CN113278073B - NKG2A nano antibody and application thereof - Google Patents

Abstract

The invention relates to an NKG2A nano antibody and application thereof, belonging to the technical field of biological engineering. The amino acid sequence of the NKG2A nano antibody is shown as SEQ ID NO. 1. The NKG2A nano antibody provided by the invention can be specifically combined with NKG2A protein, the sensitivity is 0.532 mu g/ml, and the NKG2A nano antibody can be used for detecting the expression of NKG2A in tissues by immunohistochemistry and WesternBlotting method, thereby providing a method for researching the function of NKG2A gene.

Description

Translated fromChinese

一种NKG2A纳米抗体及其应用A kind of NKG2A nanobody and its application

技术领域technical field

本发明涉及生物工程技术领域，具体涉及一种NKG2A纳米抗体及其应用。The invention relates to the technical field of bioengineering, in particular to an NKG2A nanobody and its application.

背景技术Background technique

骆驼科动物(羊驼、骆驼)和软骨鱼体内的一种天然缺失重链但仍然具有生物活性的特异性抗体称单域抗体，单域抗体的抗原结合位点(VHH)具有独立的抗原识别能力，独立表达的VHH又被称为纳米抗体。与传统的四联体抗体相比，纳米抗体的主要特点有：分子量小，结构简单，理化性质稳定等。纳米抗体的优良特性使其在多种方面具有优势：在抗体进入机体方面，纳米抗体能够穿过动物机体内的一些保护性屏障进入发病部位发挥作用，如血脑屏障、血睾屏障等；在抗原抗体结合方面，能够结合一些隐蔽的抗原表位，特别适用于比较难得到抗体的靶点，如GPCR、离子通道和酶活中心等；在降低生产成本方面，纳米抗体结构简单易于体外表达，同时体外表达不易产生包涵体，生产工艺简单。A specific antibody that naturally lacks the heavy chain but still has biological activity in camelids (alpacas, camels) and cartilaginous fish is called a single-domain antibody, and the antigen-binding site (VHH) of the single-domain antibody has an independent antigen recognition Ability, independent expression of VHH is also known as nanobodies. Compared with traditional tetrameric antibodies, the main characteristics of nanobodies are: small molecular weight, simple structure, stable physical and chemical properties, etc. The excellent characteristics of nanobodies give them advantages in many aspects: in terms of antibodies entering the body, nanobodies can pass through some protective barriers in the animal body and enter the diseased site to play a role, such as the blood-brain barrier, blood-testis barrier, etc.; In terms of antigen-antibody binding, it can bind some cryptic epitopes, and is especially suitable for targets that are difficult to obtain antibodies, such as GPCRs, ion channels, and enzyme activity centers; in terms of reducing production costs, nanobodies are simple in structure and easy to express in vitro. At the same time, in vitro expression is not easy to produce inclusion bodies, and the production process is simple.

NKG2A是NKG2家族中的“抑制性”成员，主要表达在CD56hiNK细胞、NKT细胞和CD8+αβT细胞亚群中。非经典MHC I类分子HLA-E是NKG2A-CD94的主要配体，表达水平比经典MHC I类分子低约25倍，在大多数正常组织中都有表达，NKG2A与HLA-E的相互作用能抑制NK细胞和T细胞的激活。NKG2A抑制剂能促进T细胞和NK细胞的抗肿瘤能力，有望和现有的免疫疗法互补，更好地治疗癌症患者。同时，更多肿瘤相关免疫检查点的开发对于给癌症患者提供选择尤为重要，而NKG2A是多种肿瘤如黑素瘤的免疫检查点。目前，还没有特异性结合NKG2A蛋白的纳米抗体的报道。NKG2A is an "inhibitory" member of the NKG2 family, mainly expressed in CD56hiNK cells, NKT cells and CD8+αβT cell subsets. The non-classical MHC class I molecule HLA-E is the main ligand of NKG2A-CD94, its expression level is about 25 times lower than that of the classical MHC class I molecule, and it is expressed in most normal tissues. The interaction between NKG2A and HLA-E can Inhibits the activation of NK cells and T cells. NKG2A inhibitors can promote the anti-tumor ability of T cells and NK cells, and are expected to complement existing immunotherapy and better treat cancer patients. At the same time, the development of more tumor-associated immune checkpoints is particularly important to provide options for cancer patients, and NKG2A is an immune checkpoint for various tumors such as melanoma. Currently, there are no reports of nanobodies that specifically bind to NKG2A protein.

发明内容Contents of the invention

本发明的目的在于提供一种NKG2A纳米抗体及其应用。本发明所述NKG2A纳米抗体固有的分子量小、理化特征稳定、亲和力高和结构简单易于重组表达制备，能够穿过血脑屏障，在疾病的诊断与治疗中有较大的应用前景。采用本发明提供的NKG2A纳米抗体能够特异性结合NKG2A蛋白，灵敏度为0.532μg/ml，可用于免疫组织化学和Western Blotting法检测组织中NKG2A的表达。The purpose of the present invention is to provide a NKG2A nanobody and its application. The NKG2A nanobody of the present invention has inherent small molecular weight, stable physical and chemical characteristics, high affinity, simple structure, easy recombinant expression and preparation, can pass through the blood-brain barrier, and has great application prospects in the diagnosis and treatment of diseases. The NKG2A nanobody provided by the invention can specifically bind to the NKG2A protein with a sensitivity of 0.532 μg/ml, and can be used to detect the expression of NKG2A in tissues by immunohistochemistry and Western Blotting.

本发明提供了一种NKG2A纳米抗体，所述NKG2A纳米抗体的氨基酸序列如SEQ IDNO.1所示。The present invention provides a NKG2A nanobody, the amino acid sequence of the NKG2A nanobody is shown in SEQ ID NO.1.

优选的是，编码所述NKG2A纳米抗体的基因的核苷酸序列如SEQ ID NO.2所示。Preferably, the nucleotide sequence of the gene encoding the NKG2A Nanobody is shown in SEQ ID NO.2.

本发明还提供了上述技术方案所述NKG2A纳米抗体在制备结合NKG2A蛋白的药物中的应用。The present invention also provides the application of the NKG2A nanobody described in the above technical scheme in the preparation of a drug that binds to the NKG2A protein.

本发明还提供了上述技术方案所述的NKG2A纳米抗体在制备结合NKG2A蛋白的免疫组织化学试剂中的应用。The present invention also provides the application of the NKG2A nanobody described in the above technical scheme in the preparation of immunohistochemical reagents binding to NKG2A protein.

本发明还提供了上述技术方案所述的NKG2A纳米抗体在制备结合NKG2A蛋白的WesternBlotting检测试剂中的应用。The present invention also provides the application of the NKG2A nanobody described in the above technical scheme in the preparation of a Western Blotting detection reagent that binds to the NKG2A protein.

本发明提供了一种NKG2A纳米抗体及其应用。利用噬菌体展示技术进行淘筛，噬菌体展示技术能将表达的外源多肽或蛋白以融合蛋白的形式展示在噬菌体的表面，进而通过亲和富集法筛选表达有特异性蛋白质的噬菌体。本发明采用噬菌体展示实验技术研发的NKG2A纳米抗体能够特异性结合NKG2A蛋白，对NKG2A蛋白的灵敏度为0.532μg/ml，可用于免疫组织化学和Western Blotting法检测组织中NKG2A的表达。The invention provides a NKG2A nanobody and its application. Phage display technology is used for panning. Phage display technology can display the expressed foreign polypeptide or protein on the surface of phage in the form of fusion protein, and then screen the phage expressing specific protein by affinity enrichment method. The NKG2A nanobody developed by the present invention using phage display technology can specifically bind to NKG2A protein, and has a sensitivity of 0.532 μg/ml to NKG2A protein, and can be used to detect the expression of NKG2A in tissues by immunohistochemistry and Western Blotting.

附图说明Description of drawings

图1为本发明提供的Western Blotting对纯化的NKG2A-VHH-His检测；Fig. 1 is Western Blotting provided by the present invention to the detection of purified NKG2A-VHH-His;

图2为本发明提供的考马斯亮蓝检测纯化的NKG2A-VHH检测；Fig. 2 is the NKG2A-VHH detection that the Coomassie brilliant blue detection purification provided by the present invention;

图3为本发明提供的WesternBlot法用NKG2A-VHH纳米抗体检测脾组织中NKG2A蛋白的表达；Fig. 3 is the expression of NKG2A protein in spleen tissue detected by WesternBlot method provided by the present invention with NKG2A-VHH nanobody antibody;

图4为本发明提供的免疫组织化学法用NKG2A纳米抗体检测小鼠脾脏中NKG2A表达分布。Fig. 4 is an immunohistochemical method provided by the present invention using NKG2A Nanobody to detect the expression distribution of NKG2A in mouse spleen.

具体实施方式Detailed ways

本发明提供了一种NKG2A纳米抗体，所述NKG2A纳米抗体的氨基酸序列如SEQ IDNO.1所示：QLVESGGSLVQPGGSLRLSCAASGVALDHYTLGWFRRAPGKEREAVSCISGSEGGTYYADSVKGRFTISGNNAKETVYLQMNSLKPEDTAVYYCAATPPFGAAVFDMCNPHFPFDYWGQGIQVTVSSAHH。The present invention provides a NKG2A nano -antibody that shows the amino acid sequence of NKG2A nano -antibodies as SEQ IDNO.1: QLVESGGSLVQPGGSCAASGVALGWFRAPGKEREAVSGTYADSVKGRFTIS GnnaketvylqmnslkpedTavyCaATPPFGAAVFDMCNPHFPDYWGIQGIQIQVTVSSAHH.

在本发明中，编码所述NKG2A纳米抗体的基因的核苷酸序列如SEQ ID NO.2所示：GCAGCTCGTGGAGTCAGGAGGAAGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGTCGCTTTGGATCATTATACCTTAGGCTGGTTCCGCCGGGCCCCAGGGAAGGAGCGTGAGGCGGTTTCATGTATAAGTGGTAGTGAGGGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAAACAACGCCAAGGAGACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCAACGCCCCCTTTCGGTGCAGCTGTTTTTGACATGTGTAATCCGCACTTTCCGTTTGACTACTGGGGCCAGGGGATCCAGGTCACCGTCTCCTCAGCGCACCACAG。In the present invention, the nucleotide sequence of the gene encoding the NKG2A Nanobody is shown in SEQ ID NO.2: GCAGCTCGTGGAGTCAGGAGGAAGCTTGGTGCAGCCTGGGGGGTCTCCTGAGACTCTCCTGTGCAGCCTCTGGAGTCGCTTTGGATCATTATACCTTAGGCTGGTTCCGCCGGGCCCCAGGGAAGGAGCGTGAGGCGGTTTCATGTATAAGTGG TAGTGAGGGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCGGAAACAACGCCAAGGAGACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCAACGCCCCCTTTCGGTGCAGCTGTTTTTGACATGTGTAATCCGCACTTTCCGTTTGACTACTGGGGCCAGGGGATCCAGGTCACC GTTCTCCTCAGCGCACCACAG.

