技术领域technical field
本发明涉及生物医学与分子生物学领域，涉及一种针对退行性神经系统疾病致病因子ß淀粉样蛋白的单域重链纳米抗体。The invention relates to the fields of biomedicine and molecular biology, and relates to a single-domain heavy chain nanobody targeting degenerative nervous system disease pathogenic factor β-amyloid.
背景技术Background technique
退行性神经系统疾病的代表性病症阿尔兹海默病（Alzheimer's disease，AD），俗称老年痴呆症，是一种持续性神经功能障碍，也是老年人群中痴呆症最普遍的成因。在2006年，全世界约有2600万名阿兹海默病病患，到2050年时预估全球发病率为1/85。研究显示阿兹海默病与大脑中的老年斑块（senile plaque，即amyloid plaque）和神经纤维纠结有关。目前的治疗仅能帮助缓解疾病的症状，并没有能够停止或是反转阿兹海默病病程的治疗方法。截至2012年为止，已有超过1000个临床试验研究如何治疗阿兹海默病。目前较有治愈潜力的为各种针对ß淀粉样蛋白（amyloid-beta，Aβ）所开发的基因工程抗体。Aβ多肽可包含不同数目的氨基酸，常见形式如Aβ42和Aβ40。这些针对Aβ的人源化的抗体可以通过抑制Aβ的聚集来消除其对神经元的毒性作用，或者通过清除已形成的老年斑块来达到回复神经系统功能的效果。尽管如此，这些抗体由于体积较大（150kd左右），不能轻易穿过血脑屏障，也容易失活、变性，稳定性差，溶解性低及生产成本高昂，难以在临床上应用。Alzheimer's disease (AD), commonly known as senile dementia, is a representative disease of degenerative nervous system diseases. It is a kind of persistent neurological dysfunction and the most common cause of dementia in the elderly population. In 2006, there were approximately 26 million Alzheimer's patients worldwide, and the global incidence rate is estimated to be 1 in 85 by 2050. Alzheimer's disease is linked to senile plaques (amyloid plaques) and tangles of nerve fibers in the brain, research has linked. Current treatments can only help relieve the symptoms of the disease, and there is no treatment that can stop or reverse the course of Alzheimer's disease. As of 2012, there have been more than 1,000 clinical trials investigating how to treat Alzheimer's disease. At present, there are various genetically engineered antibodies developed against ß amyloid (amyloid-beta, Aβ) that have more potential for healing. Aβ polypeptides may contain varying numbers of amino acids, common forms such as Aβ42 and Aβ40. These humanized antibodies against Aβ can eliminate its toxic effect on neurons by inhibiting the aggregation of Aβ, or restore the function of the nervous system by clearing the formed senile plaques. However, due to their large size (about 150kd), these antibodies cannot easily pass through the blood-brain barrier, are easily inactivated, denatured, have poor stability, low solubility and high production costs, making it difficult to apply them clinically.
1993 年比利时科学家首次报道驼类血液中存在不含轻链的抗体，这些缺失轻链的“单域重链抗体”（VHH）能像正常抗体一样与抗原等靶标紧密结合，而且不会像常规抗体那样互相沾粘，甚至聚集成块，另外不会因为含有传统抗体的Fc 段而引起补体反应。这种抗体只包含一个重链可变区和两个常规的CH2 与CH3 区，更重要的是表达出来的单域重链抗体具有很好的结构稳定性与抗原结合活性。由于此种VHH 的分子量只是普通抗体的1/10，大小仅有2-5 nm，因此VHH 也称纳米抗体（Nanobody）；与此同时纳米抗体化学性质也更加灵活，稳定性好，可溶性高且容易获得，同时可以轻易穿过血脑屏障，到达靶部位起作用。因此通过筛选出针对Aβ的单域重链纳米抗体（命名为NB1）可以有效克服常规抗体在治疗过程中的缺点，可开发成用于治疗Aβ为致病因子的抗体药物。此外，由于纳米抗体的化学、物理稳定性好，仅含有约130个氨基酸，便于基因工程改造或化学偶联上多种标记物，如生物素等，因此可以用于开发成针对Aβ的检测试剂盒。In 1993, Belgian scientists reported for the first time that there were antibodies without light chains in the blood of camelids. These "single-domain heavy chain antibodies" (VHH) lacking light chains can bind tightly to targets such as antigens like normal antibodies, and will not bind to targets like conventional antibodies. Antibodies stick to each other like that, and even aggregate into clumps. In addition, it will not cause complement reaction because of the Fc segment of traditional antibodies. This antibody only contains one heavy chain variable region and two conventional CH2 and CH3 regions. More importantly, the expressed single domain heavy chain antibody has good structural stability and antigen binding activity. Since the molecular weight of this kind of VHH is only 1/10 of that of ordinary antibodies, and the size is only 2-5 nm, VHH is also called nanobody (Nanobody); at the same time, the chemical properties of nanobody are more flexible, good stability, high solubility and It is easy to obtain, and at the same time, it can easily pass through the blood-brain barrier and reach the target site to take effect. Therefore, the single-domain heavy chain nanobody (named NB1) against Aβ can effectively overcome the shortcomings of conventional antibodies in the treatment process, and can be developed into an antibody drug for the treatment of Aβ as a pathogenic factor. In addition, due to the good chemical and physical stability of the nanobody, which only contains about 130 amino acids, it is convenient for genetic engineering or chemical coupling to various markers, such as biotin, etc., so it can be used to develop a detection reagent for Aβ box.
