-3’
DOWN primer 1: 5’-CTTGGTGGTCCTGGCTGCTCT-3’DOWN primer 1: 5’-CTTGGTGGTCCTGGCTGCTCT-3’
UP primer 2: 5’-AAGCTTTTGT GGTTTTGGTG TCTTGGGTTC-3’UP primer 2: 5’-AAGCTTTTGT GGTTTTGGTG TCTTGGGTTC-3’
DOWN primer 2： 5’-AAGCTTGGGG TCTTCGCTGT GGTGCG-3’DOWN primer 2: 5’-AAGCTTGGGG TCTTCGCTGT GGTGCG-3’
(2)用EcoRI和HindIII酶切上述PCR产物，即为VHH基因片段；(2) Digest the above PCR product with EcoRI and HindIII, which is the VHH gene fragment;
(3)用T4连接酶连接T7载体(10-3Cloning Kit,Merck Millipore70550-3)和VHH基因片段，将连接产物进行包装，然后进行扩增(方法见实施例2)，得噬菌体原始文库。(3) Ligate the T7 vector with T4 ligase ( 10-3 Cloning Kit, Merck Millipore 70550-3) and VHH gene fragments, the ligated product was packaged, and then amplified (see Example 2 for the method) to obtain the original phage library.
实施例2 T7噬菌体的液体扩增Example 2 Liquid Amplification of T7 Phage
(1)活化宿主菌：在50ml LB培养基(含0.1mg/mL羧苄青霉素，简称Carb-LB)中接种大肠杆菌BLT5403，37℃170r/min过夜培养，得一代宿主菌；(1) Activation of host bacteria: Inoculate Escherichia coli BLT5403 in 50ml LB medium (containing 0.1mg/mL carbenicillin, referred to as Carb-LB), and culture overnight at 37°C and 170r/min to obtain the first generation host bacteria;
(2)在Carb-LB液体培养基中接种1％的一代宿主菌，摇床培养，到OD600为0.5-1.0，得二代宿主菌；(2) inoculate 1% of the first-generation host bacteria in the Carb-LB liquid medium, and cultivate on a shaking table until theOD600 is 0.5-1.0 to obtain the second-generation host bacteria;
(3)用T7噬菌体感染二代宿主菌，37℃200r/min培养2-3h，观察溶菌情况，溶菌完全立即停止培养。将溶菌产物在8000g离心10min，并回收上清，上清即为扩增的噬菌体。测定滴度。(3) Infect the second-generation host bacteria with T7 phage, culture at 200 r/min at 37°C for 2-3 hours, observe the bacteriolysis situation, and stop the cultivation immediately if the bacteriolysis is complete. The lysate was centrifuged at 8000 g for 10 min, and the supernatant was recovered, which was the amplified phage. Determine the titer.
实施例3 T7噬菌体滴度的测定Example 3 Determination of T7 phage titer
(1)在培养皿中倒入Carb-LB底层培养基，冷却、凝固；(1) Pour the Carb-LB bottom medium into the culture dish, cool and solidify;
(2)溶解顶层培养基，冷却，加入Carb后45℃保温；(2) Dissolve the top culture medium, cool it down, add Carb and keep it warm at 45°C;
(3)用无菌LB液体培养基梯度稀释扩增的噬菌体；(3) The amplified phages are diluted in gradient with sterile LB liquid medium;
(4)300μL二代宿主菌与50μL不同梯度稀释的噬菌体混匀，加入4mL预热Carb-顶层培养基，迅速倒入有底层培养基的平皿中，晃动平皿使之分布均匀；(4) Mix 300 μL of the second-generation host bacteria with 50 μL of phage diluted in different gradients, add 4 mL of preheated Carb-top medium, quickly pour it into a plate with the bottom medium, and shake the plate to make it evenly distributed;
(5)37℃倒置培养3-4h，直到看到大小适中的噬菌斑；(5) Incubate upside down at 37°C for 3-4 hours until moderately sized plaques are seen;
(6)挑选噬菌斑数量为50-500间的平板进行噬菌斑计数，计算噬菌体滴度。(6) Select a plate with 50-500 plaques to count the plaques and calculate the phage titer.