在本发明中，所述NKG2A纳米抗体的筛选方法，优选包括以下步骤：In the present invention, the screening method of the NKG2A nanobody preferably comprises the following steps:

1)将黑素瘤纳米库进行第一轮淘洗，得到B16-NKG2A-VHH1；1) Carry out the first round of elutriation of the melanoma nano-library to obtain B16-NKG2A-VHH1;

所述第一轮淘洗的NKG2A蛋白的包被浓度为20μg/ml；The coating concentration of the NKG2A protein in the first round of elutriation is 20 μg/ml;

2)将所述步骤1)得到的B16-NKG2A-VHH1依次进行第二轮、第三轮、第四轮淘洗和第五轮淘洗，得到噬菌体溶液；2) The B16-NKG2A-VHH1 obtained in the step 1) is sequentially subjected to the second round, the third round, the fourth round of elutriation and the fifth round of elutriation to obtain a phage solution;

所述第二轮淘洗的NKG2A蛋白的包被浓度为10μg/ml；The coating concentration of the NKG2A protein in the second round of elutriation is 10 μg/ml;

所述第三轮淘洗的NKG2A蛋白的包被浓度为8μg/ml；The coating concentration of the NKG2A protein in the third round of elutriation is 8 μg/ml;

所述第四轮淘洗的NKG2A蛋白的包被浓度为5μg/ml；The coating concentration of the NKG2A protein in the fourth round of elutriation is 5 μg/ml;

所述第五轮淘洗的NKG2A蛋白的包被浓度为5μg/ml；The coating concentration of the NKG2A protein in the fifth round of elutriation is 5 μg/ml;

3)将所述步骤2)得到的噬菌体溶液与TG1菌液混合、感染后进行培养，得到菌株；3) mixing the phage solution obtained in the step 2) with the TG1 bacterial solution, and culturing after infection to obtain a bacterial strain;

4)将所述步骤3)得到的菌株与KM13辅助噬菌体混合、感染，将得到的感染物进行第一振荡培养后进行第一次离心，将得到的第一沉淀经液体培养基重悬后进行第二振荡培养后，进行第二次离心，将得到的第二上清液与封闭液混合、孵育后进行间接ELISA检测，检测第二上清液同NKG2A蛋白的反应性，以确定所述菌株与NKG2A蛋白具有反应性；4) Mix and infect the bacterial strain obtained in step 3) with the KM13 helper phage, perform the first shaking culture on the obtained infection, and then perform the first centrifugation, and resuspend the obtained first precipitate in a liquid medium, then carry out After the second shaking culture, perform the second centrifugation, mix the obtained second supernatant with the blocking solution, and perform indirect ELISA detection after incubation to detect the reactivity of the second supernatant with the NKG2A protein to determine the strain Reactive with NKG2A protein;

所述第一振荡的温度为35～42℃，所述第二振荡的温度为28～32℃；The temperature of the first oscillation is 35-42°C, and the temperature of the second oscillation is 28-32°C;

所述第一离心的离心力为7500～8500g，所述第二离心的离心力为2000～2100g；The centrifugal force of the first centrifugation is 7500-8500g, and the centrifugal force of the second centrifugation is 2000-2100g;

5)将所述步骤4)与NKG2A蛋白具有反应性的菌株进行质粒提取，以所述质粒为模版，用质粒引物对进行PCR扩增，得到纳米抗体VHH片段，将所述纳米抗体VHH片段与表达载体连接，得到重组质粒；5) performing plasmid extraction on the bacterial strain reactive with NKG2A protein in step 4), using the plasmid as a template, and performing PCR amplification with a pair of plasmid primers to obtain a Nanobody VHH fragment, and combining the Nanobody VHH fragment with The expression vector is connected to obtain a recombinant plasmid;

质粒引物包括质粒上游引物和质粒下游引物，所述质粒上游引物的核苷酸序列如SEQ ID NO.3所示，具体如下：Plasmid primers include plasmid upstream primers and plasmid downstream primers, and the nucleotide sequence of the plasmid upstream primers is as shown in SEQ ID NO.3, specifically as follows:

GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA；GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA;

所述质粒下游引物的核苷酸序列如SEQ ID NO.4所示，具体如下：The nucleotide sequence of the downstream primer of the plasmid is as shown in SEQ ID NO.4, specifically as follows:

GACCSASGTCAYCGTCTCCTCAGTCGACTCAGA；GACCSASGTCAYCGTCTCCTCAGTCGACTCAGA;

6)将所述步骤5)得到的重组质粒、pBAD18转入大肠杆菌，得到纳米抗体表达菌株，对所述纳米抗体表达菌株进行IPTG诱导后，提取得到诱导后的纳米抗体表达菌株的蛋白，将所述蛋白进行SDS-PAGE鉴定和Western Blotting鉴定，根据分子量大小和his-tag标签鉴定为NKG2A纳米抗体。6) Transforming the recombinant plasmid and pBAD18 obtained in step 5) into Escherichia coli to obtain a Nanobody expression strain, and after the Nanobody expression strain was induced by IPTG, extracting the protein of the induced Nanobody expression strain, and The protein was identified by SDS-PAGE and Western Blotting, and was identified as NKG2A nanobody according to its molecular weight and his-tag.

本发明对所述黑素瘤纳米库没有特殊限定，优选依据申请号为201910058785.0、发明名称为《一种黑素瘤纳米抗体库的构建方法》的中国专利中公开的黑素瘤纳米库的构建方法构建得到即可。The present invention has no special limitations on the melanoma nano-library, preferably according to the construction of the melanoma nano-library disclosed in the Chinese patent with application number 201910058785.0 and title of invention "A method for constructing a melanoma nano-antibody library" The method can be constructed.

本发明对所述黑素瘤纳米库进行第一轮淘洗，得到B16-NKG2A-VHH1，分装冻存于-80℃。In the present invention, the first round of elutriation is performed on the melanoma nano-bank to obtain B16-NKG2A-VHH1, which is subpackaged and frozen at -80°C.

淘洗时用50mM碳酸钠/碳酸氢钠缓冲液作为包被缓冲液，包被浓度20μg/ml，包被体积2ml，用NKG2A蛋白包被免疫管。During elutriation, 50 mM sodium carbonate/sodium bicarbonate buffer was used as the coating buffer, the coating concentration was 20 μg/ml, the coating volume was 2 ml, and the immunotube was coated with NKG2A protein.

淘洗方法优选如下：The washing method is preferably as follows:

1)将500μl黑素瘤纳米文库接种于100ml 2×YTAG培养基，37℃200rmp振荡培养1小时至OD₆₀₀为0.4；1) Inoculate 500 μl of melanoma nano-library in 100 ml of 2×YTAG medium, culture at 200 rpm at 37°C for 1 hour with shaking until OD₆₀₀ is 0.4;

2)加入KM13辅助噬菌体，100ml菌液加100μl KM13辅助噬菌体，37℃静置感染30min，而后振荡培养30min；2) Add KM13 helper phage, add 100μl KM13 helper phage to 100ml bacterial solution, let stand for infection at 37°C for 30min, then shake and culture for 30min;

3)4000×g离心10min，去除培养基上清，用100ml 2×YTAK培养基重悬菌体沉淀，30℃200rmp振荡培养过夜；3) Centrifuge at 4000×g for 10 minutes, remove the supernatant of the medium, resuspend the bacterial pellet with 100ml 2×YTAK medium, and culture at 30°C and 200rmp overnight;

4)次日上午11000×g，4℃离心过夜培养菌液10min，将上清转至新的离心瓶并加入20ml PEG/NaCl溶液，混匀冰浴90min；4) The next morning, 11000×g, centrifuge overnight culture solution at 4°C for 10 minutes, transfer the supernatant to a new centrifuge bottle and add 20ml PEG/NaCl solution, mix well and bathe in ice for 90 minutes;

5)11000×g，4℃离心30min，弃上清，而后再次离心2min，彻底吸尽上清；5) Centrifuge at 11000×g for 30 minutes at 4°C, discard the supernatant, then centrifuge again for 2 minutes, and thoroughly absorb the supernatant;

6)使用1.3ml PBS缓冲液重悬沉淀，11600×g离心10分钟；6) Use 1.3ml PBS buffer to resuspend the pellet and centrifuge at 11600×g for 10 minutes;

7)回收上清，命名为SR-B16-NKG2A-VHH1，取100μl待用于滴度测定，剩余同1.2mlMPBS溶液混合，室温共孵育1h，得到混合液(MPBS溶液处理过的NKG2A-VHH1)，待用。7) Recover the supernatant, name it SR-B16-NKG2A-VHH1, take 100 μl for titer determination, mix the rest with 1.2ml MPBS solution, and incubate at room temperature for 1 hour to obtain a mixed solution (NKG2A-VHH1 treated with MPBS solution) ,stand-by.

包被蛋白处理优选如下：Coat protein treatment is preferably as follows:

1)包被蛋白次日，将免疫管内的液体倒出，使用PBS缓冲液洗管3次。1) The next day after coating the protein, pour out the liquid in the immunotube, and wash the tube 3 times with PBS buffer.

2)在每管中加满MPBS，室温封闭2h后使用PBS缓冲液洗管3次。2) Each tube was filled with MPBS, blocked at room temperature for 2 hours, and then washed with PBS buffer 3 times.

3)在免疫管中加入2ml上述淘洗步骤7)得到的混合液，室温孵育2h后使用PBST溶液洗管10次，而后用PBS缓冲液洗管10次。3) Add 2ml of the mixture obtained in the elutriation step 7) to the immunotube, incubate at room temperature for 2 hours, wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer solution.

4)在每管中加入2ml 100mM TEA溶液，室温轻摇15min洗脱结合的噬菌体，而后加入2ml Tris-HCl溶液中和。4) Add 2ml 100mM TEA solution to each tube, shake gently at room temperature for 15min to elute the bound phage, and then add 2ml Tris-HCl solution to neutralize.

