CN106831991A - The nano antibody of anti-c Myc labels - Google Patents

Abstract

The invention belongs to the field of genetic engineering, specifically a single-domain heavy chain antibody directed against the c-Myc tag, which has the amino acid sequence shown in SEQ ID NO.: 1, and can be used in the fields of immunoassay, antigen enrichment and purification, and the like. The amino acid sequence provided by the present invention can be used as a precursor to be modified by random or site-directed mutagenesis techniques to obtain mutants with better properties (affinity, specificity, stability, etc.) for development and further use in medicine, Industrial, agricultural proteins or peptides.

Description

Nanobodies against c-Myc tag

technical field

The present invention relates to single domain heavy chain antibody technology (also known as nanobody technology), and genetic engineering antibody technology, especially single domain heavy chain antibody or polypeptide directed at c-Myc tag.

technical background

The discovery of the c-Myc tagged protein originated in 1985 when Evan prepared a monoclonal antibody 9E10 against the human proto-oncogene product Myc protein. Later studies found that the epitope recognized by the antibody consists of 10 amino acid residues, and its sequence It is Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and these 10 amino acids can still maintain strong antigenic activity after fusion expression with other proteins, and can be recognized by corresponding antibodies without being affected by The effect of the protein framework. Therefore, the c-Myc tag system is widely used in the fields of immunological detection, cell imaging, affinity purification and protein engineering.

The invention discloses a single-domain heavy chain antibody (ie nanobody, the same below) that can specifically bind to a c-Myc tag, which can be used for detection and purification of c-Myc tag fusion proteins.

At present, there are monoclonal or polyclonal antibodies against c-Myc tag in the market for detection, but the development and production process of monoclonal antibodies is extremely cumbersome and complicated, and the sources of polyclonal antibodies are limited. In contrast, single-domain heavy-chain antibodies consist of only one domain, and have the advantages of acid and alkali resistance, high temperature resistance, high specificity, small molecular weight, and large-scale production. The purification medium has the advantages of low cost and reusability, and has broad application prospects.

Contents of the invention

The purpose of the present invention is to provide a single domain heavy chain antibody against c-Myc tag, which can be used to prepare reagents and tools for detecting and purifying c-Myc tag.

The present invention provides a single-domain heavy chain antibody against c-Myc tag (that is, a nanobody against c-Myc tag according to the present invention, the same below), which has the amino acid sequence shown in SEQ ID NO.:1. The IMGT numbering of its amino acid sequence and the division of structural domains include four framework regions (Framework region, FR) and three complementarity-determining regions (Complementarity-determining region, CDR).

The present invention provides a nucleic acid molecule, which is characterized in that it encodes SEQ ID NO.: 1, and the specific sequence of the nucleic acid molecule can be obtained at any time through the genetic code.

The present invention also provides a nucleic acid molecule, which is characterized in that it encodes a partial structural domain of SEQ ID NO.: 1, and the specific sequence of the nucleic acid molecule can be obtained at any time through the genetic code. It may be the nucleic acid molecule of SEQ ID NO.:2.

The nucleotide sequence or at least a part of the sequence provided by the present invention can be expressed through a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include bacteria, yeast, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.

The present invention also provides a vector comprising the nucleic acid sequence. Due to the degeneracy of the genetic code, the nucleic acid sequence may vary according to different application purposes.

The present invention also provides a host cell including the protein or expression vector.

The present invention also provides a method for detecting c-Myc tag, comprising the single domain heavy chain antibody against c-Myc tag of the present invention. A c-Myc tag detection method is established based on the ability of the c-Myc tag-specific single domain heavy chain antibody provided by the present invention to specifically bind to the c-Myc tag. Among them, preferred methods include enzyme-linked immunosorbent assay (Enzyme-linkedimmunosorbent assay, ELISA), fluorescence immunoassay (Fluoroimmunoassay, FIA), immunochip method, affinity chromatography and immunochromatography, etc.

The amino acid sequence provided by the present invention can be used as a precursor to be modified by random or site-directed mutagenesis to obtain mutants with better properties (water solubility, stability, affinity and specificity, etc.), which can interact with c-Myc Tag-specific binding.