实施例4纳米抗体的筛选Example 4 Screening of Nanobodies
(1)将Aβ用TBS稀释成10μg/mL，调pH至7.0，100μL/孔进行酶标板包被；(1) Dilute Aβ with TBS to 10 μg/mL, adjust the pH to 7.0, 100 μL/well for enzyme plate coating;
(2)室温摇床反应2h后用TBS洗板3次；(2) After reacting in a shaker at room temperature for 2 hours, wash the plate with TBS 3 times;
(3)用1％无蛋白封闭液(购自生工生物工程有限公司)进行封闭，室温摇床1.5h后用TBST洗板6次；(3) Block with 1% protein-free blocking solution (purchased from Sangon Bioengineering Co., Ltd.), shake the plate at room temperature for 1.5 hours, and wash the plate with TBST for 6 times;
(4)加入扩增的噬菌体，100μL/孔，室温摇床孵育30min后用TBST洗板10次，充分去除未结合的噬菌体；(4) Add amplified phage, 100 μL/well, incubate on a shaker at room temperature for 30 min, then wash the plate with TBST 10 times to fully remove unbound phage;
(5)加入T7洗脱缓冲液(1％SDS)洗脱噬菌体，室温摇床30min，并将洗脱液扩增，得下一轮筛选用噬菌体，进入下一轮筛选；(5) Add T7 elution buffer (1% SDS) to elute the phage, shake it at room temperature for 30 minutes, and amplify the eluate to obtain the phage for the next round of screening, and enter the next round of screening;
(6)经过四轮筛选之后，特异性的噬菌体得到富集。(6) After four rounds of screening, specific phages were enriched.
实施例5单克隆噬菌体的序列分析The sequence analysis of embodiment 5 monoclonal phage
经第四轮筛选后，按照实施例3的方法得到单克隆噬菌斑。PCR法扩增后进行序列分析，方法如下：After the fourth round of screening, monoclonal phage plaques were obtained according to the method in Example 3. Sequence analysis was carried out after PCR amplification, as follows:
(1)PCR扩增(1) PCR amplification
①用灭菌牙签挑取噬菌斑，同一噬菌斑一部分放入EDTA溶胶缓冲液中用于PCR，另一部分进行扩增，用于后续ELISA；①Use a sterilized toothpick to pick up the phage plaques, put a part of the same phage plaques into EDTA sol buffer for PCR, and amplify the other part for subsequent ELISA;
②放入EDTA溶胶缓冲液中的部分65℃处理10min；②The part put into EDTA sol buffer was treated at 65°C for 10min;
③冷却至室温，14000g离心5min，取上清98℃热击处理5min；③ Cool to room temperature, centrifuge at 14000g for 5min, take the supernatant and heat shock at 98°C for 5min;
④取2μL作为模板进行PCR扩增；④ Take 2 μL as a template for PCR amplification;
反应条件为：94℃预变性5min后，98℃变性10s，55℃退火30s，72℃延伸60s，30个循环，最后72℃延伸7min；The reaction conditions are: 94°C pre-denaturation for 5 minutes, 98°C denaturation for 10 seconds, 55°C annealing for 30 seconds, 72°C extension for 60 seconds, 30 cycles, and finally 72°C extension for 7 minutes;
(2)将PCR产物送交华大基因测序；(2) Send the PCR product to BGI for sequencing;
(3)将得到的核酸序列转化为氨基酸序列并进行比对，其氨基酸序列为SEQ IDNO.8，由4个框架区(FR)、3个互补决定区(CDR)组成。所述纳米抗体的氨基酸序列以及其组成序列如下。(3) The obtained nucleic acid sequence is converted into an amino acid sequence and compared. The amino acid sequence is SEQ ID NO.8, which consists of 4 framework regions (FR) and 3 complementarity determining regions (CDR). The amino acid sequence of the nanobody and its constituent sequences are as follows.
氨基酸序列如SEQ ID NO.8所示，其中4个框架区FR1、FR2、FR3和FR4的氨基酸序列依次分别为SEQ ID NO.1～4所示；3个互补决定区CDR1、CDR2、CDR3氨基酸序列依次分别为SEQ ID NO.5～7所示；所述框架区、互补决定区在SEQ ID NO.8中位置关系如图1所示。The amino acid sequence is shown in SEQ ID NO.8, wherein the amino acid sequences of the four framework regions FR1, FR2, FR3 and FR4 are respectively shown in SEQ ID NO.1-4; the amino acids of the three complementarity determining regions CDR1, CDR2 and CDR3 The sequences are respectively shown in SEQ ID NO.5-7; the positional relationship of the framework region and complementarity determining region in SEQ ID NO.8 is shown in FIG. 1 .