5)将洗脱的噬菌体(命名为SC-B16-NKG2A-VHH1)转至50ml离心管，并加入16mlOD₆₀₀为0.4的TG1菌液，37℃水浴30分钟，使洗脱的噬菌体感染TG1菌液。(并在免疫管内加入4ml的OD₆₀₀为0.4的TG1菌液进行感染，最后合并，总共24ml的体积)5) Transfer the eluted phage (named SC-B16-NKG2A-VHH1) to a 50ml centrifuge tube, add 16ml of TG1 bacterial solution with an_OD600 of 0.4, bathe in water at 37°C for 30 minutes, and infect the eluted phage with TG1 bacterial solution . (And add 4ml of TG1 bacterial solution with OD₆₀₀ of 0.4 to the immune tube for infection, and finally merge, with a total volume of 24ml)

6)取100μl菌液待用于滴度测定，剩余菌液于4000g离心10min。6) Take 100 μl of bacterial liquid for titer determination, and centrifuge the remaining bacterial liquid at 4000 g for 10 min.

7)使用1ml 2×YT培养基重悬菌体沉淀，将重悬后的菌液涂布于5个2×YTAG固体培养板(150mm平板)，置于30℃孵箱培养过夜。7) Use 1ml 2×YT medium to resuspend the bacterial pellet, spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150mm plates), and culture in a 30°C incubator overnight.

8)次日用2×YT培养基收集平板上长出的菌落，加入60％的甘油至终浓度为15％，其即为一级文库菌，命名为B16-NKG2A-VHH1，分装冻存于-80℃。8) The next day, use 2×YT medium to collect the colony grown on the plate, add 60% glycerol to a final concentration of 15%, which is the first-level library bacteria, named B16-NKG2A-VHH1, subpackage and freeze at -80°C.

测定拯救噬菌体滴度：SR-B16-NKG2A-VHH1进行梯度稀释，稀释度从10^-7～10^-13；每个稀释度取10μl噬菌体感染190μl OD₆₀₀为0.4的TG1菌液；每个稀释度取100μl菌液涂布2×YTAG固体培养板，置于30℃培养箱培养过夜；对测定板上的菌落计数，计算SR-B16-NKG2A-VHH1滴度。Measure rescue phage titer: SR-B16-NKG2A-VHH1 was serially diluted from 10^-7 to 10^-13 ; for each dilution, 10 μl of phage was used to infect 190 μl of TG1 bacterial solution with OD₆₀₀ of 0.4; for each dilution Take 100 μl of bacterial liquid to coat 2×YTAG solid culture plate, and place it in a 30°C incubator for overnight cultivation; count the colonies on the assay plate, and calculate the titer of SR-B16-NKG2A-VHH1.

测定洗脱噬菌体滴度：将用于滴度测定的菌液梯度稀释，稀释度从10^-1～10^-5；每个稀释度取100μl菌液涂布2×YTAG固体培养板，置于30℃培养箱培养过夜；对测定板上的菌落计数，计算SC-B16-NKG2A-VHH1滴度；进而计算第一轮淘洗的输入输出比I/O。Measure the titer of the eluted phage: dilute the bacterial solution used for titer determination in a gradient from 10^-1 to 10^-5 ; take 100 μl of the bacterial solution for each dilution and spread it on a 2×YTAG solid culture plate, place it at 30 Cultivate overnight in an incubator at ℃; count the colonies on the assay plate, and calculate the SC-B16-NKG2A-VHH1 titer; and then calculate the input-output ratio I/O of the first round of elutriation.

在一轮淘洗的基础上，依次进行二至五轮淘洗，所述第二轮淘洗的NKG2A蛋白的包被浓度为10μg/ml，所述第三轮淘洗的NKG2A蛋白的包被浓度为8μg/ml，所述第四轮淘洗的NKG2A蛋白的包被浓度为5μg/ml，所述第五轮淘洗的NKG2A蛋白的包被浓度为5μg/ml。On the basis of one round of elutriation, two to five rounds of elutriation were carried out sequentially. The coating concentration of the NKG2A protein in the second round of elutriation was 10 μg/ml, and the coating concentration of the NKG2A protein in the third round of elutriation was The concentration is 8 μg/ml, the coating concentration of the NKG2A protein in the fourth round of elutriation is 5 μg/ml, and the coating concentration of the NKG2A protein in the fifth round of elutriation is 5 μg/ml.

在本发明中，所述步骤3)培养的温度优选为25～35℃，所述步骤4)感染的时间优选为25～35min，所述步骤4)孵育的时间优选为50～70min。In the present invention, the temperature of the cultivation in step 3) is preferably 25-35°C, the infection time in step 4) is preferably 25-35 minutes, and the incubation time in step 4) is preferably 50-70 minutes.

在本发明中，所述NKG2A纳米抗体的分子量为13KD。In the present invention, the molecular weight of the NKG2A Nanobody is 13KD.

本发明还提供了上述技术方案所述NKG2A纳米抗体在制备结合NKG2A蛋白的药物中的应用。The present invention also provides the application of the NKG2A nanobody described in the above technical scheme in the preparation of a drug that binds to the NKG2A protein.

本发明还提供了上述技术方案所述的NKG2A纳米抗体在制备结合NKG2A蛋白的免疫组织化学试剂中的应用。The present invention also provides the application of the NKG2A nanobody described in the above technical scheme in the preparation of immunohistochemical reagents binding to NKG2A protein.

本发明还提供了上述技术方案所述的NKG2A纳米抗体在制备结合NKG2A蛋白的WesternBlotting检测试剂中的应用。The present invention also provides the application of the NKG2A nanobody described in the above technical scheme in the preparation of a Western Blotting detection reagent that binds to the NKG2A protein.

下面结合具体实施例对本发明所述的一种NKG2A纳米抗体及其应用做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。A NKG2A Nanobody and its applications of the present invention will be further described in detail below in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

实施例1Example 1

NKG2A纳米抗体的筛选方法，包括以下步骤：The screening method of NKG2A nanobody comprises the following steps:

1)将黑素瘤纳米库(根据中国专利CN201910058785.0公开的方法制备即可)进行第一轮淘洗，得到B16-NKG2A-VHH1；1) Perform the first round of elutriation on the melanoma nanobank (prepared according to the method disclosed in Chinese patent CN201910058785.0) to obtain B16-NKG2A-VHH1;

第一轮淘洗的NKG2A蛋白的包被浓度为20μg/ml；The coating concentration of NKG2A protein in the first round of elutriation was 20 μg/ml;

2)将步骤1)得到的B16-NKG2A-VHH1依次进行第二轮、第三轮、第四轮淘洗和第五轮淘洗，得到噬菌体溶液；2) The B16-NKG2A-VHH1 obtained in step 1) is sequentially subjected to the second round, the third round, the fourth round of elutriation and the fifth round of elutriation to obtain a phage solution;

第二轮淘洗的NKG2A蛋白的包被浓度为10μg/ml；The coating concentration of NKG2A protein in the second round of elutriation was 10 μg/ml;

第三轮淘洗的NKG2A蛋白的包被浓度为8μg/ml；The coating concentration of NKG2A protein in the third round of elutriation was 8 μg/ml;

第四轮淘洗的NKG2A蛋白的包被浓度为5μg/ml；The coating concentration of NKG2A protein in the fourth round of elutriation was 5 μg/ml;

第五轮淘洗的NKG2A蛋白的包被浓度为5μg/ml；The coating concentration of NKG2A protein in the fifth round of elutriation was 5 μg/ml;

3)将步骤2)得到的噬菌体液与TG1菌液混合、感染后进行培养，得到菌株；3) mixing the phage liquid obtained in step 2) with the TG1 bacterial liquid, and culturing after infection to obtain bacterial strains;

4)将步骤3)得到的菌株与KM13辅助噬菌体混合、感染，将得到的感染物进行第一振荡培养后进行第一离心，将得到的第一沉淀经液体培养基重悬后进行第二振荡培养后，进行第二离心，将得到的第二上清液与封闭液混合、孵育后进行间接ELISA检测，检测第二上清液同NKG2A蛋白的反应性，以确定所述菌株与NKG2A蛋白具有反应性；4) Mix and infect the strain obtained in step 3) with the KM13 helper phage, perform the first shaking culture on the obtained infection, and then perform the first centrifugation, and resuspend the obtained first precipitate in liquid medium and then perform the second shaking After culturing, perform a second centrifugation, mix the obtained second supernatant with the blocking solution, and perform an indirect ELISA test after incubation to detect the reactivity of the second supernatant with the NKG2A protein to determine that the strain has the NKG2A protein reactivity;

第一振荡的温度为35～42℃，第二振荡的温度为28～32℃；The temperature of the first oscillation is 35-42°C, and the temperature of the second oscillation is 28-32°C;

第一离心的离心力为7500～8500g，第二离心的离心力为2000～2100g；The centrifugal force of the first centrifugal is 7500-8500g, and the centrifugal force of the second centrifugal is 2000-2100g;

5)将步骤4)与NKG2A蛋白具有反应性的菌株进行质粒提取，以质粒为模版，用质粒引物对进行PCR扩增，得到纳米抗体VHH片段，将纳米抗体VHH片段与表达载体连接，得到重组质粒；5) Extract the plasmid from the strain reactive with NKG2A protein in step 4), use the plasmid as a template, and perform PCR amplification with a pair of plasmid primers to obtain a nanobody VHH fragment, and connect the nanobody VHH fragment to an expression vector to obtain a recombinant plasmid;

质粒引物包括质粒上游引物(SEQ ID NO.3)和质粒下游引物(SEQ ID NO.4)；Plasmid primers include plasmid upstream primers (SEQ ID NO.3) and plasmid downstream primers (SEQ ID NO.4);

6)将步骤5)得到的重组质粒、pBAD18转入大肠杆菌，得到纳米抗体表达菌株，对所述纳米抗体表达菌株进行IPTG诱导后，提取得到诱导后的纳米抗体表达菌株的蛋白，将所述蛋白进行SDS-PAGE鉴定和Western Blotting鉴定，根据分子量大小和his-tag标签鉴定为NKG2A纳米抗体。6) Transform the recombinant plasmid and pBAD18 obtained in step 5) into Escherichia coli to obtain a Nanobody expression strain, and after the Nanobody expression strain is induced by IPTG, extract the protein of the induced Nanobody expression strain, and the The protein was identified by SDS-PAGE and Western Blotting, and it was identified as NKG2A Nanobody according to its molecular weight and his-tag.