The present invention also relates to the application of the aforementioned single domain heavy chain antibody against c-Myc tag in immunodetection, enrichment and purification. These immunoassays refer to immunoassays for non-disease diagnosis and treatment purposes.

The present invention also relates to an immunoaffinity adsorption material for c-Myc tag, including a carrier, and a ligand carried on the carrier, which is characterized in that the material uses a nanobody for c-Myc tag as a ligand, and the c-Myc tag is used as a ligand. The Myc-tagged nanobody has the amino acid sequence shown in SEQ ID NO.:1. The carrier material is not limited to agarose gel, and silicon spheres, nano magnetic beads, etc. can also be used.

Some terms described in the present invention have the following meanings:

Domain: The basic structural unit of the tertiary structure of a protein, usually with a certain function.

IMGT numbering: A standardized antibody amino acid sequence numbering method in the IMGT database (The International ImMunoGeneTics Datbase). For the specific numbering method, please refer to the literature (Ehrenman, F., Q.Kaas, et.al. (2010). IMGT/3D structure-DB and IMGT/DomainGapAlign: adatabase and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF.Nucleic Acids Res 38(Database issue):D301-307.Lefranc,M.P.,C.Pommie,et al.(2003).IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains Dev comp Immunol27 (1):55-77.) described in.

Codon: also known as triplet codon (triplet code), refers to a nucleotide triplet corresponding to a certain amino acid. The insertion of this amino acid into the growing polypeptide chain is determined during translation.

detailed description

The present invention will be further described through the preparation, analysis and application of single domain heavy chain antibody (polypeptide) below, and these specific examples should not be interpreted as limiting the scope of application of the present invention in any way.

Example 1:

Construction of an immune library of anti-c-Myc tag single domain heavy chain antibody (ie single domain heavy chain antibody against c-Myc tag)

The c-Myc tag was covalently coupled to bovine serum albumin (BSA) to obtain the c-Myc artificial antigen c-Myc-BSA. After emulsifying 300 μg of c-Myc-BSA with Freund's complete adjuvant, Alpacas (Lama pacos) were immunized by subcutaneous multipoint injection. For booster immunization, 150 μg c-Myc-BSA was emulsified with Freund's incomplete adjuvant at intervals of 2 weeks. Blood was collected from the vein 7 days after each immunization, and the serum titer was determined by indirect ELISA method. The sample with the highest serum titer was selected to separate lymphocytes. cells, RNA was extracted.

The extraction of RNA was carried out according to the instruction manual of RNAiso reagent from TAKARA company. Using RNA as a template and oligo dT as a primer, the first strand of cDNA was synthesized according to the instructions of TAKARA reverse transcriptase.

The gene encoding the variable region of the heavy chain antibody was obtained by nested PCR using PrimeSTAR high-fidelity DNA polymerase (see Table 1 for the primers used). In the first round of PCR, primers AlpVh-LD and CH2-R were used to amplify cDNA respectively, and the reaction conditions were 98°C, 10s, 55°C, 20s, 72°C, 1min, 20 cycles, 98°C, 10s, 68°C, 1min , 72°C for 10 min.

Use 1.2% agarose gel electrophoresis on the first round of PCR products to recover DNA fragments of 600bp to 750bp as templates for the second round of PCR, using primers AlpVh-SfiI and AlpVHHR1-NotI, AlpVh-SfiI and AlpVHHR2-NotI respectively , amplified, the reaction conditions are, 98°C, 10s, 50°C, 20s, 72°C, 40s, 5 cycles, 98°C, 10s, 68°C, 40s, 30 cycles, 72°C extension 10min. The DNA fragments were recovered and quantified by a DNA fragment recovery kit, and stored at -20°C for future use. The phagemid pHEN1 and the PCR amplification product were digested with Sfi I and Not I respectively, recovered and quantified by agarose gel, then ligated overnight at 16°C at a molar ratio of 1:3.

Table 1 Primers used for library construction and identification

Note: The underline indicates the restriction endonuclease recognition sequence

After ethanol precipitation, the ligated product was dissolved in 10 μL sterile water, and electroporated ten times to transform Escherichia coli TG1. Take 10 μL of the electroshocked and cultured bacterial solution and dilute it in multiples, spread ampicillin on a 2×YT culture plate, and calculate the storage capacity. All the rest were spread on a 24cm×24cm ampicillin 2×YT culture plate, cultured upside down at 37°C for 13-16 hours. Scrape and wash the bacterial lawn on the culture plate with 10 mL of 2×YT medium, add glycerol with a final concentration of 20-30%, aliquot, and store at -80°C for later use.