实施例6 Aβ纳米抗体的表达与分离纯化Example 6 Expression and separation and purification of Aβ nanobody
(1)纳米抗体的表达：(1) Expression of Nanobodies:
①将纳米抗体的基因序列(如SEQ ID NO.9)插入表达载体pET23a，并转化入大肠杆菌BL21(DE3)细胞；① Insert the gene sequence of the nanobody (such as SEQ ID NO.9) into the expression vector pET23a, and transform it into Escherichia coli BL21(DE3) cells;
②用LB培养基，在温度37℃的条件下培养，待菌体OD600达到1-1.5时，加入终浓度为1mmol的IPTG进行诱导；②Use LB medium to culture at a temperature of 37°C. When the OD600 of the bacteria reaches 1-1.5, add IPTG with a final concentration of 1 mmol for induction;
③继续培养3-4小时后弃上清，收集全菌；③ After continuing to cultivate for 3-4 hours, discard the supernatant and collect the whole bacteria;
④用裂解缓冲液重悬菌体后超声裂解细胞，在10000rpm离心5min，收集上清液以及沉淀。以15％SDS-PAGE电泳检测纳米抗体的表达情况，结果如图2所示。④Use the lysis buffer to resuspend the cells, then ultrasonically lyse the cells, centrifuge at 10,000rpm for 5min, and collect the supernatant and precipitate. The expression of the nanobody was detected by 15% SDS-PAGE electrophoresis, and the results are shown in FIG. 2 .
图2中M为蛋白质分子量标准；泳道3为未加IPTG诱导的全菌蛋白；泳道1与2为IPTG诱导后的全菌蛋白，在16.5kDa处有明显的本发明纳米抗体的蛋白表达斑点。比较泳道3与泳道1或2发现，加入IPTG后纳米抗体表达量增加。In Fig. 2, M is the protein molecular weight standard; Swimming lane 3 is the whole bacteria protein induced without IPTG; Swimming lanes 1 and 2 are the whole bacteria protein induced by IPTG, and there are obvious protein expression spots of the Nanobody of the present invention at 16.5kDa. Comparing lane 3 with lane 1 or 2, it was found that the expression of Nanobody increased after adding IPTG.
(2)纳米抗体的纯化：采用Ni-NTA亲和层析柱纯化融合蛋白：(2) Purification of Nanobodies: Ni-NTA affinity chromatography column was used to purify the fusion protein:
①层析柱用上样缓冲液(15mmol PBS，50mmol咪唑，500mmol NaCl)清洗后，加入上述(1)中收集的上清液；① After washing the chromatography column with loading buffer (15mmol PBS, 50mmol imidazole, 500mmol NaCl), add the supernatant collected in (1) above;
②先用清洗缓冲液(15mmol PBS，150mmol咪唑，500mmol NaCl)清洗去除杂蛋白，再用洗脱缓冲液(15mmol PBS，500mmol咪唑，500mmol NaCl)洗脱并收集洗脱液；② Wash with washing buffer (15mmol PBS, 150mmol imidazole, 500mmol NaCl) to remove foreign proteins, then elute with elution buffer (15mmol PBS, 500mmol imidazole, 500mmol NaCl) and collect the eluate;
③纯化后纳米抗体以15％SDS-PAGE电泳检测融合蛋白的纯化情况，结果如图3所示。③ After purification, the nanobody was electrophoresed with 15% SDS-PAGE to detect the purification of the fusion protein, and the results are shown in Figure 3.
图3中SDS-PAGE电泳检测金属螯合层析分离纯化纳米抗体情况。M为蛋白质分子量标准，泳道1为流穿液；泳道2-4为50mM咪唑洗脱；泳道5-11为150mM咪唑洗脱；泳道12-13为500mM咪唑洗脱。图3的结果显示，纳米抗体经镍柱亲和层析处理后，获得条带较为单一的纳米抗体，其分子量约为16.5kDa。In Fig. 3, SDS-PAGE electrophoresis detects the separation and purification of Nanobodies by metal chelation chromatography. M is protein molecular weight standard, lane 1 is flow-through; lanes 2-4 are 50mM imidazole elution; swimming lanes 5-11 are 150mM imidazole elution; swimming lanes 12-13 are 500mM imidazole elution. The results in Figure 3 show that after the nanobody was treated by nickel column affinity chromatography, a nanobody with a relatively single band was obtained, and its molecular weight was about 16.5 kDa.