从已制备的黑素瘤纳米库进行第一轮淘洗，得到B16-NKG2A-VHH1，分装冻存于-80℃。The first round of elutriation was performed from the prepared melanoma nano-library to obtain B16-NKG2A-VHH1, which was aliquoted and frozen at -80°C.

淘洗时用50mM碳酸钠/碳酸氢钠缓冲液作为包被缓冲液，包被浓度20μg/ml，包被体积2ml，以NKG2A蛋白包被免疫管。During elutriation, 50 mM sodium carbonate/sodium bicarbonate buffer was used as the coating buffer, the coating concentration was 20 μg/ml, the coating volume was 2 ml, and the immunotube was coated with NKG2A protein.

淘洗方法如下：The washing method is as follows:

1)将500μl黑素瘤纳米库接种于100ml 2×YTAG培养基，37℃200rmp振荡培养1小时至OD₆₀₀为0.4；1) Inoculate 500 μl of melanoma nanobanks in 100 ml of 2×YTAG medium, culture with shaking at 200 rpm at 37°C for 1 hour until OD₆₀₀ is 0.4;

2)加入KM13辅助噬菌体，100ml菌液加100μl KM13辅助噬菌体，37℃静置感染30min，而后振荡培养30min；2) Add KM13 helper phage, add 100μl KM13 helper phage to 100ml bacterial solution, let stand for infection at 37°C for 30min, then shake and culture for 30min;

3)4000×g离心10min，去除培养基上清，用100ml 2×YTAK培养基重悬菌体沉淀，30℃200rmp振荡培养过夜；3) Centrifuge at 4000×g for 10 minutes, remove the supernatant of the medium, resuspend the bacterial pellet with 100ml 2×YTAK medium, and culture at 30°C and 200rmp overnight;

4)次日上午11000×g，4℃离心过夜培养菌液10min，将上清转至新的离心瓶并加入20ml PEG/NaCl溶液，混匀冰浴90min；4) The next morning, 11000×g, centrifuge overnight culture solution at 4°C for 10 minutes, transfer the supernatant to a new centrifuge bottle and add 20ml PEG/NaCl solution, mix well and bathe in ice for 90 minutes;

5)11000×g，4℃离心30分钟，弃上清，而后再次离心2min，彻底吸尽上清；5) Centrifuge at 11000×g for 30 minutes at 4°C, discard the supernatant, then centrifuge again for 2 minutes, and completely suck up the supernatant;

6)使用1.3ml PBS缓冲液重悬沉淀，而后将其分装于2个1.5ml离心管中，11600×g离心10min；6) Use 1.3ml PBS buffer to resuspend the pellet, then divide it into two 1.5ml centrifuge tubes, and centrifuge at 11600×g for 10min;

7)回收上清，命名为SR-B16-NKG2A-VHH1，取100μl待用于滴度测定，剩余同1.2mlMPBS溶液混合，室温共孵育1h，得到混合液(MPBS溶液处理过的NKG2A-VHH1)，待用。7) Recover the supernatant, name it SR-B16-NKG2A-VHH1, take 100 μl for titer determination, mix the rest with 1.2ml MPBS solution, and incubate at room temperature for 1 hour to obtain a mixed solution (NKG2A-VHH1 treated with MPBS solution) ,stand-by.

包被蛋白处理：Coating Protein Treatment:

1)包被蛋白次日，将免疫管内的液体倒出，使用PBS缓冲液洗管3次。1) The next day after coating the protein, pour out the liquid in the immunotube, and wash the tube 3 times with PBS buffer.

2)在每管中加满MPBS，室温封闭2h后使用PBS缓冲液洗管3次。2) Each tube was filled with MPBS, blocked at room temperature for 2 hours, and then washed with PBS buffer 3 times.

3)在免疫管中加入2ml上述淘洗步骤7)得到的混合液，室温孵育2h后使用PBST溶液洗管10次，而后用PBS缓冲液洗管10次。3) Add 2ml of the mixture obtained in the elutriation step 7) to the immunotube, incubate at room temperature for 2 hours, wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer solution.

4)在每管中加入2ml 100mM TEA溶液，室温轻摇15min洗脱结合的噬菌体，而后加入2ml Tris-HCl溶液中和。4) Add 2ml 100mM TEA solution to each tube, shake gently at room temperature for 15min to elute the bound phage, and then add 2ml Tris-HCl solution to neutralize.

5)将洗脱的噬菌体(命名为SC-B16-NKG2A-VHH1)转至50ml离心管，并加入16mlOD₆₀₀为0.4的TG1菌液，37℃水浴30分钟，使洗脱的噬菌体感染TG1菌液。(并在免疫管内加入4ml的OD₆₀₀为0.4的TG1菌液进行感染，最后合并，总共24ml的体积)。5) Transfer the eluted phage (named SC-B16-NKG2A-VHH1) to a 50ml centrifuge tube, add 16ml of TG1 bacterial solution with an_OD600 of 0.4, bathe in water at 37°C for 30 minutes, and infect the eluted phage with TG1 bacterial solution . (And add 4ml of TG1 bacterium solution with_OD600 of 0.4 to the immune tube for infection, and finally merge, with a total volume of 24ml).

6)取100μl菌液待用于滴度测定，剩余菌液于4000g离心10min。6) Take 100 μl of bacterial liquid for titer determination, and centrifuge the remaining bacterial liquid at 4000 g for 10 min.

7)使用1ml 2×YT培养基重悬菌体沉淀，将重悬后的菌液涂布于5个2×YTAG固体培养板(150mm平板)，置于30℃孵箱培养过夜。7) Use 1ml 2×YT medium to resuspend the bacterial pellet, spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150mm plates), and culture in a 30°C incubator overnight.

8)次日用2×YT培养基收集平板上长出的菌落，加入60％的甘油至终浓度为15％，其即为一级文库菌，命名为B16-NKG2A-VHH1，分装冻存于-80℃。8) The next day, use 2×YT medium to collect the colony grown on the plate, add 60% glycerol to a final concentration of 15%, which is the first-level library bacteria, named B16-NKG2A-VHH1, subpackage and freeze at -80°C.

测定拯救噬菌体滴度：SR-B16-NKG2A-VHH1进行梯度稀释，稀释度从10^-7～10^-13；每个稀释度取10μl噬菌体感染190μl OD₆₀₀为0.4的TG1菌液；每个稀释度取100μl菌液涂布2×YTAG固体培养板，置于30℃培养箱培养过夜；对测定板上的菌落计数，计算SR-B16-NKG2A-VHH1滴度。Measure rescue phage titer: SR-B16-NKG2A-VHH1 was serially diluted from 10^-7 to 10^-13 ; for each dilution, 10 μl of phage was used to infect 190 μl of TG1 bacterial solution with OD₆₀₀ of 0.4; for each dilution Take 100 μl of bacterial liquid to coat 2×YTAG solid culture plate, and place it in a 30°C incubator for overnight cultivation; count the colonies on the assay plate, and calculate the titer of SR-B16-NKG2A-VHH1.

测定洗脱噬菌体滴度：将用于滴度测定的菌液梯度稀释，稀释度从10^-1～10^-5；每个稀释度取100μl菌液涂布2×YTAG固体培养板，置于30℃培养箱培养过夜；对测定板上的菌落计数，计算SC-B16-NKG2A-VHH1滴度；进而计算第一轮淘洗的输入输出比I/O。Measure the titer of the eluted phage: dilute the bacterial solution used for titer determination in a gradient from 10^-1 to 10^-5 ; take 100 μl of the bacterial solution for each dilution and spread it on a 2×YTAG solid culture plate, place it at 30 Cultivate overnight in an incubator at ℃; count the colonies on the assay plate, and calculate the SC-B16-NKG2A-VHH1 titer; and then calculate the input-output ratio I/O of the first round of elutriation.

在一轮淘洗的基础上，依次进行二至五轮淘洗：NKG2A蛋白包被浓度分别为10μg/ml、8μg/ml、5μg/ml、5μg/ml；拯救噬菌体滴度测定稀释度分别为10^-7～10^-12、10^-8～10^-11、10^-8～10^-11、10^-8～10^-11；洗脱噬菌体M13-NKG2A的滴度测定稀释度分别为10^-1～10^-6、10^-1～10^-6、10^-8～10^-11、10^-8～10^-11；洗脱的噬菌体用Tris-HCl溶液(1M，pH值为7.4)中和后，取200μl噬菌体感染800μl OD₆₀₀为0.4的TG1菌液(取100μl进行梯度稀释，剩余的进行保菌)，而后做10^-3～10^-6共4个稀释度，每个稀释度涂布3个2×YTAG固体培养板(150mm平板)，每板100μl菌液，置于30℃培养过夜；对培养板菌落计数，计算滴度，并将培养板标记为平板，置于4℃冰箱待用。On the basis of one round of elutriation, two to five rounds of elutriation were carried out sequentially: NKG2A protein coating concentrations were 10 μg/ml, 8 μg/ml, 5 μg/ml, 5 μg/ml; 10^-7 ～10^-12 , 10^-8 ～10^-11 , 10^-8 ～10^-11 , 10^-8 ～10^-11 ; the titer determination dilutions of eluted phage M13-NKG2A are 10^-1 ～ 10^-6 , 10^-1 ～10^-6 , 10^-8 ～10^-11 , 10^-8 ～10^-11 ; the eluted phages were neutralized with Tris-HCl solution (1M, pH value 7.4), and the Infect 800 μl of TG1 bacterial solution with OD₆₀₀ of 0.4 with 200 μl phage (take 100 μl for gradient dilution, and keep the rest for bacteria preservation), then make 4 dilutions from 10^-3 to 10^-6 , and apply three 2× for each dilution YTAG solid culture plate (150mm plate), 100 μl of bacterial solution per plate, cultured overnight at 30°C; count the colonies on the culture plate, calculate the titer, mark the culture plate as a plate, and place it in a refrigerator at 4°C until use.