发明内容Contents of the invention
本发明所解决的技术问题是：提供一种针对Aβ的单域重链纳米抗体及其氨基酸序列，同时提供该单域重链纳米抗体的DNA编码序列，并提出该抗体在制备针对Aβ检测试剂和治疗阿兹海默病的药物方面的应用。The technical problem solved by the present invention is to provide a single-domain heavy chain nanobody against Aβ and its amino acid sequence, and at the same time provide the DNA coding sequence of the single-domain heavy chain nanobody, and propose that the antibody can be used in the preparation of Aβ detection reagents. and drugs for the treatment of Alzheimer's disease.
为了解决上述技术问题，本发明所采用的技术方案是：In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:
提出了一种针对Aβ的单域重链纳米抗体，其VHH链包括框架区FR和互补决定区CDR，其特征在于，所述框架区FR包括FR1、FR2、FR3、FR4的氨基酸序列，其氨基酸序列分别为SEQ ID NO:1所示FR1，SEQ ID NO:2所示FR2，SEQ ID NO:3所示的FR3，SEQ ID NO:4所示的FR4；所述互补决定区CDR包括 CDR1、CDR2、CDR3的氨基酸序列，其氨基酸序列分别为SEQ ID NO:5所示的CDR1，SEQ ID NO:6所示的CDR2，SEQ ID NO：7所示的CDR3。A single-domain heavy chain nanobody for Aβ is proposed, its VHH chain includes a framework region FR and a complementarity determining region CDR, characterized in that the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4, and its amino acid The sequences are FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 2, FR3 shown in SEQ ID NO: 3, and FR4 shown in SEQ ID NO: 4; the complementarity determining region CDR includes CDR1, The amino acid sequences of CDR2 and CDR3 are respectively CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7.
其中，所述的针对Aβ的单域重链纳米抗体的VHH链具有SEQ ID NO：8所示的氨基酸序列。Wherein, the VHH chain of the single domain heavy chain nanobody against Aβ has the amino acid sequence shown in SEQ ID NO:8.
还提供一种DNA分子，它用以编码上述针对Aβ的单域重链纳米抗体的VHH链，其核苷酸序列如SEQ ID NO：9所示。Also provided is a DNA molecule, which is used to encode the VHH chain of the above-mentioned single domain heavy chain nanobody against Aβ, the nucleotide sequence of which is shown in SEQ ID NO:9.
除此之外，还提供一种表达载体，它包含上述DNA分子的核苷酸序列，一种宿主细胞，它可以表达上述针对Aβ的单域重链纳米抗体。In addition, it also provides an expression vector, which contains the nucleotide sequence of the above-mentioned DNA molecule, and a host cell, which can express the above-mentioned single-domain heavy chain nanobody against Aβ.
本发明还提供了针对Aβ的单域重链纳米抗体用于制备检测Aβ试剂中的用途以及用于制备治疗阿兹海默病药物中的用途。The present invention also provides the use of the single-domain heavy chain nanobody against Aβ in the preparation of reagents for detecting Aβ and the use in the preparation of drugs for treating Alzheimer's disease.
最后，提出一种用于检测β淀粉样蛋白的检测试剂盒，该试剂盒包括上面所述的针对β淀粉样蛋白的单域重链纳米抗体。Finally, a detection kit for detecting β-amyloid is proposed, which includes the above-mentioned single-domain heavy chain nanobody against β-amyloid.