According to the calculated pool capacity results, inoculate 10 times the pool volume of living cells in 20 mL of 2×YT (containing 2% glucose, 100 μg/mL ampicillin), and culture at 37°C and 200 r/min until the OD ₆₀₀ reaches 0.5, according to the multiplicity of infection Add helper phage 20:1, 37°C, 200r/min, 60min. Centrifuge the culture, resuspend the pellet with 50mL of 2×YT (containing 100μg/mL ampicillin and 50μg/mL kanamycin), cultivate overnight at 37°C, 200r/min, centrifuge at 8000rpm to take the supernatant, add 5× PEG/NaCl solution, put it on ice for 1.5h or overnight at 4°C, centrifuge at 8000rpm for 30min, resuspend the pellet in phosphate buffer solution (PBS, 0.01M, pH 7.4) containing 10% glycerol, and obtain the anti-c-Myc tag single domain For the heavy chain antibody immune library, take 10 μL to measure the titer, and store the rest at -80°C for future use.

Example 2:

Panning and Identification of Anti-c-Myc Tag Single Domain Heavy Chain Antibody

The single domain heavy chain antibody against c-Myc tag was panned from the anti-c-Myc tag single domain heavy chain antibody immune library obtained in Example 1 by solid phase affinity panning. Add 120 μL of Myc-GST fusion protein (a protein fused with Myc tag and glutathione) diluted with PBS to each enzyme-labeled well, and coat overnight at 4°C. The coating concentration for each round of panning is 100, 75, 50 μg/mL; aspirate the coating solution, wash the plate 5 times with PBS, add 300 μL 3% BSA-PBS to each well, block for 2 hours at 37°C; wash the plate 5 times with PBS, add 100 μL phage antibody library (about 1×10 ¹¹ CFU), 37°C, incubate for 2.0 h; aspirate unbound phage, wash the plate with PBST (containing 0.5% Tween-20) for 3-5 times (increase 5 times for each round), and then wash the plate with PBS for 15-25 times ;Elute the phage adsorbed in the enzyme-labeled well with 100 μL eluent (glycine-hydrochloric acid, pH 2.2), neutralize the eluate with 35 μL Tris-HCl (1mol/L, pH 8.0), take 10 μL for drop The remaining 125 μL of the eluate was amplified and used for the next round of panning.

After four rounds of panning, the helper phage KM13 was used to rescue the randomly selected monoclonals to obtain phage particles displaying antibody variable regions, and then the binding activity and specificity of the phage particles were determined by indirect phage-ELISA. For the control, see Table 2 for the specific sample addition steps.

Table 2 Indirect phage-ELISA loading table

The ELISA positive clones were sent to the Biotechnology Service Company for sequence determination to obtain the DNA sequence of the insert fragment, which encodes a single domain heavy chain antibody against the c-Myc tag.

DNA sequence (SEQ ID NO.: 2):

CAGTTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGCCTGGGGAGTCACTGACGCTCACCTGTGTAGCCTCAGGCGGCACTTTGGAGAATTGGGATATAGCCTGGTTCCGTCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCGCATATTTTAGTAGTGATGGTATTAGAAATTATGGAGACTCCATGAGGGGCCGATTCAAAATCTCTAGAGACAACGCCAAGAACACAGCGTGGCTGCAGATGAATCGCCTGAGCGTTGAAGACACGTCCACATATTATTGTGCGGCCAAAGAAAGGGTCGGACGTGCATGGCGAAATGATGGACTTTACCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

The encoding has the amino acid sequence shown in SEQ ID NO.:1:

QLQLVESGGGLVQPGESLTLTTCVASGGTLENWDIAWFRQAPGKEREGVAYFSSDGIRNYGDSMRGRFKISRDNAKNTAWLQMNRLSVEDTSTYYCAAKERVGRAWRNDGLYRYWGQGTQVTVSS.

