CN106831992B

CN106831992B - Nanobodies against c-Myc tags

Info

Publication number: CN106831992B
Application number: CN201710100766.0A
Authority: CN
Inventors: 吴红静; 付金衡; 涂追
Original assignee: Lattice Power Jiangxi Corp
Current assignee: Lattice Power Jiangxi Corp
Priority date: 2017-02-23
Filing date: 2017-02-23
Publication date: 2020-06-16
Anticipated expiration: 2037-02-23
Also published as: CN106831992A

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a single-domain heavy chain antibody aiming at a c-Myc label, which has an amino acid sequence shown in SEQ ID No. 1 and can be used in the fields of immunodetection, antigen enrichment and purification and the like. The amino acid sequence provided by the invention can be used as a precursor, and can be transformed by random or site-directed mutagenesis technology to obtain mutants with better properties (affinity, specificity, stability and the like) for developing proteins or polypeptides for further use in medicine, industry and agriculture.

Description

Nanobodies against c-Myc tags

Technical Field

The invention relates to a single-domain heavy chain antibody technology (also called nano antibody technology) and a genetic engineering antibody technology, in particular to a single-domain heavy chain antibody or polypeptide aiming at a c-Myc label.

Technical Field

The discovery of c-Myc tag protein is derived from a monoclonal antibody 9E10 which is prepared by Evan in 1985 and is aimed at human proto-oncogene product Myc protein, and the research finds that the epitope recognized by the antibody consists of 10 amino acid residues, the sequence of the epitope is Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and the 10 amino acids can still keep strong antigen activity after being fused and expressed with other proteins, can be recognized by corresponding antibodies and are not influenced by protein frameworks. Therefore, the c-Myc label system is widely applied to the fields of immunological detection, cell imaging, affinity purification, protein engineering and the like.

The invention discloses a single-domain heavy chain antibody (namely a nano antibody, the same below) capable of being specifically combined with a c-Myc label, which can be used for detecting and purifying a c-Myc label fusion protein.

Monoclonal or polyclonal antibodies aiming at the c-Myc label are used for detection in the market at present, but the development and production processes of the monoclonal antibodies are complicated and complicated, and the sources of the polyclonal antibodies are limited. Compared with the prior art, the single-domain heavy chain antibody only consists of one structural domain, has the advantages of acid and alkali resistance, high temperature resistance, high specificity, small molecular weight, large-scale production and the like, and the purification medium prepared by using the single-domain heavy chain antibody as the ligand has the advantages of low cost, reusability and the like, and has wide application prospect.

Disclosure of Invention

The invention aims to provide a single-domain heavy chain antibody aiming at a c-Myc label, which can be used for preparing reagents and tools for detecting and purifying the c-Myc label.

The invention provides a single-domain heavy chain antibody (namely the invention is directed to the nano antibody of the c-Myc label, the same below) with an amino acid sequence shown in SEQ ID NO. 1. The division of the IMGT numbering and domains of its amino acid sequence includes four Framework Regions (FRs) and three complementary-determining regions (CDRs).

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

The invention also provides a nucleic acid molecule, which is characterized in that the nucleic acid molecule codes a part of structural domain of SEQ ID No. 1, and a specific sequence of the nucleic acid molecule can be obtained at any time through a genetic codon. Can be a nucleic acid molecule of SEQ ID No. 2.

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

The invention also provides a vector comprising the nucleic acid sequence. Due to the degeneracy of the genetic code, the nucleic acid sequence can vary depending on the intended use.

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

The invention also provides a method for detecting the c-Myc label, which contains the single-domain heavy chain antibody aiming at the c-Myc label. The detection method of the c-Myc label is established based on the specific binding capacity of the single-domain heavy chain antibody aiming at the c-Myc label and the c-Myc label. Among them, preferred methods include Enzyme-linked immunosorbent assay (ELISA), Fluoroimmunoassay (FIA), immuno-chip assay, affinity chromatography, immunochromatography, and the like.

The amino acid sequence provided by the invention can be used as a precursor, and can be modified by a random or site-directed mutagenesis technology to obtain a mutant with better properties (water solubility, stability, affinity, specificity and the like), and the mutant can be specifically combined with a c-Myc label.

The invention also relates to application of the single-domain heavy chain antibody aiming at the c-Myc label in immunodetection, enrichment and purification. These immunoassays are referred to as immunoassays for purposes other than disease diagnosis and treatment.