本发明的有益效果是：与现有技术相比，本发明的优点如下：The beneficial effects of the present invention are: compared with prior art, the present invention has the following advantages:
本发明采用Aβ肽段免疫新疆双峰驼，随后利用该骆驼外周血淋巴细胞建立了针对Aβ的单域重链纳米抗体基因库，将Aβ偶联在酶标板上，以此形式的抗原利用噬菌体展示技术筛选免疫性的单域重链纳米抗体基因库，从而获得了针对Aβ特异性的单域重链纳米抗体基因，将此基因转入大肠杆菌中，建立了能在大肠杆菌中高效表达的单域重链纳米抗体株，根据序列比对软件分析各个克隆株的基因序列，获得针对Aβ单域重链纳米抗体VHH链的氨基酸序列，并证明了该单域重链纳米抗体可以同Aβ特异性结合，从而可以应用到Aβ检测试剂和治疗阿兹海默病药物的开发。In the present invention, Aβ peptides are used to immunize Xinjiang Bactrian camels, and then the peripheral blood lymphocytes of the camels are used to establish a single-domain heavy chain nanobody gene library for Aβ, and Aβ is coupled to an enzyme-labeled plate to utilize the antigen in this form Phage display technology screened the immune single-domain heavy chain nanobody gene library, thus obtaining the Aβ-specific single-domain heavy chain nanobody gene, which was transferred into E. coli, and the high-efficiency expression in E. coli was established The single-domain heavy chain nanobody strain was analyzed according to the sequence comparison software to analyze the gene sequences of each clone, and the amino acid sequence of the VHH chain of the Aβ single-domain heavy-chain nanobody was obtained, and it was proved that the single-domain heavy-chain nanobody could be identical to Aβ Specific binding, so that it can be applied to the development of Aβ detection reagents and drugs for the treatment of Alzheimer's disease.
附图说明Description of drawings
图1是对于所构建的噬菌体展示单域重链纳米抗体文库进行插入率检测的菌落PCR电泳图，其中泳道1是DNA分子marker，泳道2-25是所构建的Aβ单域重链纳米抗体文库中随机挑取的克隆PCR检测电泳图；Figure 1 is a colony PCR electrophoresis image for the detection of insertion rate of the constructed phage display single domain heavy chain nanobody library, wherein lane 1 is a DNA molecular marker, and lanes 2-25 are the constructed Aβ single domain heavy chain nanobody library PCR detection electropherograms of randomly selected clones;
图2是表达的针对Aβ单域重链纳米抗体，经镍柱亲和层析纯化前后的SDS-PAGE的电泳图，M: 蛋白分子标记，单位为KDa；1: 样品与镍柱结合前，破菌后蛋白总的粗提液样品；2: 总的蛋白粗提液过镍柱后的样品（15KDa处无条带说明单域重链纳米抗体吸附停留在镍柱上）；3-7: 共5步含500毫摩尔咪唑洗脱液洗脱的样品（3-7递减显示至第5轮基本完全洗脱）。Figure 2 is the SDS-PAGE electrophoresis of the expressed Aβ single domain heavy chain nanobody before and after purification by nickel column affinity chromatography, M: protein molecular marker, the unit is KDa; 1: before the sample is combined with the nickel column, The total protein crude extract sample after breaking the bacteria; 2: The total protein crude extract sample after passing through the nickel column (the absence of a band at 15KDa indicates that the single domain heavy chain nanobody is adsorbed on the nickel column); 3-7: Samples eluted in a total of 5 steps containing 500 mM imidazole eluent (decreases 3-7 show almost complete elution to round 5).
图3是以Aβ单域重链纳米抗体NB1为核心材料制作的基于ELISA技术的Aβ检测试剂盒原理示意图。Figure 3 is a schematic diagram of the principle of an Aβ detection kit based on ELISA technology made with the Aβ single domain heavy chain nanobody NB1 as the core material.
附图标记： 1.包被的Aβ单域重链纳米抗体NB1；2. 抗原Aβ；3. Aβ抗体；4. 酶标抗体；5. 辣根过氧化物酶。Reference signs: 1. Coated Aβ single domain heavy chain nanobody NB1; 2. Antigen Aβ; 3. Aβ antibody; 4. Enzyme-labeled antibody; 5. Horseradish peroxidase.