特异性纳米抗体的筛选：Screening of specific nanobodies:

单克隆噬菌体上清的制备：从平板各挑取96个单克隆菌株共接种1块96孔深孔培养板，每孔中均含1ml 2×YTAG培养基，培养板分别标记为NKG2A文库菌株，于30℃振荡培养。8h后，从每孔中吸取50μl菌液接种于500ul 2×YTAG培养基，于37℃振荡培养，原平板的剩余菌液中则加入60μl 60％的甘油至终浓度为15％，冻存于-80℃。转接平板于37℃振荡培养1h后，在每孔中加入50μl KM13(60μl KM13+12ml 2×YTAG培养基)辅助噬菌体，37℃静置感染30min，而后37℃振荡培养40min。1800×g离心深孔板10min，弃上清并在每孔中加入400ul 2×YTAK培养基重悬沉淀，30℃振荡培养过夜。次日，最大转速2020xg离心20分钟，从各孔中吸250μl噬菌体上清转移至新的深孔板中，并在每孔中加入250μl封闭液(含3％BSA的PBS缓冲溶液)常温共孵育1小时，待用于间接ELISA检测。Preparation of monoclonal phage supernatant: pick 96 monoclonal strains from the plate and inoculate a 96-well deep-well culture plate, each well contains 1ml 2×YTAG medium, and the culture plates are respectively marked as NKG2A library strains, Shake culture at 30°C. After 8 hours, draw 50 μl of bacterial solution from each well and inoculate it into 500ul 2×YTAG medium, culture it with shaking at 37°C, add 60 μl of 60% glycerol to the remaining bacterial solution on the original plate to a final concentration of 15%, and freeze it in -80°C. After the transfer plate was cultured with shaking at 37°C for 1 hour, 50 μl KM13 (60 μl KM13+12ml 2×YTAG medium) helper phage was added to each well, and the infection was allowed to stand at 37°C for 30 minutes, and then cultured with shaking at 37°C for 40 minutes. Centrifuge the deep-well plate at 1800×g for 10 minutes, discard the supernatant and add 400ul 2×YTAK medium to each well to resuspend the pellet, shake and culture at 30°C overnight. The next day, centrifuge at a maximum speed of 2020xg for 20 minutes, transfer 250 μl of phage supernatant from each well to a new deep-well plate, and add 250 μl of blocking solution (PBS buffer solution containing 3% BSA) to each well for co-incubation at room temperature 1 hour, to be used for indirect ELISA detection.

特异性单克隆噬菌体的鉴定：通过间接ELISA试验检测噬菌体上清同NKG2A蛋白的反应性，具体方法如下：设计实验组、阴性对照组和BSA对照组，实验组与阴性对照组使用NKG2A蛋白，包被96孔酶标板，包被浓度为2μg/ml，BSA对照组使用BSA蛋白包被96孔酶标板，包被浓度为2ug/ml，每孔100μl，置于4℃过夜。次日弃孔内包被液体，在每孔中加入100μl封闭液于37℃封闭1h。弃孔内封闭液，实验组和BSA对照组在每孔分别中加入100μl封闭液处理过的五轮筛选得到的噬菌体上清作为一抗，阴性对照加入等量PBS，37℃孵育1h。用PBST洗液洗板6次。在每孔中加入100μl二抗(HRP-M13 Antibody，稀释度1:6000)，37℃孵育1h。用PBST洗液洗板8次。在每孔中加入100μl显色底物，避光反应5～15min，而后在每孔中加入50μl终止液终止反应。将96孔酶标板置于读板机上读取OD₄₅₀吸收值。对ELISA结果进行分析并确定阳性菌株。Identification of specific monoclonal phage: Detect the reactivity of phage supernatant with NKG2A protein by indirect ELISA test. A 96-well ELISA plate was coated with a coating concentration of 2 μg/ml. The BSA control group was coated with a 96-well ELISA plate with a coating concentration of 2 μg/ml, 100 μl per well, and placed at 4° C. overnight. The next day, the coating liquid in the wells was discarded, and 100 μl of blocking solution was added to each well for blocking at 37°C for 1 h. The blocking solution in the well was discarded, and 100 μl of the phage supernatant obtained from the five rounds of screening treated with the blocking solution was added to each well of the experimental group and the BSA control group as the primary antibody, and the negative control was added with the same amount of PBS, and incubated at 37°C for 1 h. Plates were washed 6 times with PBST wash solution. Add 100 μl of secondary antibody (HRP-M13 Antibody, dilution 1:6000) to each well, and incubate at 37°C for 1 hour. Plates were washed 8 times with PBST wash solution. Add 100 μl of chromogenic substrate to each well, and react in the dark for 5-15 minutes, then add 50 μl of stop solution to each well to stop the reaction. Place the 96-well ELISA plate on a plate reader to read the OD₄₅₀ absorbance value. Analyze the ELISA results and determine the positive strains.

将阳性孔对应的甘油菌接种于5ml 2×YTAG培养基，37℃振荡培养后将菌液送测序公司测序。待测序结果返回后，对测序结果进行分析，选择测序正确的菌株再次重复上述实验，验证阳性菌株，根据NKG2A单克隆阳性菌株ELISA鉴定结果(见表1)确定重组质粒构建菌株。Inoculate the glycerol bacteria corresponding to the positive wells into 5ml 2×YTAG medium, culture with shaking at 37°C, and then send the bacteria solution to the sequencing company for sequencing. After the sequencing results were returned, the sequencing results were analyzed, and the strains with the correct sequencing were selected to repeat the above experiment to verify the positive strains. According to the ELISA identification results of the NKG2A monoclonal positive strains (see Table 1), the recombinant plasmid construction strains were determined.

NKG2A纳米抗体活性和亲和性：NKG2A Nanobody Activity and Affinity:

原核表达重组质粒的构建：将上述测序结果正确的克隆株的甘油菌接种5ml 2×YTAG培养基培养，并利用质粒小量提取试剂盒提取质粒作为原核表达的模板质粒。之后设计用于原核表达的引物，并在引物的5’端和3'端分别引入BamHⅠ和SalI酶切位点。利用设计的引物扩增纳米抗体VHH序列，并通过上述酶切位点将其连接入pQE30原核表达载体，构建纳米抗体原核表达重组质粒以进行纳米抗体的NKG2A特异性鉴定。Construction of recombinant plasmids for prokaryotic expression: inoculate 5 ml of 2×YTAG medium with the glycerol bacteria of the cloned strains with correct sequencing results, and use the plasmid mini-extraction kit to extract the plasmids as template plasmids for prokaryotic expression. Afterwards, primers for prokaryotic expression were designed, and BamHI and SalI restriction sites were introduced at the 5' and 3' ends of the primers, respectively. The designed primers were used to amplify the VHH sequence of the nanobody, and then ligated it into the pQE30 prokaryotic expression vector through the above restriction sites to construct a recombinant plasmid for prokaryotic expression of the nanobody for NKG2A-specific identification of the nanobody.

原核表达的引物：Primers for prokaryotic expression:

F(SEQ ID NO.3):GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA；F (SEQ ID NO. 3): GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA;

R(SEQ ID NO.4):GACCSASGTCAYCGTCTCCTCAGTCGACTCAGA；R (SEQ ID NO.4):GACCSASGTCAYCGTCTCCTCAGTCGACTCAGA;

筛选步骤如下：The screening steps are as follows:

将重组质粒和pBAD18空载转化入BL21(DE3)菌株并获得相应的纳米抗体表达菌株。而后对纳米抗体进行诱导表达，具体方法为：The recombinant plasmid and pBAD18 were transformed into BL21(DE3) strain and the corresponding nanobody expression strain was obtained. Then, the expression of the nanobody is induced, and the specific method is as follows:

将转化后涂板后的菌液进行过夜培养，次日挑取培养板上的单克隆菌落过夜培养。将次日培养的菌液进行保菌。The transformed bacterial solution was cultured overnight, and the next day, the monoclonal colonies on the culture plate were picked and cultured overnight. Bacteria preservation was carried out on the cultured bacteria solution the next day.

吸取10μl甘油菌接种于5mlAmp抗性的LB培养基，37℃振荡培养过夜；Take 10 μl of glycerol bacteria and inoculate them in 5ml of Amp-resistant LB medium, shake and culture overnight at 37°C;

第二天吸取50μl菌液接种5mlAmp抗性的LB培养，各接种2管，37℃振荡培养至OD₆₀₀为0.6；On the second day, inoculate 5ml of Amp-resistant LB culture with 50μl of bacterial liquid, inoculate 2 tubes each, and culture with shaking at 37°C until the_OD600 is 0.6;

在其中1管菌液中加入IPTG诱导(终浓度0.4mM)，另1管不加IPTG做为未诱导对照，15℃振荡培养过夜；Add IPTG to one of the tubes for induction (final concentration 0.4mM), and the other tube without IPTG as the uninduced control, shake culture at 15°C overnight;

同时做BL21(DE3)空菌株对照，空菌株对照培养使用无抗性的LB培养基。At the same time, do BL21 (DE3) empty strain control, and the empty strain control culture uses LB medium without resistance.

纳米抗体的SDS-PAGE鉴定：SDS-PAGE identification of Nanobodies:

将对纳米抗体的表达进行SDS-PAGE鉴定，具体方法为：The expression of nanobodies will be identified by SDS-PAGE, the specific method is:

吸取1ml菌液于1.5ml离心管，13000rpm离心2min；Pipette 1ml of bacterial solution into a 1.5ml centrifuge tube, centrifuge at 13000rpm for 2min;

弃上清，使用PBS缓冲液洗涤菌体沉淀2次；Discard the supernatant, and wash the cell pellet twice with PBS buffer;

用20μl PBS缓冲液重悬菌体沉淀，而后加入5μl 5×蛋白上样缓冲液，并于沸水中煮样5分钟。用10％的聚丙烯酰胺凝胶对样品进行电泳。待电泳结束后，用考马斯亮蓝染液染胶1h，而后用脱色液进行脱色。Resuspend the bacterial pellet with 20 μl PBS buffer, then add 5 μl 5× protein loading buffer, and boil the sample in boiling water for 5 minutes. The samples were electrophoresed on a 10% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue staining solution for 1 h, and then decolorized with decolorizing solution.