实施例7 ELISA检测纳米抗体的结合特异性Example 7 ELISA detection of the binding specificity of Nanobodies
(1)将100μl不同序列和类型的Aβ(0.5mg/L)加入到ELISA板上，37℃温育1h后，于4℃冰箱中放置过夜；(1) Add 100 μl of Aβ (0.5 mg/L) of different sequences and types to the ELISA plate, incubate at 37°C for 1 hour, and place in a refrigerator at 4°C overnight;
(2)倒尽板孔中液体，加满PBST洗涤液(NaCl 8g、KH2PO40.2g、Na2HPO4·12H2O2.9g、Tween-200.5ml，用蒸馏水定容至1000ml)，静置3-5min，反复3次，最后将板倒置在吸水纸上，使孔中洗涤液流尽；(2) Pour out the liquid in the wells of the plate and fill up with PBST washing solution (NaCl 8g, KH2 PO4 0.2g, Na2 HPO4 12H2 O 2.9g, Tween-200.5ml, dilute to 1000ml with distilled water), Stand for 3-5min, repeat 3 times, and finally turn the plate upside down on the absorbent paper to drain the washing liquid in the hole;
(3)加封闭液(0.5％牛血清白蛋白，pH7.4的PBS)200μl，37℃放置1h后洗涤，洗涤同步骤(2)；(3) Add 200 μl of blocking solution (0.5% bovine serum albumin, PBS at pH 7.4), place at 37° C. for 1 hour and then wash, the washing is the same as step (2);
(4)每孔加入纳米抗体200μl，1小时后洗涤，洗涤同步骤(2)；(4) Add 200 μl of Nanobody to each well, wash after 1 hour, and wash with step (2);
(5)将anti-6×His Tag antibody(0.5mg/ml)按1:1000稀释，每孔加100μl，37℃放置1h后洗涤，洗涤同步骤(2)；(5) Dilute anti-6×His Tag antibody (0.5mg/ml) at 1:1000, add 100 μl to each well, place at 37°C for 1 hour and wash, the washing is the same as step (2);
(6)将羊抗兔IgG-HRP(0.5mg/ml)按1:1000稀释，每孔加200μl，37℃放置1h；(6) Dilute goat anti-rabbit IgG-HRP (0.5 mg/ml) at 1:1000, add 200 μl to each well, and place at 37°C for 1 hour;
(7)加TMB工作液200μl，于室温下在暗处放置10-15min，加终止液，每孔50μl；(7) Add 200 μl of TMB working solution, place in the dark at room temperature for 10-15 minutes, add stop solution, 50 μl per well;
(8)用酶标仪记录450nm处的读数，结果如图4所示；(8) Record the reading at 450nm with a microplate reader, and the results are as shown in Figure 4;
图4中纳米抗体对不同聚集状态的Aβ(单体、可溶聚集体和纤维)和不同序列的Aβ(Aβ12-35、Aβ40、Aβ42)都有结合能力。The nanobodies in Fig. 4 have the ability to bind Aβ in different aggregation states (monomer, soluble aggregate and fiber) and Aβ in different sequences (Aβ12-35 , Aβ40 , Aβ42 ).
实施例8 MTT检测抗体对Aβ处理细胞的保护作用Example 8 MTT detects the protective effect of antibodies on Aβ-treated cells
(1)人神经母瘤细胞SH-SY5Y以106个/ml接种于25cm2细胞培养瓶，培养液为含有10％胎牛血清(NQBB,Australia)和1％双抗(Thermo Fisher Scientific,USA)的DMEM高糖培养基(Gibco,USA)，培养条件为37℃，5％二氧化碳；(1) Human neuroblastoma cells SH-SY5Y were inoculated in a 25 cm2 cell culture flask at 106 cells/ml, and the culture medium contained 10% fetal bovine serum (NQBB, Australia) and 1% double antibody (Thermo Fisher Scientific, USA) ) DMEM high-glucose medium (Gibco, USA), culture condition is 37 ℃, 5% carbon dioxide;
(2)细胞达到80％汇合度后用0.25％胰蛋白酶消化并传代，取对数生长期的细胞以5000个/孔进行铺板，继续于细胞培养箱中培养24h以使细胞贴壁；(2) After the cells reached 80% confluence, they were digested with 0.25% trypsin and subcultured, and the cells in the logarithmic growth phase were plated at 5000 cells/well, and continued to be cultured in a cell incubator for 24 hours to allow the cells to adhere to the wall;
(3)细胞贴壁后每孔经过PBS洗1次，更换为100μL DMEM高糖培养基/N2添加物(Gibco,USA)，继续培养1h；(3) After the cells adhered to the wall, each well was washed once with PBS, replaced with 100 μL DMEM high-glucose medium/N2 supplement (Gibco, USA), and cultured for 1 hour;
(4)每孔加入终浓度为15μM的Aβ聚集体或Aβ聚集体和不同浓度的纳米抗体(nanobody)，继续培养48h；(4) Aβ aggregates or Aβ aggregates and different concentrations of nanobodies (nanobodies) at a final concentration of 15 μM were added to each well, and cultured for 48 hours;