具体实施方式Detailed ways
下面将结合说明书附图，对本发明作进一步的说明。The present invention will be further described below in conjunction with the accompanying drawings.
本发明首先将经过KLH蛋白偶联的人工合成Aβ作为抗原免疫一只新疆双峰驼，经过4次免疫之后提取该双峰驼外周血淋巴细胞并构建了Aβ特异的单域重链抗体文库。将Aβ偶联在酶标板上，展示正确的空间结构，使得Aβ的抗原表位得以暴露出来，以此形式的抗原利用噬菌体展示技术筛选Aβ免疫性的纳米抗体基因库（骆驼重链抗体噬菌体展示基因库），而获得了能在大肠杆菌中高效表达的单域重链纳米抗体株。In the present invention, a Xinjiang Bactrian camel is first immunized with artificially synthesized Aβ coupled with KLH protein as an antigen, and after 4 times of immunization, peripheral blood lymphocytes of the Bactrian camel are extracted and an Aβ-specific single domain heavy chain antibody library is constructed. The Aβ is coupled to the microtiter plate, and the correct spatial structure is displayed, so that the antigenic epitope of the Aβ can be exposed, and the antigen in this form is screened by the phage display technology to screen the Aβ immune nanobody gene library (camel heavy chain antibody phage display gene library), and obtained a single-domain heavy chain nanobody strain that can be highly expressed in Escherichia coli.
下面结合具体实施例，进一步阐述本发明。Below in conjunction with specific embodiment, further illustrate the present invention.
实施例1：针对Aβ的单域重链抗体NB1 文库的构建Example 1: Construction of single domain heavy chain antibody NB1 library against Aβ
（1）由KLH蛋白偶联的合成Aβ肽段20mg(购自南京金斯瑞生物科技有限公司)与弗氏佐剂等体积混合，免疫一只新疆双峰驼（购自句容圣龙家畜养殖厂），每周一次，共免疫4 次，除第一次使用完全的弗氏佐剂，剩余几次全部使用弗式不完全佐剂，免疫过程中刺激B淋巴细胞表达抗原特异性的针对Aβ的单域重链纳米抗体VHH。（2）4 次免疫结束后，提取骆驼外周血淋巴细胞100ml 并提取总RNA。（3）将提取的RNA 反转录成cDNA。（3）利用套式PCR 扩增VHH 链，结果如图1 显示，该片段的大小约为400 bp。（4）使用限制性的内切酶PstI 及NotI 酶切20μg pComb3 噬菌体展示载体（Biovector 供应）及10μg VHH基因片段的PCR纯化产物， 并连接两个片段。（5）将连接产物电转化至电转感受态细胞TG1（购自北京神州红叶科技有限公司）中，构建Aβ的单域重链纳米抗体VHH 噬菌体展示文库并测定库容，库容的大小为6×108 。文库构建完成后，为检测文库的插入率，我们随机的选取24 颗克隆做菌落PCR（图一）。结果显示我们的插入率近100% 。(1) 20 mg of synthetic Aβ peptide coupled with KLH protein (purchased from Nanjing GenScript Biotechnology Co., Ltd.) was mixed with an equal volume of Freund’s adjuvant to immunize a Xinjiang Bactrian camel (purchased from Jurong Shenglong Livestock Breeding plant), once a week, a total of 4 times of immunization, except for the first use of complete Freund's adjuvant, the rest of the few times all use Freund's incomplete adjuvant, stimulate B lymphocytes to express antigen-specific targeting Single domain heavy chain nanobody VHH of Aβ. (2) After the 4 times of immunization, extract 100ml of camel peripheral blood lymphocytes and extract total RNA. (3) Reverse transcribe the extracted RNA into cDNA. (3) Nested PCR was used to amplify the VHH chain. As shown in Figure 1, the size of the fragment was about 400 bp. (4) Use restriction endonucleases PstI and NotI to digest 20 μg of pComb3 phage display vector (supplied by Biovector) and 10 μg of PCR purified product of VHH gene fragment, and connect the two fragments. (5) The ligation product was electrotransformed into electroporation-competent cells TG1 (purchased from Beijing Shenzhou Hongye Technology Co., Ltd.), and the single-domain heavy chain nanobody VHH phage display library of Aβ was constructed and the storage capacity was determined. The storage capacity was 6×108 . After the library was constructed, in order to test the insertion rate of the library, we randomly selected 24 clones for colony PCR (Figure 1). The results show that our insertion rate is nearly 100%.