The invention also relates to an immunoaffinity adsorption material aiming at the c-Myc label, which comprises a carrier and a ligand carried on the carrier, and is characterized in that the material takes a nano antibody aiming at the c-Myc label as the ligand, and the nano antibody aiming at the c-Myc label has an amino acid sequence shown in SEQ ID NO. 1. The carrier material is not limited to agarose gel, and silicon spheres, nano magnetic beads and the like can also be selected.

Some of the terms described herein have the following meanings:

domain (b): the basic structural unit of the tertiary structure of a protein generally has a certain function.

IMGT number: a standardized numbering scheme for The amino acid sequence of antibodies in The IMGT database (The International ImmunoGeneTiCs Datbase). Specific numbering methods can be described in the literature (Ehrenman, F., Q. Kaas, et. al. (2010); IMGT/3D structure-DB and IMGT/DomainGapAlign: adataabase and a tool for immunoglobulin or antibodies, T cell receptors, MHC, IgSF and MhcSF. nucleic Acids Res 38 (Database. major. E.: D301-307.Lefranc, M.P., C.Pommer, et. al. (2003), IMGT unique number for immunoglobulin and T cell receptor domains and Igsufactual V-domains Dev. 27(1): 55-77).

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

Detailed Description

The present invention is further illustrated below by the preparation, analysis and use of single domain heavy chain antibodies (polypeptides), and these specific examples should not be construed in any way as limiting the scope of the invention.

Example 1:

construction of an immune library of anti-c-Myc-tag single-domain heavy-chain antibodies (i.e., single-domain heavy-chain antibodies directed against the c-Myc-tag)

Covalently coupling the c-Myc label with Bovine Serum Albumin (BSA) to obtain c-Myc artificial antigen c-Myc-BSA, emulsifying 300 mu g c-Myc-BSA with Freund's complete adjuvant, and performing subcutaneous multi-point injection immunization on alpaca (Lama pacos). The boosting immunization adopts 150 mu g c-Myc-BSA to emulsify with Freund's incomplete adjuvant, the emulsification is carried out at intervals of 2 weeks, blood is taken from veins after 7 days of each immunization, the serum titer is measured by adopting an indirect ELISA method, the sample with the highest serum titer is selected to separate lymphocytes, and RNA is extracted.

RNA extraction was performed according to TAKARA RNAiso reagent instructions. First strand cDNA was synthesized using RNA as a template and oligo dT as a primer, according to the reverse transcriptase instructions of TAKARA.

The genes encoding the variable regions of the heavy chain antibodies were obtained by nested PCR using PrimeSTAR high fidelity DNA polymerase (primers used are shown in Table 1). The first round of PCR was performed by amplifying cDNA with primers AlpVh-LD and CH2-R, respectively, under the conditions of 98 deg.C, 10s, 55 deg.C, 20s, 72 deg.C, 1min, 20 cycles, 98 deg.C, 10s, 68 deg.C, 1min, and 72 deg.C for 10min of extension.

The first round PCR product is electrophoresed by 1.2% agarose gel, DNA fragments of 600 bp-750 bp are recovered and used as templates of the second round PCR, primers AlpVh-SfiI and AlpVHHR1-NotI, AlpVh-SfiI and AlpVHHR2-NotI are respectively used for amplification, and the reaction conditions are 98 ℃, 10 seconds, 50 ℃, 20 seconds, 72 ℃, 40 seconds, 5 cycles, 98 ℃, 10 seconds, 68 ℃, 40 seconds, 30 cycles and 72 ℃ extension for 10 min. And (4) recovering and quantifying by using a DNA fragment recovery kit, and storing at-20 ℃ for later use. The phagemid pHEN1 and the PCR amplification product were digested with Sfi I and Not I, respectively, and then recovered and quantified by agarose gel, and then ligated at 16 ℃ overnight at a molar ratio of 1: 3.