具有抗NKG2A中和活性的纳米抗体的筛选：将筛选出的纳米抗体对应甘油菌株接种于5mlAmp抗性的LB培养基，37℃振荡培养10h后转接到500mlAmp抗性的LB培养基中，37℃振荡培养至OD₆₀₀为0.6时加入IPTG(终浓度0.4mM)诱导表达，15℃振荡培养过夜。次日，对上述纳米抗体进行小量纯化。Screening of nanobodies with anti-NKG2A neutralizing activity: Inoculate the glycerol strains corresponding to the selected nanobodies in 5ml of Amp-resistant LB medium, shake and culture at 37°C for 10h, then transfer to 500ml of Amp-resistant LB medium, 37 Cultivate with shaking at ℃ until OD₆₀₀ is 0.6, add IPTG (final concentration 0.4mM) to induce expression, and culture at 15°C overnight with shaking. The next day, a small amount of purification of the above-mentioned Nanobodies was carried out.

NKG2A纳米抗体的亲和性：用5μg/ml的NKG2A包被ELISA板；经BSA封闭后将纯化稀释后的NKG2A纳米抗体作为一抗，分别梯度稀释到5μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml、0.3125μg/ml，进行ELISA鉴定。Affinity of NKG2A nanobody: ELISA plate was coated with 5 μg/ml NKG2A; after blocking with BSA, the purified and diluted NKG2A nanobody was used as the primary antibody, which was serially diluted to 5 μg/ml, 2.5 μg/ml, and 1.25 μg respectively /ml, 0.625μg/ml, 0.3125μg/ml, for ELISA identification.

纯化产物的鉴定：经ELISA鉴定，筛选出亲和性最好的NKG2A纳米抗体，并对该抗体用WesternBlotting法进行his标签鉴定：经SDS-PAGE电泳后，转到NC膜，直接用his二抗进行标记，经显影术显示抗体。Identification of the purified product: After ELISA identification, the NKG2A nanobody with the best affinity was screened out, and the antibody was identified by his tag by Western Blotting method: after SDS-PAGE electrophoresis, it was transferred to NC membrane, and his secondary antibody was used directly Labeling is performed and antibodies are visualized by imaging.

结果：result:

测序和特异性单克隆噬菌体ELISA筛选结果Results of sequencing and ELISA screening of specific monoclonal phages

通过测序公司进行测序，选择能够正确表达VHH片段克隆菌株的进行间接ELISA方法检测单克隆对应的噬菌体上清同NKG2A蛋白的反应性，这些单克隆均同NKG2A蛋白有不同程度的反应性(表1)。Sequencing was performed by a sequencing company, and the cloned strains that could correctly express VHH fragments were selected for indirect ELISA to detect the reactivity of the phage supernatants corresponding to the monoclonals with the NKG2A protein. These monoclonals all had varying degrees of reactivity with the NKG2A protein (Table 1 ).

测序正确并预测的氨基酸序列为：The correct and predicted amino acid sequence is:

SEQ ID NO.1(NKG2A-VHH-A3-1)：SEQ ID NO.1(NKG2A-VHH-A3-1):

QLVESGGSLVQPGGSLRLSCAASGVALDHYTLGWFRRAPGKEREAVSCISGSEGGTYYADSVKGRFTISGNNAKETVYLQMNSLKPEDTAVYYCAATPPFGAAVFDMCNPHFPFDYWGQGIQVTVSSAHH；QLVESGGSLVQPGGSLRLSCAASGVALDHYTLGWFRRAPGKEREAVSCISGSEGGTYYADSVKGRFTISGNNAKETVYLQMNSLKPEDTAVYYCAATPPFGAAVFDMCNPHFPFDYWGQGIQVTVSSAHH;

SEQ ID NO.5(NKG2A-VHH-D2-1)：SEQ ID NO.5(NKG2A-VHH-D2-1):

QLVESGGGLVQAGRSLRLSCTASGFTLEHYAIGWFRQAPGKGREGISCISSNDDTPYYADFVKGRFTISRDNAKNTVFLQMNSLKCEDIVVVHSAIIRARGPRSPSPERTT；QLVESGGGLVQAGRSLRLSCTASGFTLEHYAIGWFRQAPGKGREGISCISSNDDTPYYADFVKGRFTISRDNAKNTVFLQMNSLKCEDIVVVHSAIIRARGPRSPSPERTT;

SEQ ID NO.6(NKG2A-VHH-D3-1)：SEQ ID NO.6(NKG2A-VHH-D3-1):

QLVESGGGLVQPGGSLRLSCAVAGATLDYYKMGWFRQVPGKEREGVSCISRSGLGDGSGLRDGSTAYLDSVKGRFTISRDNTKSTVHLHMNSLKPEDTAIYFCAAAAPRFGERLCRLDEDDFGSWGQGTQVTVSSAHH；QLVESGGGLVQPGGSLRLSCAVAGATLDYYKMGWFRQVPGKEREGVSCISRSGLGDGSGLRDGSTAYLDSVKGRFTISRDNTKSTVHLHMNSLKPEDTAIYFCAAAAAPRFGERLCRLDEDDFGSWGQGTQVTVSSAHH;

SEQ ID NO.7(NKG2A-VHH-H4-1)：SEQ ID NO.7(NKG2A-VHH-H4-1):

QLVESGGGSVPPGSSLRLSCATSGFDLDGYAIMWFRQVPGKRREGVSCIRGSEGTKEYEDSVKGRFTISRDKSKNTVNLQMYNLKPEDTGTYYCAANPYRLCVFEDPQVFDYWGQGTQVIVASAHH；QLVESGGGSVPPGSSLRLSCATSGFDLDGYAIMWFRQVPGKRREGVSCIRGSEGTKEYEDSVKGRFTISRDKSKNTVNLQMYNLKPEDTGTYYCAANPYRLCVFEDPQVFDYWGQGTQVIVASAHH;

SEQ ID NO.8(NKG2A-VHH-G7-2)：SEQ ID NO.8(NKG2A-VHH-G7-2):

QLVESGGGLVPPGGSLRLSCAVSGFTLDDYAIGWFRQVPGKEREGVSCIRSSEGTKEYEDSVKGRFTISRDKSTNTVNLQMYHQKPEDTGTYYCASNPYRLCVFEDPQVFHYWGQGIQVIVASAHHQLVESGGGLVPPGGSLRLSCAVSGFTLDDYAIGWFRQVPGKEREGVSCIRSEGTKEYEDSVKGRFTISRDKSTNTVNLQMYHQKPEDTGTYYCASNPYRLCVFEDPQVFHYWGQGIQVIVASAHH

SEQ ID NO.9(NKG2A-VHH-G8-2)：SEQ ID NO.9(NKG2A-VHH-G8-2):

QLVESGGGLVQPGGSLRLSCVASGGDLSYYAIGWFRQVPGKEREGVACIPYEDGYAKIYADSVKGRFTISRDIAKNTWYLEMDSLKTWDTAVYYCAASARVGFLFRQCTEKGYDYWGQGTQVTVASAHH；QLVESGGGLVQPGGSLRLSCVASGGDLSYYAIGWFRQVPGKEREGVACIPYEDGYAKIYADSVKGRFTISRDIAKNTWYLEMDSLKTWDTAVYYCAASARVGFLFRQCTEKGYDYWGQGTQVTVASAHH;

SEQ ID NO.10(NKG2A-VHH-H2-2)：SEQ ID NO.10(NKG2A-VHH-H2-2):

QLVESGGGLVQAGRSLRLSCTASGFTLEHYAIGWFRQAPGKGREGISCISSNDDTPYYADFVKGRFTISRDNAKNTVFLQMNSLKCEDIVVVHSAIIRARGPRSPSPERTTAKTPAPGHYGQRAAAGAPVPYPDPLEPRAA；QLVESGGGLVQAGRSLRLSCTASGFTLEHYAIGWFRQAPGKGREGISCISSNDDTPYYADFVKGRFTISRDNAKNTVFLQMNSLKCEDIVVVHSAIIRARGPRSPSPERTTAKTPAPGHYGQRAAAGAPVPYPDPLEPRAA;

SEQ ID NO.11(NKG2A-VHH-H7-2)：SEQ ID NO.11(NKG2A-VHH-H7-2):

QLVESGGGMVQSGGSLRLSCEASGFTFSMYGMRWVRQAPGKGPEWVSGISSDGESTYYSESVKGRFTISRDNAKNTLYLQMSNLRPEDTAQCYCVRRVPSSYAMDYWGKGSPVTVSSAHHQLVESGGGMVQSGGSLRLSCEASGFTFSMYGMRWVRQAPGKGPEWVSGISSDGESTYYSEVKGRFTISRDNAKNTLYLQMSNLRPEDTAQCYCVRRVPSSYAMDYWGKGSPVTVSSAHH

SEQ ID NO.12(NKG2A-VHH-H12-2)：SEQ ID NO.12(NKG2A-VHH-H12-2):

QLVESGGGLVQSGGSLNLSCAASGSITTMWAQRWYRQVPGKQRDLVASITDSGSTNYDDSVKGRFTISEDTTANTLDLQMDSLKPEDTAMYYCAASRADRSSSEGDFGMDYWGKGALVIVSSAHHQLVESGGGLVQSGGSLNLSCAASGSITTMWAQRWYRQVPGKQRDLVASITDSGSTNYDDSVKGRFTISEDTTANTLDLQMDSLKPEDTAMYYCAASRADRSSSEGDFGMDYWGKGALVIVSSAHH

表1 NKG2A单克隆ELISA筛选结果Table 1 NKG2A monoclonal ELISA screening results

NKG2A纳米抗体亲和性检测：NKG2A Nanobody Affinity Detection:

ELISA检测后，利用Western blotting实验技术筛选带有His标签菌株，筛选得到的四株单克隆菌株：NKG2A-VHH-A3-1(SEQ ID NO.1)、NKG2A-VHH-D2-1(SEQ ID NO.5)、NKG2A-VHH-D3-1(SEQ ID NO.6)和NKG2A-VHH-H2-2(SEQ ID NO.10)做为特异性单克隆阳性菌株，进行后续试验。After ELISA detection, use Western blotting assay technology to screen strains with His tag, and screen four monoclonal strains obtained: NKG2A-VHH-A3-1 (SEQ ID NO.1), NKG2A-VHH-D2-1 (SEQ ID NO.5), NKG2A-VHH-D3-1 (SEQ ID NO.6) and NKG2A-VHH-H2-2 (SEQ ID NO.10) were used as specific monoclonal positive strains for subsequent tests.