实施例2：针对Aβ的单域重链纳米抗体NB1的筛选过程：Example 2: Screening process for single domain heavy chain nanobody NB1 against Aβ:
（1） 取200微升转化TG1细胞加入到100毫升2×TY培养基中，37℃培养3小时。（2）加入40 微升 VCSM13辅助噬菌体，室温静置30分钟。（3）离心10分钟，将离心沉淀下来的细胞重悬接入250 毫升 2×TY培养基，37℃培养过夜；（4）将培养液8000 rpm离心30分钟，取上清液，用PEG/NaCl沉淀扩增后的噬菌体，并将沉淀后的噬菌体溶解在PBS溶液中。（5）将溶解在100毫摩尔 pH 8.2 NaHCO3 中的Aβ 200微克偶联在酶标板上，4℃放置过夜，同时设立负对照。（26）第二天两个孔中分别加入100微升 0.1%酪蛋白，室温封闭2小时。（7）2小时后，加入100微升采用上述步骤4所述方法收集的噬菌体，在室温下作用1小时。(8）用 PBST（PBS中含有0.05%吐温20）洗5遍，洗去不结合的噬菌体。(9）用三乙基胺 （100 毫摩尔 ）将与Aβ特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选，相同筛选过程重复3-4轮。在不断地筛选的过程中，阳性的克隆将不断的被富集，从而达到了利用噬菌体展示技术筛取阳性克隆的目的。(1) Add 200 microliters of transformed TG1 cells into 100 milliliters of 2×TY medium and incubate at 37°C for 3 hours. (2) Add 40 microliters of VCSM13 helper phage and let stand at room temperature for 30 minutes. (3) Centrifuge for 10 minutes, resuspend the centrifuged cells into 250 ml of 2×TY medium, and incubate overnight at 37°C; (4) Centrifuge the culture solution at 8000 rpm for 30 minutes, take the supernatant, and use PEG/ The amplified phages were precipitated with NaCl, and the precipitated phages were dissolved in PBS solution. (5) Couple 200 micrograms of Aβ dissolved in 100 millimolar pH 8.2 NaHCO3 to a microtiter plate, place it at 4°C overnight, and set up a negative control at the same time. (26) On the second day, add 100 microliters of 0.1% casein to the two wells respectively, and block for 2 hours at room temperature. (7) After 2 hours, add 100 microliters of phages collected by the method described in step 4 above, and let them react for 1 hour at room temperature. (8) Wash 5 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phages. (9) Use triethylamine (100 mmol) to dissociate the phage that specifically binds to Aβ, and infect Escherichia coli TG1, which is in logarithmic growth, to produce and purify the phage for the next round of screening, the same The screening process was repeated for 3-4 rounds. In the process of continuous screening, positive clones will be continuously enriched, thus achieving the purpose of screening positive clones using phage display technology.
实施例3：用噬菌体的酶联免疫方法（ELISA）筛选特异性单个阳性克隆：Example 3: Using phage enzyme-linked immunosorbent method (ELISA) to screen specific single positive clones:
（1）从上述4轮筛选后含有噬菌体的细胞培养皿中，挑选96个单个菌落并接种于含有100微克氨苄青霉素每毫升的TB培养基(1升TB培养基中含有2.3克磷酸二氢钾，12.52克磷酸氢二钾，12克蛋白胨，24克酵母提取物，4毫升甘油)中，生长至对数期后，加入终浓度为1毫摩尔的IPTG，28℃培养过夜。（2）利用渗透法获得粗提抗体，并将抗体转移到经抗原包被的ELISA板中，在室温下放置1小时。（3）用PBST洗去未结合的抗体，加入一抗mouse anti-HA tag antibody（鼠抗HA抗体，购自北京康为世纪生物科技有限公司），在室温下放置1小时。(1) From the cell culture dish containing phage after the above four rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 micrograms of ampicillin per milliliter (1 liter of TB medium contains 2.3 grams of potassium dihydrogen phosphate , 12.52 grams of dipotassium hydrogen phosphate, 12 grams of peptone, 24 grams of yeast extract, 4 milliliters of glycerol), after growing to the logarithmic phase, adding IPTG with a final concentration of 1 mmol, and culturing overnight at 28°C. (2) Use the infiltration method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour. (3) Wash off unbound antibodies with PBST, add primary mouse anti-HA tag antibody (mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), and place at room temperature for 1 hour.