TABLE 1 primers used for library construction and identification

Note: restriction enzyme recognition sequences are underlined

After the ligation product was precipitated with ethanol, it was dissolved in 10. mu.L of sterile water and transformed into E.coli TG1 by electroporation ten times. 10 mu L of electric shock is taken, the cultured bacterium solution is diluted in a multiple ratio, an ampicillin 2 XYT culture plate is coated, and the storage capacity is calculated. The rest part of the culture medium is completely coated on a 24cm × 24cm ampicillin 2 × YT culture plate, and is subjected to inverted culture at 37 ℃ for 13-16 h. Scraping and washing the lawn on the culture plate by using 10mL of 2 XYT culture medium, adding glycerol with the final concentration of 20-30%, subpackaging and storing at-80 ℃ for later use.

According to the calculated library volume results, 10-fold library volume of viable cells were inoculated into 20mL of 2 XYT (containing 2% glucose, 100. mu.g/mL ampicillin) and cultured at 37 ℃ and 200r/min to OD₆₀₀When the infection complex number reaches 0.5, the helper phage is added according to the ratio of 20: 1, the temperature is 37 ℃, 200r/min and 60 min. Centrifuging the culture, resuspending the precipitate with 50mL of 2 XYT (containing 100. mu.g/mL ampicillin and 50. mu.g/mL kanamycin), culturing overnight at 37 ℃ at 200r/min, then centrifuging at 8000rpm to obtain supernatant, adding 5 XPEG/NaCl solution, standing on ice for 1.5h or overnight at 4 ℃, centrifuging at 8000rpm for 30min, resuspending the precipitate in phosphate buffer (PBS, 0.01M, pH7.4) containing 10% glycerol to obtain the anti-c-Myc label single-domain heavy chain antibody immune library, taking 10. mu.L of the measured titer, and packaging the rest at-80 ℃ for later use.

Example 2:

panning and identification of anti-c-Myc tag single-domain heavy chain antibody

The anti-c-Myc-tag single-domain heavy-chain antibody immune library obtained in example 1 was subjected to solid-phase affinity panning to pan single-domain heavy-chain antibodies against the c-Myc-tag. Adding 120 mu L of Myc-GST fusion protein (Myc label and glutathione fusion protein) diluted by PBS into each enzyme labeling hole, coating at 4 ℃ overnight, wherein the coating concentration of each round of panning is 100, 75 and 50 mu g/mL respectively; sucking out the coating solution, washing the plate with PBS for 5 times, adding 300 μ L of 3% BSA-PBS into each well, sealing at 37 deg.C for 2 h; the plate was washed 5 times with PBS and 100. mu.L of phage antibody library (containing approximately 1X 10)¹¹CFU), incubating for 2.0h at 37 ℃; unbound phage were aspirated and washed with PBST (0.5% T)wenn-20) washing the plate 3-5 times (increasing 5 times by round), and then washing the plate 15-25 times with PBS; the phage adsorbed in the wells were eluted with 100. mu.L of eluent (glycine-hydrochloric acid, pH 2.2), the eluate was neutralized with 35. mu.L of Tris-HCl (1mol/L, pH8.0), 10. mu.L of the eluate was taken for titer determination, and the remaining 125. mu.L of the eluate was amplified for the next round of panning.

After four rounds of panning, randomly picked monoclonals are rescued by using an auxiliary phage KM13 to respectively obtain phage particles displaying antibody variable regions, the binding activity and specificity of the phage particles are measured by using indirect phase-ELISA, experiments are set for comparison, and specific sample adding steps are shown in Table 2.

TABLE 2 Indirect phase-ELISA sample application Table

And (3) sending the ELISA positive clone to a biotechnology service company for sequence determination to obtain a DNA sequence of the insert, wherein the DNA sequence encodes a single-domain heavy chain antibody aiming at the c-Myc label.

DNA sequence (SEQ ID NO: 2):

CAGTTGCAGCTCGTGGAGTCAGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTAACACTCTCCTGCTCAGCCTCTGGATTCAATATATCACAATATTCCGTGGGGTGGTTCCGCGAGGCCCCAGGGGAAGAGCGTGAGGGGATCTCATGTCTGGACATTGATGGCAAAATTACCACCTTCTCAGACGCCATACAGGGCCGATTCACCATCGCCCGAGACAATGCTGCAAATATGATATACTTACACATGGACGCCCTGAACCCTCTGGATACGGCCGTTTATCGGTGTGTCGCCAGATGGGACTGTTCGCGACACGATTTTATCACCCAGAAAACCGCTACCGGCATCTGGGGCCCGGGGACCCAGGTCACCGTGTCAGCA

encodes a polypeptide having the amino acid sequence shown in SEQ ID NO. 1:

QLQLVESGGGSVQPGGSLTLSCSASGFNISQYSVGWFREAPGEEREGISCLDIDGKITTFSDAIQGRFTIARDNAANMIYLHMDALNPLDTAVYRCVARWDCSRHDFITQKTATGIWGPGTQVTVSA。

example 3:

scale preparation of anti-c-Myc tag single-domain heavy chain antibody

Acquisition of a DNA fragment encoding an anti-c-Myc tag single domain heavy chain antibody: 1. adopting restriction endonuclease SfiI/NotI, performing double digestion on phagemid pHEN-anti-c-Myc single-domain heavy chain antibody gene, and performing agarose gel electrophoresis to recover the anti-c-Myc label single-domain heavy chain antibody gene; 2. directly sending the coding sequence of the anti-c-Myc label single-domain heavy chain antibody to a biotechnology service company for chemical synthesis; 3. specific primers were designed and amplified from alpaca (Lama pacos) derived cDNA libraries by PCR technique.

Cloning the obtained gene fragment of the anti-c-Myc tag single-domain heavy chain antibody to an expression vector pET25-Flag (the c-Myc tag carried by the vector is replaced by Flag tag: DYKDDDDK), and constructing the escherichia coli expression plasmid of the anti-c-Myc tag single-domain heavy chain antibody through PCR and enzyme digestion identification.

The expression plasmid is transformed into escherichia coli rosetta, and a single colony is picked for induced expression. Inoculating the single colony into 5mL LB-A (Luria-Bertani broth with 100. mu.g/mL ampicillin) liquid medium, and performing shake culture at 37 ℃ and 220r/min for 12 h; transferring the strain into 50mL LB-A liquid culture medium with the inoculum size of 1% of the culture medium volume, and performing shaking culture at 37 ℃ and 220r/min until OD is reached₆₀₀Reaching 0.5 (about 3-3.5 h), adding IPTG with the final concentration of 0.1mM, and inducing and culturing at 30 ℃ and 200 r/min.

Inducing the culture to centrifuge at 8000r/min, adding 25mL phosphate buffer (pH 7.4) into the cell precipitate, mixing, centrifuging at 8000r/min, removing supernatant, and retaining the cell precipitate; adding 15mL of the same buffer solution into the cell sediment, uniformly mixing, carrying out ultrasonic cell disruption treatment on ice under the conditions of 200W, 2s disruption and 5s pause for 250 cycles, centrifuging the cell disruption material at 8000r/min for 15min at 4 ℃, taking the supernatant, carrying out affinity chromatography purification and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, or adding 20% of glycerol into the supernatant, uniformly mixing, and storing in a freezer at-20 ℃ for later use.

The expression quantity of the target protein (single-domain heavy chain antibody) can be further improved by optimizing induction expression conditions (such as host bacteria, expression vectors, induction culture time, temperature, IPTG concentration and the like), and a way is provided for preparing a large amount of anti-c-Myc label single-domain heavy chain antibodies.

Example 4:

fusion expression of anti-c-Myc tag single-domain heavy chain antibody

The gene of the anti-c-Myc label single-domain heavy chain antibody is cloned to a fusion expression vector pAP (containing alkaline phosphatase gene), and the alkaline phosphatase fusion expression plasmid of the anti-c-Myc label single-domain heavy chain antibody is constructed through PCR and enzyme digestion identification.

Alkaline phosphatase can non-specifically catalyze the hydrolysis of phosphate monoesters to produce inorganic phosphate and corresponding alcohol, phenol, or carbohydrate compounds. The enzyme is often used as a signal element for ELISA, immunoblotting, histochemistry and other detection methods. The fusion expression plasmid fuses the anti-c-Myc label single-domain heavy chain antibody to the N end of alkaline phosphatase, and the fusion protein AP-anti-c-Myc label single-domain heavy chain antibody can be expressed and purified in escherichia coli by referring to the expression method in the application example 3.