对特异性单克隆进行后续原核表达载体的构建，成功构建NKG2A-VHH-A3-1，NKG2A-VHH-D2-1，NKG2A-VHH-D3-1，NKG2A-VHH-H2-2蛋白表达菌株，NKG2A纳米抗体诱导表达并纯化后，利用ELISA技术进行蛋白亲和检测，筛得一株阳性菌株，检测结果为：NKG2A-VHH-A3-1对NKG2A蛋白的灵敏度为0.532μg/ml(其余菌株与对照组区别较小，灵敏度差)；His标签检测(如图1所示)：用Western Blotting法对纯化的抗体进行His标签检测，具体为将纯化的纳米抗体作为待检蛋白进行western-blot鉴定，使用HRP Anti His-Tag Mouse抗体检测鉴定纯化的纳米抗体，结果发现分子量大小约为13kDa，符合纳米抗体大小，通过分子量大小和特异性结合判断为NKG2A纳米抗体，此NKG2A纳米抗体为灵敏度最高的纳米抗体，序列如SEQ ID NO.1所示。Subsequent construction of prokaryotic expression vectors was carried out on specific monoclonals, and NKG2A-VHH-A3-1, NKG2A-VHH-D2-1, NKG2A-VHH-D3-1, NKG2A-VHH-H2-2 protein expression strains were successfully constructed, After the NKG2A nanobody was induced, expressed and purified, ELISA was used for protein affinity detection, and a positive strain was screened out. The detection result was: the sensitivity of NKG2A-VHH-A3-1 to NKG2A protein was 0.532 μg/ml (the rest of the strains were compared with The difference between the control group is small and the sensitivity is poor); His tag detection (as shown in Figure 1): use the Western Blotting method to perform His tag detection on the purified antibody, specifically using the purified nanobody as the protein to be tested for western-blot identification , using HRP Anti His-Tag Mouse antibody to detect and identify the purified nanobody, the result found that the molecular weight is about 13kDa, which is in line with the size of the nanobody, and it is judged as NKG2A nanobody by molecular weight and specific binding, and this NKG2A nanobody is the most sensitive The nanobody has a sequence as shown in SEQ ID NO.1.

将纯化的纳米抗体作为待检蛋白进行考马斯亮蓝鉴定，判断纯化的纳米抗体是否单一。图2为考马斯亮蓝检测纯化的纳米抗体。从图2可看出纯化得到的抗体比较单一，杂蛋白较少。Use the purified nanobody as the protein to be tested for Coomassie brilliant blue identification to determine whether the purified nanobody is single. Figure 2 shows the purified Nanobodies detected by Coomassie Brilliant Blue. It can be seen from Figure 2 that the purified antibody is relatively simple and has less impurities.

图3为Western blotting法用NKG2A-VHH纳米抗体(即序列如SEQ ID NO.1所示的NKG2A纳米抗体)检测脾组织中NKG2A蛋白的表达。选取小鼠脾脏组织中蛋白样品，将NKG2AVHH作一抗，HRPAnti His-Tag Mouse用作二抗，计算得出NKG2A蛋白分子量26kDa，通过Western blotting实验结果，发现在25kDa的Maker稍上方一点有明显条带，为本发明的目的条带(HEK-293T细胞系用作阴性对照组)。实验结果充分说明纯化的纳米抗体可用于western-blot一抗，且实验效果较为理想。Figure 3 is a Western blotting method using NKG2A-VHH nanobody (that is, the NKG2A nanobody whose sequence is shown in SEQ ID NO.1) to detect the expression of NKG2A protein in spleen tissue. Select the protein sample in the mouse spleen tissue, use NKG2AVHH as the primary antibody, and HRPAnti His-Tag Mouse as the secondary antibody, and calculate the molecular weight of NKG2A protein to be 26kDa. According to the results of Western blotting experiments, it is found that there is a clear bar just above the 25kDa Maker The band is the target band of the present invention (HEK-293T cell line is used as negative control group). The experimental results fully demonstrate that the purified nanobody can be used as the primary antibody of western-blot, and the experimental effect is ideal.

图4为免疫组织化学法用NKG2A纳米抗体检测小鼠脾脏中NKG2A表达分布，箭头标注的部位为用NKG2A-VHH标注的小鼠脾脏中表达NKG2A的部位。选取组织中蛋白样品，将NKG2A纳米抗体用作一抗，对照组用PBS替代，HRP Anti His-Tag Mouse用作二抗，实验结果充分说明纯化的纳米抗体可用于免疫组化一抗，且实验效果理想。Figure 4 shows the expression distribution of NKG2A in the spleen of mice detected by immunohistochemical method with NKG2A nanobody, and the parts marked by arrows are the parts expressing NKG2A in the spleen of mice marked with NKG2A-VHH. The protein samples in the tissue were selected, the NKG2A nanobody was used as the primary antibody, the control group was replaced by PBS, and the HRP Anti His-Tag Mouse was used as the secondary antibody. The experimental results fully demonstrated that the purified nanobody can be used as the primary antibody for immunohistochemistry, and the experiment The effect is ideal.

以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

序列表sequence listing

<110> 山西农业大学<110> Shanxi Agricultural University

<120> 一种NKG2A纳米抗体及其应用<120> A kind of NKG2A nanobody and its application

<160> 12<160> 12

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 130<211> 130

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 1<400> 1

Gln Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly Ser LeuGln Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Val Ala Leu Asp His Tyr Thr LeuArg Leu Ser Cys Ala Ala Ser Gly Val Ala Leu Asp His Tyr Thr Leu

            20                  25                  3020 25 30

Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Ala Val Ser CysGly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Ala Val Ser Cys

        35                  40                  4535 40 45

Ile Ser Gly Ser Glu Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys GlyIle Ser Gly Ser Glu Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly

    50                  55                  6050 55 60

Arg Phe Thr Ile Ser Gly Asn Asn Ala Lys Glu Thr Val Tyr Leu GlnArg Phe Thr Ile Ser Gly Asn Asn Ala Lys Glu Thr Val Tyr Leu Gln

65                  70                  75                  8065 70 75 80

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala AlaMet Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala

                85                  90                  9585 90 95

Thr Pro Pro Phe Gly Ala Ala Val Phe Asp Met Cys Asn Pro His PheThr Pro Pro Phe Gly Ala Ala Val Phe Asp Met Cys Asn Pro His Phe

            100                 105                 110100 105 110

Pro Phe Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr Val Ser Ser AlaPro Phe Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr Val Ser Ser Ala

        115                 120                 125115 120 125

His HisHis His

    130130

<210> 2<210> 2

<211> 393<211> 393

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 2<400> 2

gcagctcgtg gagtcaggag gaagcttggt gcagcctggg gggtctctga gactctcctg 60gcagctcgtg gagtcaggag gaagcttggt gcagcctggg gggtctctga gactctcctg 60

tgcagcctct ggagtcgctt tggatcatta taccttaggc tggttccgcc gggccccagg 120tgcagcctct ggagtcgctt tggatcatta taccttaggc tggttccgcc gggccccagg 120

gaaggagcgt gaggcggttt catgtataag tggtagtgag ggtggcacat actatgcaga 180gaaggagcgt gaggcggttt catgtataag tggtagtgag ggtggcacat actatgcaga 180

ctccgtgaag ggccgattca ccatctccgg aaacaacgcc aaggagacgg tgtatctgca 240ctccgtgaag ggccgattca ccatctccgg aaacaacgcc aaggagacgg tgtatctgca 240

aatgaacagc ctgaaacctg aggacacagc cgtttattac tgtgcagcaa cgcccccttt 300aatgaacagc ctgaaacctg aggacacagc cgtttattac tgtgcagcaa cgcccccttt 300

cggtgcagct gtttttgaca tgtgtaatcc gcactttccg tttgactact ggggccaggg 360cggtgcagct gtttttgaca tgtgtaatcc gcactttccg tttgactact ggggccaggg 360

gatccaggtc accgtctcct cagcgcacca cag 393gatccaggtc accgtctcct cagcgcacca cag 393

<210> 3<210> 3

<211> 33<211> 33

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 3<400> 3

gtgaggatcc gagtctggag grrgcttggt gca 33gtgaggatcc gagtctggag grrgcttggt gca 33

<210> 4<210> 4

<211> 33<211> 33

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 4<400> 4

gaccsasgtc aycgtctcct cagtcgactc aga 33gaccsasgtc aycgtctcct cagtcgactc aga 33

<210> 5<210> 5

<211> 111<211> 111

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 5<400> 5

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Arg Ser LeuGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Arg Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Glu His Tyr Ala IleArg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Glu His Tyr Ala Ile

            20                  25                  3020 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Ile Ser CysGly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Ile Ser Cys

        35                  40                  4535 40 45

Ile Ser Ser Asn Asp Asp Thr Pro Tyr Tyr Ala Asp Phe Val Lys GlyIle Ser Ser Asn Asp Asp Thr Pro Tyr Tyr Ala Asp Phe Val Lys Gly

    50                  55                  6050 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Phe Leu GlnArg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Phe Leu Gln

65                  70                  75                  8065 70 75 80

Met Asn Ser Leu Lys Cys Glu Asp Ile Val Val Val His Ser Ala IleMet Asn Ser Leu Lys Cys Glu Asp Ile Val Val Val His Ser Ala Ile

                85                  90                  9585 90 95

Ile Arg Ala Arg Gly Pro Arg Ser Pro Ser Pro Glu Arg Thr ThrIle Arg Ala Arg Gly Pro Arg Ser Pro Ser Pro Glu Arg Thr Thr

            100                 105                 110100 105 110

<210> 6<210> 6

<211> 138<211> 138

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 6<400> 6

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser LeuGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Ala Val Ala Gly Ala Thr Leu Asp Tyr Tyr Lys MetArg Leu Ser Cys Ala Val Ala Gly Ala Thr Leu Asp Tyr Tyr Lys Met

            20                  25                  3020 25 30

Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ser CysGly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ser Cys

        35                  40                  4535 40 45

Ile Ser Arg Ser Gly Leu Gly Asp Gly Ser Gly Leu Arg Asp Gly SerIle Ser Arg Ser Gly Leu Gly Asp Gly Ser Gly Leu Arg Asp Gly Ser

    50                  55                  6050 55 60

Thr Ala Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg AspThr Ala Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65                  70                  75                  8065 70 75 80

Asn Thr Lys Ser Thr Val His Leu His Met Asn Ser Leu Lys Pro GluAsn Thr Lys Ser Thr Val His Leu His Met Asn Ser Leu Lys Pro Glu

                85                  90                  9585 90 95

Asp Thr Ala Ile Tyr Phe Cys Ala Ala Ala Ala Pro Arg Phe Gly GluAsp Thr Ala Ile Tyr Phe Cys Ala Ala Ala Ala Pro Arg Phe Gly Glu