（4）用PBST洗去未结合的抗体，加入anti-mouse alkaline phosphatase conjugate（山羊抗小鼠碱性磷酸酶标记抗体，购自艾美捷科技有限公司），在室温下放置1小时。（5）用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，于ELISA仪上，在405 nm波长读取吸收值。（6）当样品孔OD值大于对照孔OD值2倍以上时，判为阳性克隆孔。（7）将阳性克隆接种至5毫升含100微克氨苄青霉素每毫升的LB液体中以便提取质粒并进行测序。(4) Wash off unbound antibody with PBST, add anti-mouse alkalinePhosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody, purchased from Aimeijie Technology Co., Ltd.), placed at room temperature for 1 hour. (5) Wash off the unbound antibody with PBST, add alkaline phosphatase chromogenic solution, and read the absorbance at a wavelength of 405 nm on the ELISA instrument. (6) When the OD value of the sample well is more than 2 times the OD value of the control well, it is judged as a positive clone well. (7) Inoculate positive clones into 5 ml of LB liquid containing 100 micrograms of ampicillin per ml for plasmid extraction and sequencing.
根据序列比对软件Vector NTI分析各个克隆株的基因序列，把CDR1，CDR2，CDR3序列相同的株视为同一克隆株，而其序列不同的株视为不同克隆株，最终共有1株抗体。其抗体的氨基酸序列为SEQ ID NO：8所示的FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4区，构成整个VHH。According to the sequence comparison software Vector NTI, the gene sequences of each clone were analyzed, and the strains with the same CDR1, CDR2, and CDR3 sequences were regarded as the same clone, while the strains with different sequences were regarded as different clones, and finally there was one antibody strain. The amino acid sequence of its antibody is FR1-CDR1-FR2-CDR2- shown in SEQ ID NO: 8The FR3-CDR3-FR4 region constitutes the entire VHH.
实施例4：抗Aß单域重链纳米抗体NB1在宿主菌大肠杆菌中表达、纯化：Example 4: Expression and purification of the anti-Aß single domain heavy chain nanobody NB1 in the host bacteria Escherichia coli:
（1）将前面测序分析所获得不同克隆株的质粒电转化到大肠杆菌WK6中，并将其涂布在含有100微克每毫升氨苄青霉素和2%葡萄糖 LB固体培养基的板上，37℃过夜，（2）挑选单个菌落接种在15毫升含有氨苄青霉素的LB培养液中，37℃摇床培养过夜，（3）接种1毫升的过夜菌种至330 毫升 TB培养基中，37℃摇床培养，培养到OD值达到0.6-1.0时，加入IPTG，28℃摇床培养过夜，（4）第二天，离心收菌，（5）将菌体利用渗透法使菌体蛋白释放出来以获得抗体粗提液，（6）经镍柱亲和层析纯化抗体蛋白，为获得高纯度的抗体，采用咪唑梯度洗脱法，低浓度咪唑洗脱液（50毫摩尔）用于洗去杂带，高浓度咪唑洗脱液（500毫摩尔）最终可制备纯度达90%以上的蛋白。图2是表达的针对Aβ的单域重链抗体NB1在镍柱亲和层析纯化过程中各步骤的SDS-PAGE的电泳图；其中泳道1是与镍柱结合前，破菌后蛋白总的粗提液样品，泳道2是总的蛋白粗提液过镍柱后的样品（15KDa处无条带说明该抗体吸附停留在镍柱上），泳道3-7是共5轮含500毫摩尔咪唑洗脱液洗脱的样品，其中3-7递减说明至第5轮基本完全洗脱。(1) Electrotransform the plasmids of different clones obtained from the previous sequencing analysis into Escherichia coli WK6, and spread it on a plate containing 100 micrograms per milliliter of ampicillin and 2% glucose LB solid medium, overnight at 37°C , (2) Pick a single colony and inoculate it in 15 ml of LB medium containing ampicillin, and culture it on a shaker at 37°C overnight, (3) inoculate 1 ml of the overnight strain into 330 ml of TB medium, and culture it on a shaker at 37°C , when the OD value reaches 0.6-1.0, add IPTG, shake overnight at 28°C, (4) the next day, collect the bacteria by centrifugation, (5) use the osmosis method to release the protein of the bacteria to obtain antibodies Crude extract, (6) Purify antibody protein by nickel column affinity chromatography. In order to obtain high-purity antibody, adopt imidazole gradient elution method, and low-concentration imidazole eluent (50 mmol) is used to wash away impurity bands, A high-concentration imidazole eluent (500 mmol) can finally prepare a protein with a purity of more than 90%. Figure 2 is the SDS-PAGE electrophoresis of each step of the expressed single domain heavy chain antibody NB1 against Aβ in the purification process of nickel column affinity chromatography; where lane 1 is the total protein content before binding to the nickel column and after breaking the bacteria Crude extract sample, lane 2 is the sample after the total protein crude extract passed through the nickel column (the absence of a band at 15KDa indicates that the antibody is adsorbed on the nickel column), lane 3-7 is a total of 5 rounds containing 500 mmol imidazole Samples eluted with eluent, where the 3-7 decrease indicates that the elution is basically complete by the fifth round.