(5)将培养液吸出，用PBS洗一次，每孔加入90μL DMEM高糖培养基和10μL 5mg/mlMTT(Amresco,USA)溶液，放置于细胞培养箱中继续培养4h；(5) Aspirate the culture solution, wash once with PBS, add 90 μL DMEM high-glucose medium and 10 μL 5 mg/ml MTT (Amresco, USA) solution to each well, and place in a cell culture incubator for 4 hours;
(6)小心吸出每孔中的培养液，加入150μL DMSO，水平震荡使DMSO充分溶解蓝紫色甲臢结晶，测定570nm吸光度，参比波长为630nm。结果如图5所示。(6) Carefully suck out the culture medium in each well, add 150 μL DMSO, shake horizontally to fully dissolve the blue-purple formazan crystals in DMSO, measure the absorbance at 570 nm, and the reference wavelength is 630 nm. The result is shown in Figure 5.
图5中纳米抗体不仅可以逆转Aβ聚集体引起的细胞损伤，并且对SH-SY5Y没有细胞毒性，且还能一定程度上促进SH-SY5Y的增值，且上述效应具有剂量依赖性。The nanobody in Figure 5 can not only reverse the cell damage caused by Aβ aggregates, but also has no cytotoxicity to SH-SY5Y, and can also promote the proliferation of SH-SY5Y to a certain extent, and the above effects are dose-dependent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112649615A (en)* | 2020-12-21 | 2021-04-13 | 大连理工大学 | Method for detecting soluble Abeta oligomers with different sizes and application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101076543A (en)* | 2004-10-13 | 2007-11-21 | 阿布林克斯公司 | Single domain camelide anti-amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenerative neural diseases such as alzheimer's disease |
| CN101970484A (en)* | 2007-12-11 | 2011-02-09 | 葛兰素集团有限公司 | Antigen binding proteins |
| WO2014007982A2 (en)* | 2012-07-03 | 2014-01-09 | Janssen Alzheimer Immunotherapy | C-terminal and central epitope a-beta antibodies |
| CN104558172A (en)* | 2015-01-04 | 2015-04-29 | 东南大学 | Single-domain heavy-chain nanobody for amyloid-beta and application of single-domain heavy-chain nanobody |
| WO2015071856A1 (en)* | 2013-11-13 | 2015-05-21 | F. Hoffmann-La Roche Ag | Camelid single-domain antibody directed against amyloid bêta and methods for producing conjugates thereof |
| CN104662423A (en)* | 2012-03-13 | 2015-05-27 | 杨森阿尔茨海默氏症免疫治疗公司 | Oligomeric Abeta in the diagnosis, prognosis, and monitoring of alzheimer's disease |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101076543A (en)* | 2004-10-13 | 2007-11-21 | 阿布林克斯公司 | Single domain camelide anti-amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenerative neural diseases such as alzheimer's disease |
| CN101970484A (en)* | 2007-12-11 | 2011-02-09 | 葛兰素集团有限公司 | Antigen binding proteins |
| CN104662423A (en)* | 2012-03-13 | 2015-05-27 | 杨森阿尔茨海默氏症免疫治疗公司 | Oligomeric Abeta in the diagnosis, prognosis, and monitoring of alzheimer's disease |
| WO2014007982A2 (en)* | 2012-07-03 | 2014-01-09 | Janssen Alzheimer Immunotherapy | C-terminal and central epitope a-beta antibodies |
| WO2015071856A1 (en)* | 2013-11-13 | 2015-05-21 | F. Hoffmann-La Roche Ag | Camelid single-domain antibody directed against amyloid bêta and methods for producing conjugates thereof |
| CN104558172A (en)* | 2015-01-04 | 2015-04-29 | 东南大学 | Single-domain heavy-chain nanobody for amyloid-beta and application of single-domain heavy-chain nanobody |
| Publication number | Publication date |
|---|---|
| CN105542005A (en) | 2016-05-04 |
| Publication | Publication Date | Title |
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