            100                 105                 110100 105 110

Arg Leu Cys Arg Leu Asp Glu Asp Asp Phe Gly Ser Trp Gly Gln GlyArg Leu Cys Arg Leu Asp Glu Asp Asp Phe Gly Ser Trp Gly Gln Gly

        115                 120                 125115 120 125

Thr Gln Val Thr Val Ser Ser Ala His HisThr Gln Val Thr Val Ser Ser Ala His His

    130                 135130 135

<210> 7<210> 7

<211> 126<211> 126

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 7<400> 7

Gln Leu Val Glu Ser Gly Gly Gly Ser Val Pro Pro Gly Ser Ser LeuGln Leu Val Glu Ser Gly Gly Gly Ser Val Pro Pro Gly Ser Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Ala Thr Ser Gly Phe Asp Leu Asp Gly Tyr Ala IleArg Leu Ser Cys Ala Thr Ser Gly Phe Asp Leu Asp Gly Tyr Ala Ile

            20                  25                  3020 25 30

Met Trp Phe Arg Gln Val Pro Gly Lys Arg Arg Glu Gly Val Ser CysMet Trp Phe Arg Gln Val Pro Gly Lys Arg Arg Glu Gly Val Ser Cys

        35                  40                  4535 40 45

Ile Arg Gly Ser Glu Gly Thr Lys Glu Tyr Glu Asp Ser Val Lys GlyIle Arg Gly Ser Glu Gly Thr Lys Glu Tyr Glu Asp Ser Val Lys Gly

    50                  55                  6050 55 60

Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Val Asn Leu GlnArg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Val Asn Leu Gln

65                  70                  75                  8065 70 75 80

Met Tyr Asn Leu Lys Pro Glu Asp Thr Gly Thr Tyr Tyr Cys Ala AlaMet Tyr Asn Leu Lys Pro Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Ala

                85                  90                  9585 90 95

Asn Pro Tyr Arg Leu Cys Val Phe Glu Asp Pro Gln Val Phe Asp TyrAsn Pro Tyr Arg Leu Cys Val Phe Glu Asp Pro Gln Val Phe Asp Tyr

            100                 105                 110100 105 110

Trp Gly Gln Gly Thr Gln Val Ile Val Ala Ser Ala His HisTrp Gly Gln Gly Thr Gln Val Ile Val Ala Ser Ala His His

        115                 120                 125115 120 125

<210> 8<210> 8

<211> 126<211> 126

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 8<400> 8

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly Gly Ser LeuGln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Pro Gly Gly Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Asp Asp Tyr Ala IleArg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Asp Asp Tyr Ala Ile

            20                  25                  3020 25 30

Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ser CysGly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ser Cys

        35                  40                  4535 40 45

Ile Arg Ser Ser Glu Gly Thr Lys Glu Tyr Glu Asp Ser Val Lys GlyIle Arg Ser Ser Glu Gly Thr Lys Glu Tyr Glu Asp Ser Val Lys Gly

    50                  55                  6050 55 60

Arg Phe Thr Ile Ser Arg Asp Lys Ser Thr Asn Thr Val Asn Leu GlnArg Phe Thr Ile Ser Arg Asp Lys Ser Thr Asn Thr Val Asn Leu Gln

65                  70                  75                  8065 70 75 80

Met Tyr His Gln Lys Pro Glu Asp Thr Gly Thr Tyr Tyr Cys Ala SerMet Tyr His Gln Lys Pro Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Ser

                85                  90                  9585 90 95

Asn Pro Tyr Arg Leu Cys Val Phe Glu Asp Pro Gln Val Phe His TyrAsn Pro Tyr Arg Leu Cys Val Phe Glu Asp Pro Gln Val Phe His Tyr

            100                 105                 110100 105 110

Trp Gly Gln Gly Ile Gln Val Ile Val Ala Ser Ala His HisTrp Gly Gln Gly Ile Gln Val Ile Val Ala Ser Ala His His

        115                 120                 125115 120 125

<210> 9<210> 9

<211> 129<211> 129

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 9<400> 9

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser LeuGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Val Ala Ser Gly Gly Asp Leu Ser Tyr Tyr Ala IleArg Leu Ser Cys Val Ala Ser Gly Gly Asp Leu Ser Tyr Tyr Ala Ile

            20                  25                  3020 25 30

Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala CysGly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Cys

        35                  40                  4535 40 45

Ile Pro Tyr Glu Asp Gly Tyr Ala Lys Ile Tyr Ala Asp Ser Val LysIle Pro Tyr Glu Asp Gly Tyr Ala Lys Ile Tyr Ala Asp Ser Val Lys

    50                  55                  6050 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Thr Trp Tyr LeuGly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Thr Trp Tyr Leu

65                  70                  75                  8065 70 75 80

Glu Met Asp Ser Leu Lys Thr Trp Asp Thr Ala Val Tyr Tyr Cys AlaGlu Met Asp Ser Leu Lys Thr Trp Asp Thr Ala Val Tyr Tyr Cys Ala

                85                  90                  9585 90 95

Ala Ser Ala Arg Val Gly Phe Leu Phe Arg Gln Cys Thr Glu Lys GlyAla Ser Ala Arg Val Gly Phe Leu Phe Arg Gln Cys Thr Glu Lys Gly

            100                 105                 110100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ala Ser Ala HisTyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ala Ser Ala His

        115                 120                 125115 120 125

HisHis

<210> 10<210> 10

<211> 141<211> 141

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 10<400> 10

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Arg Ser LeuGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Arg Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Glu His Tyr Ala IleArg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Glu His Tyr Ala Ile

            20                  25                  3020 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Ile Ser CysGly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Ile Ser Cys

        35                  40                  4535 40 45

Ile Ser Ser Asn Asp Asp Thr Pro Tyr Tyr Ala Asp Phe Val Lys GlyIle Ser Ser Asn Asp Asp Thr Pro Tyr Tyr Ala Asp Phe Val Lys Gly

    50                  55                  6050 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Phe Leu GlnArg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Phe Leu Gln

65                  70                  75                  8065 70 75 80

Met Asn Ser Leu Lys Cys Glu Asp Ile Val Val Val His Ser Ala IleMet Asn Ser Leu Lys Cys Glu Asp Ile Val Val Val His Ser Ala Ile

                85                  90                  9585 90 95

Ile Arg Ala Arg Gly Pro Arg Ser Pro Ser Pro Glu Arg Thr Thr AlaIle Arg Ala Arg Gly Pro Arg Ser Pro Ser Pro Glu Arg Thr Thr Ala

            100                 105                 110100 105 110

Lys Thr Pro Ala Pro Gly His Tyr Gly Gln Arg Ala Ala Ala Gly AlaLys Thr Pro Ala Pro Gly His Tyr Gly Gln Arg Ala Ala Ala Gly Ala

        115                 120                 125115 120 125

Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala AlaPro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala

    130                 135                 140130 135 140

<210> 11<210> 11

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 11<400> 11

Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Ser Gly Gly Ser LeuGln Leu Val Glu Ser Gly Gly Gly Met Val Gln Ser Gly Gly Ser Leu

1               5                   10                  151 5 10 15

Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Met Tyr Gly MetArg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Met Tyr Gly Met

            20                  25                  3020 25 30

Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser GlyArg Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Gly

        35                  40                  4535 40 45

Ile Ser Ser Asp Gly Glu Ser Thr Tyr Tyr Ser Glu Ser Val Lys GlyIle Ser Ser Asp Gly Glu Ser Thr Tyr Tyr Ser Glu Ser Val Lys Gly

    50                  55                  6050 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu GlnArg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln

65                  70                  75                  8065 70 75 80

Met Ser Asn Leu Arg Pro Glu Asp Thr Ala Gln Cys Tyr Cys Val ArgMet Ser Asn Leu Arg Pro Glu Asp Thr Ala Gln Cys Tyr Cys Val Arg

                85                  90                  9585 90 95

Arg Val Pro Ser Ser Tyr Ala Met Asp Tyr Trp Gly Lys Gly Ser ProArg Val Pro Ser Ser Tyr Ala Met Asp Tyr Trp Gly Lys Gly Ser Pro

            100                 105                 110100 105 110

Val Thr Val Ser Ser Ala His HisVal Thr Val Ser Ser Ala His His

        115                 120115 120

<210> 12<210> 12

<211> 125<211> 125

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 12<400> 12

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly Ser LeuGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly Ser Leu

1               5                   10                  151 5 10 15

Asn Leu Ser Cys Ala Ala Ser Gly Ser Ile Thr Thr Met Trp Ala GlnAsn Leu Ser Cys Ala Ala Ser Gly Ser Ile Thr Thr Met Trp Ala Gln

            20                  25                  3020 25 30

Arg Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Asp Leu Val Ala SerArg Trp Tyr Arg Gln Val Pro Gly Lys Gln Arg Asp Leu Val Ala Ser

        35                  40                  4535 40 45

Ile Thr Asp Ser Gly Ser Thr Asn Tyr Asp Asp Ser Val Lys Gly ArgIle Thr Asp Ser Gly Ser Thr Asn Tyr Asp Asp Ser Val Lys Gly Arg

    50                  55                  6050 55 60

Phe Thr Ile Ser Glu Asp Thr Thr Ala Asn Thr Leu Asp Leu Gln MetPhe Thr Ile Ser Glu Asp Thr Thr Ala Asn Thr Leu Asp Leu Gln Met

65                  70                  75                  8065 70 75 80

Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala SerAsp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ser

                85                  90                  9585 90 95

Arg Ala Asp Arg Ser Ser Ser Glu Gly Asp Phe Gly Met Asp Tyr TrpArg Ala Asp Arg Ser Ser Ser Glu Gly Asp Phe Gly Met Asp Tyr Trp

            100                 105                 110100 105 110

Gly Lys Gly Ala Leu Val Ile Val Ser Ser Ala His HisGly Lys Gly Ala Leu Val Ile Val Ser Ser Ala His His

        115                 120                 125115 120 125

Claims (3)

1. An NKG2A nano antibody, which is characterized in that the amino acid sequence of the NKG2A nano antibody is shown in SEQ ID NO. 1.

2. Use of the NKG2A nanobody of claim 1 for the preparation of an immunohistochemical agent binding to NKG2A protein.

3. The NKG2A nanobody of claim 1 is used for preparing a Western Blotting detection reagent combined with NKG2A protein.

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