Example 3:

Scale Production of Anti-c-Myc Tag Single Domain Heavy Chain Antibody

Obtaining the DNA fragment encoding the anti-c-Myc tag single-domain heavy-chain antibody: 1. Using restriction endonuclease SfiI/NotI, double-digest the phagemid pHEN-anti-c-Myc single-domain heavy-chain antibody gene, and agar Glycogel electrophoresis to recover the anti-c-Myc tag single domain heavy chain antibody gene; 2. Directly send the anti-c-Myc tag single domain heavy chain antibody coding sequence to a biotechnology service company for chemical synthesis; 3. Design specific primers, through PCR technology amplifies from a cDNA library derived from alpaca (Lama pacos).

The obtained anti-c-Myc tag single domain heavy chain antibody gene fragment was cloned into the expression vector pET25-flag (the c-Myc tag carried by the vector itself was replaced with the Flag tag: DYKDDDDK), identified by PCR and enzyme digestion, and constructed Complete the E. coli expression plasmid of anti-c-Myc tag single domain heavy chain antibody.

The expression plasmid was transformed into Escherichia coli rosetta, and a single colony was picked for induced expression. Insert a single colony into 5mL LB-A (Luria-Bertani broth with 100μg/mL ampicillin) liquid medium, shake at 37°C and 220r/min for 12h; transfer it to 50mL In LB-A liquid medium, shake culture at 37°C and 220r/min until OD ₆₀₀ reaches 0.5 (about 3-3.5h), add IPTG with a final concentration of 0.1mM, induce culture at 30°C and 200r/min.

Induce the culture to centrifuge at 8000r/min, add 25mL of phosphate buffer (pH 7.4) to the cell pellet and mix well, then centrifuge at 8000r/min, remove the supernatant and keep the cell pellet; add 15mL of the same buffer to the cell pellet, mix well, Ultrasonic cell disruption treatment on ice, the condition of ultrasonic disruption is 200W, 2s, 5s, 250 cycles in total, centrifuge the cell disrupted product at 8000r/min for 15min at 4°C, take the supernatant for affinity chromatography purification and SDS- For PAGE electrophoresis analysis, or add glycerol with a final concentration of 20% to the supernatant, mix well, and store in a -20°C freezer until use.

By optimizing the induced expression conditions (such as host bacteria, expression vectors, induction culture time, temperature, and IPTG concentration, etc.), the expression of the target protein (single domain heavy chain antibody) can be further increased, and it is possible to prepare a large number of anti-c-Myc tag single domain heavy chain antibodies. Chain antibodies provide access.

Example 4:

Fusion Expression of Anti-c-Myc Tag Single Domain Heavy Chain Antibody

The anti-c-Myc tag single domain heavy chain antibody gene of the present invention is cloned into the fusion expression vector pAP (containing alkaline phosphatase gene), identified by PCR and enzyme digestion, and the base of the anti c-Myc tag single domain heavy chain antibody is constructed Sex phosphatase fusion expression plasmid.

Alkaline phosphatase can non-specifically catalyze the hydrolysis of phosphate monoesters to generate inorganic phosphate and corresponding alcohols, phenols or sugar compounds. This enzyme is often used as a signal element in detection methods such as ELISA, western blot, and histochemistry. Fusion expression plasmid The anti-c-Myc tag single domain heavy chain antibody is fused to the N-terminal of alkaline phosphatase, referring to the expression method in Application Example 3, the fusion protein AP-anti-c- Myc tag single domain heavy chain antibody.

Example 5:

Preparation of Anti-c-Myc Tag Single Domain Heavy Chain Antibody for Affinity Purification

1) Small-scale preparation of c-Myc-labeled immunoaffinity magnetic beads

Using nano-magnetic beads as the carrier, after coupling the anti-c-Myc tag single domain heavy chain antibody, the c-Myc tag immunomagnetic beads are obtained. The specific preparation method is as follows:

Take 1mg of carboxy-modified magnetic beads in a centrifuge tube, add 500μl activation buffer (10mM, NaH ₂ PO ₄ , pH 6.0), vortex to mix evenly, recover the magnetic beads with a magnetic stand, and wash 3 times with activation buffer. Add 2 mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) respectively, vortex and mix, and let stand for 25 min. Wash the magnetic beads 3 times with coupling buffer (10 mM, Na ₂ HPO ₄ , pH 7.4), add 1 mg of anti-c-Myc tag single domain heavy chain antibody dissolved in coupling buffer, react at room temperature for 3.5 h, use coupling Wash the magnetic beads 5 times with coupling buffer, add 500 μl coupling buffer containing 1% (w/v) bovine serum albumin (BSA) or 1% (w/v) ovalbumin (OVA) to block unreacted activity Group, react at room temperature for 35 minutes. The magnetic beads were washed 5 times with coupling buffer, resuspended in PBS solution (10 mM, pH 7.2, 0.02% w/v, Na ₃ N) and stored at 4°C.

2) Preparation of c-Myc-labeled immunoaffinity adsorption material and affinity column

Using agarose microspheres as a carrier, coupled with anti-c-Myc tag single domain heavy chain antibody, the specific preparation method is as follows:

The CNBr-activated dry gel was washed 10 times with 0.1M HCl, equilibrated for 5 min each time. Wash 10 times with coupling buffer (10 mM, Na ₂ HPO ₄ , pH 7.2), add anti-c-Myc tag single domain heavy chain antibody (2 mg/gram of agarose microspheres), react at room temperature for 3.5 h, and allow anti-c-Myc - Myc-tagged single-domain heavy-chain antibody covalently coupled to CNBr-activated Sepharose microspheres. After washing 3 times with coupling buffer (10 mM, Na ₂ HPO ₄ , pH 7.2), a blocking solution was added to react at room temperature for 2.5 h to block unreacted active groups. Wash 3 times alternately with phosphate buffer (10 mM, pH 7.2) and acetate buffer (0.1 M, pH 4.5) with 6 gel volumes to obtain an immunophile covalently coupled to the anti-c-Myc tag single domain heavy chain antibody. and adsorbent materials. Take 0.2ml of the above immunoaffinity adsorption material on a chromatographic column with a capacity of 1ml, wash with 8 to 10 times the column bed volume of PBS (10mM, pH 7.2), add 20% ethanol solution, and store at 4°C.

3) Preparation of c-Myc-labeled immunoaffinity adsorption material and affinity column

Using silica gel microspheres as a carrier, coupled with anti-c-Myc tag single domain heavy chain antibody, the specific preparation method is as follows:

Take 2 g of silica gel microspheres and wash them alternately with pure water and phosphate buffer (PBS, 10 mM, pH 6.5) for 6 to 10 times, suspend the silica gel microspheres with 10 ml of PBS buffer, add 5 mg of anti-c-Myc tag single domain heavy chain antibody, mix Add carbodiimide (EDC) at a final concentration of 5 mg/ml, mix quickly, and react with stirring at 4°C for 12-20 hours to obtain an immunoaffinity adsorption covalently coupled to an anti-c-Myc tag single domain heavy chain antibody Material. Take 0.2ml of the above-mentioned immunoaffinity adsorption material on a chromatographic column with a capacity of 1ml, wash with PBS (10mM, pH 6.5) of 58 to 10 times the column bed volume, and then add PBS containing 0.02% (w/v) Na ₃ N (10mM, pH 6.5), stored at 4°C.

4) Determination of the adsorption capacity and repeated use of the c-Myc tag affinity chromatography column

The column was washed with 6 times the column bed volume of PBS (10mM, pH 7.2), the protein sample solution was added, and the effluent was passed through the column again. Then rinse with 3 times the column bed volume of pure water, and then use glycine hydrochloric acid (pH 2.2) to elute the specifically adsorbed recombinant protein containing the c-Myc tag, and the collected eluate is the purified protein solution. The experimental results show that the affinity column/magnetic beads prepared with 1 mL of 1), 2), and 3) can specifically adsorb the target protein. After repeated use 10 times, the recovery rate is still greater than 80%. The single-domain heavy-chain antibody can be cultured and produced in large quantities through biological methods, avoiding cumbersome production methods such as artificial antibodies, greatly reducing production costs, and can be used repeatedly, with broad application prospects.