实施例5：经纯化后的Aβ单域重链纳米抗体在制备用于检测样品中Aβ含量的试剂盒方面的应用。Example 5: Application of the purified Aβ single domain heavy chain nanobody in the preparation of a kit for detecting the Aβ content in a sample.
如图3所示，将Aβ单域重链纳米抗体NB1首先包被在ELISA 板中，加入不同梯度浓度的抗原Aβ标准品（如1ng/ml至1mg/ml），平行操作加入待检测Aβ的样品，在室温下轻摇30分钟。用PBST 洗去未结合的抗原Aβ后，再加入Aβ单域重链抗体或其它Aβ抗体，以及用于显色反应的、可以识别Aβ单域重链抗体或其他常规Aβ抗体的标记二抗（如可以识别兔源抗Aβ常规抗体的辣根过氧化物酶标记抗体，艾美捷科技有限公司）。在室温下放置1 小时，用PBST 洗去未结合的抗体后，加入碱性磷酸酶显色液，于ELISA 仪上，在405nm 波长，读取吸收值。首先将各浓度梯度的Aβ标准品的吸收值做出浓度标准曲线，然后将待检测样品的吸收值读数带入浓度标准曲线，即可判断出样品中的Aβ含量。As shown in Figure 3, the Aβ single-domain heavy chain nanobody NB1 was first coated on the ELISA plate, and antigen Aβ standards of different gradient concentrations (such as 1ng/ml to 1mg/ml) were added, and the Aβ to be detected was added in parallel. Samples were shaken gently for 30 min at room temperature. After washing away the unbound antigen Aβ with PBST, add the Aβ single domain heavy chain antibody or other Aβ antibodies, and the labeled secondary antibody for color reaction that can recognize the Aβ single domain heavy chain antibody or other conventional Aβ antibodies ( For example, the horseradish peroxidase-labeled antibody that can recognize the rabbit-derived anti-Aβ conventional antibody, Aimeijie Technology Co., Ltd.). Leave it at room temperature for 1 hour, wash off the unbound antibody with PBST, add alkaline phosphatase chromogenic solution, and read the absorbance value at 405nm wavelength on the ELISA instrument. Firstly, the absorption value of the Aβ standard substance in each concentration gradient is used to make a concentration standard curve, and then the absorption value reading of the sample to be tested is brought into the concentration standard curve to determine the Aβ content in the sample.
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements are possible, which fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.
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| CN201510001584.9ACN104558172A (en) | 2015-01-04 | 2015-01-04 | Single-domain heavy-chain nanobody for amyloid-beta and application of single-domain heavy-chain nanobody |
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| CN101555477A (en)* | 2009-04-22 | 2009-10-14 | 北京交通大学 | Heavy and light chain variable region gene of monoclonal antibody resisting human amyloid |
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| CN105542005A (en)* | 2016-02-03 | 2016-05-04 | 大连理工大学 | A nanobody against human amyloid beta peptide and its application |
| CN105542005B (en)* | 2016-02-03 | 2018-11-09 | 大连理工大学 | A nanobody against human amyloid beta peptide and its application |
| CN106282214A (en)* | 2016-08-03 | 2017-01-04 | 康众（北京）生物科技有限公司 | A kind of method of quick acquisition nano antibody and application thereof |
| 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 |
| CN114578066A (en)* | 2022-05-07 | 2022-06-03 | 北京第一生物化学药业有限公司 | Products and methods for detecting amyloid beta |
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