Embodiment 6:

Heat resistance experiment of nanobodies against c-Myc tag

The nanobody thermal stability is determined by ELISA experiment, and the experimental method is as follows:

Take the Myc-GST protein at a concentration of 5 μg/mL, add 100 μL per well into a 96-well ELISA plate, and coat overnight at 4°C; wash the plate three times with 0.05% PBST; block with 3% skimmed milk at 37°C for 1 h; c-Myc nanobody, 100 μL per well, 37°C, incubate for 1 h; add 1:2000 working concentration of HRP-labeled anti-His tag secondary antibody, add 100 μL per well, 37°C, incubate for 1 h; add TMB chromogenic solution, each Add 100 μL to the well; incubate at different temperatures for 30 min in multiple groups; after the reaction, add 2M sulfuric acid to terminate the reaction, add 50 μL to each well, mix well to terminate the reaction, and measure the absorbance at 450 nm.

The results showed that even when the temperature was as high as 70°C, 80°C, and 90°C, the protein activity still had biological activity, and the OD ₄₅₀ values were 1.5, 1.3, and 1.1, respectively, showing good heat resistance.

SEQUENCE LISTING

<110> Nanchang University

<120> Nanobodies against c-Myc tag

<130> 2017

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 124

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

Ser Leu Thr Leu Thr Cys Val Ala Ser Gly Gly Thr Leu Glu Asn Trp

20 25 30

Asp Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Tyr Phe Ser Ser Asp Gly Ile Arg Asn Tyr Gly Asp Ser Met Arg

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Glu Arg Val Gly Arg Ala Trp Arg Asn Asp Gly Leu Tyr Arg

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 2

<211> 372

<212>DNA

<213> Lama pacos

<400> 2

cagttgcagc tcgtggagtc agggggaggc ttggtgcagc ctggggagtc actgacgctc 60

acctgtgtag cctcaggcgg cactttggag aattgggata tagcctggtt ccgtcaggcc 120

ccagggaagg agcgtgaggg ggtcgcatat tttagtagtg atggtattag aaattatgga 180

gactccatga ggggccgatt caaaatctct agagacaacg ccaagaacac agcgtggctg 240

cagatgaatc gcctgagcgt tgaagacacg tccacatatt attgtgcggc caaagaaagg 300

gtcggacgtg catggcgaaa tgatggactt taccgctact ggggccagggg gacccaggtc 360

accgtctcct ca 372

<210> 3

<211> 18

<212>DNA

<213> Artificial sequence

<400> 3

cttggtggtc ctggctgc 18

<210> 4

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (44)..(44)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (47)..(47)

<223> n is a, c, g, or t

<400> 4

tcgcggccca gccggccatg gcccagktgc agctcgtgga gtcnggngg 49

<210> 5

<211> 33

<212>DNA

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cgagtgcggc cgcggggtct tcgctgtggt gcg 33

<210> 6

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<212>DNA

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cgagtgcggc cgcttgtggt tttggtgtct tggg 34

<210> 7

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ggtacgtgct gttgaactgt tcc 23

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agcggataac aatttcacac agga 24

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gccccattca gatcctcttc 20

Claims (9)

1. A nanobody directed at the c-Myc tag, having the amino acid sequence shown in SEQ ID NO.:1. 2. A nucleic acid molecule, characterized in that it encodes the amino acid sequence described in claim 1. 3. The nucleic acid molecule according to claim 2, characterized in that the sequence is as SEQ ID NO.:2. 4. A vector comprising the nucleic acid sequence of claim 2. 5. A host cell comprising the vector of claim 4. 6. The application of the Nanobody against c-Myc tag described in claim 1 in immunodetection, enrichment and purification of c-Myc tag. 7. The application of the nanobody against c-Myc tag described in claim 1 in the preparation of c-Myc tag immunodetection, enrichment and purification reagents or materials. 8. The nanobody against c-Myc tag according to claim 1 is modified by random or site-directed mutagenesis technology and can specifically bind to c-Myc tag. 9. An immunoaffinity adsorption material directed at c-Myc label, comprising a carrier, a ligand carried on the carrier, characterized in that the material uses a nanobody directed at the c-Myc label as the ligand, and the c-Myc label is directed against The Myc-tagged nanobody has the amino acid sequence shown in SEQ ID NO.:1.