Example 5:

preparation of anti-c-Myc label single-domain heavy chain antibody for affinity purification material

1) Miniprep of c-Myc-tagged immunoaffinity magnetic beads

The preparation method comprises the following steps of coupling an anti-c-Myc label single-domain heavy chain antibody by using nano magnetic beads as a carrier to obtain c-Myc label immunomagnetic beads:

1mg of carboxyl-modified magnetic beads were placed in a centrifuge tube and 500. mu.l of activation buffer (10mM, NaH) was added₂PO₄Ph6.0), vortex and mix well, magnetic beads were recovered by magnetic rack, and washed 3 times with activation buffer. 2mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added separately, mixed by vortexing, and allowed to stand for 25 min. With coupling buffer (10mM, Na)₂HPO₄pH7.4), adding 1mg of anti-c-Myc label single-domain heavy chain antibody dissolved in coupling buffer solution, reacting for 3.5h at room temperature, washing the magnetic beads for 5 times by using the coupling buffer solution, adding 500 mu l of coupling buffer solution containing 1% (w/v) Bovine Serum Albumin (BSA) or 1% (w/v) Ovalbumin (OVA) to block unreacted active groups, and reacting for 35min at room temperature. The beads were washed 5 times with coupling buffer, PBS solution (10mM, pH 7.2, 0.02% w/v, Na)₃N) resuspension and storage at 4 ℃.

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

The agarose microspheres are used as carriers to couple the anti-c-Myc label single-domain heavy chain antibody, and the specific preparation method is as follows:

the CNBr activated dry gel was washed 10 times with 0.1M HCl and equilibrated for 5min each time. With coupling buffer (10mM, Na)₂HPO₄pH 7.2) was washed 10 times, and an anti-c-Myc-tagged single domain heavy chain antibody (2 mg/g agarose microspheres) was added and reacted at room temperature for 3.5h to covalently couple the anti-c-Myc-tagged single domain heavy chain antibody to CNBr-activated agarose gel microspheres. With coupling buffer (10mM, Na)₂HPO₄pH 7.2) was washed 3 times, and then a blocking solution was added to the reaction mixture to react at room temperature for 2.5 hours to block unreacted active groups. The immunoaffinity adsorption material covalently coupled with the anti-c-Myc label single-domain heavy chain antibody was obtained by alternately washing 3 times with 6 gel volumes of phosphate buffer (10mM, pH 7.2) and acetate buffer (0.1M, pH 4.5). 0.2ml of the immunoaffinity adsorption material is put into a chromatographic column with the capacity of 1ml, washed by PBS (10mM, pH 7.2) with the volume of 8-10 times of the volume of a column bed, added with 20% ethanol solution and stored at 4 ℃.

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

Silica gel microspheres are used as carriers to couple the anti-c-Myc label single-domain heavy chain antibody, and the specific preparation method is as follows:

and (2) alternately washing 2g of silica gel microspheres with pure water and phosphate buffer (PBS, 10mM, pH 6.5) for 6-10 times, suspending the silica gel microspheres with 10ml of PBS buffer, adding 5mg of anti-c-Myc label single-domain heavy chain antibody, uniformly mixing, adding carbodiimide (EDC) with the final concentration of 5mg/ml, rapidly and uniformly mixing, and stirring at 4 ℃ for reaction for 12-20 hours to obtain the immunoaffinity adsorption material covalently coupled with the anti-c-Myc label single-domain heavy chain antibody. Washing 0.2ml of the immunoaffinity adsorption material in a 1ml column with 58-10 times column volume of PBS (10mM, pH 6.5), and adding Na 0.02% (w/v)₃N in PBS (10mM, pH 6.5) and stored at 4 ℃.

4) Measurement of adsorption amount and reuse of c-Myc tag affinity column

The column was washed with 6 bed volumes of PBS (10mM, pH 7.2), protein sample solution was added and the effluent was re-passed through the column. And then eluting with pure water with the volume of 3 times of that of the column bed, eluting the specifically adsorbed recombinant protein containing the c-Myc label by using glycine hydrochloric acid (pH 2.2), and collecting the eluent, namely the purified protein solution. The experimental result shows that the affinity column/magnetic bead filled with 1mL1), 2) and 3) can specifically adsorb the target protein. After 10 times of repeated use, the recovery rate is still more than 80%. The aglucon can be cultured and produced in a large amount by a biological method to be a single-domain heavy chain antibody, so that the complicated production methods such as artificial antibodies are avoided, the production cost is greatly reduced, and the aglucon can be repeatedly used, so that the application prospect is wide.

Example 6:

heat resistance experiment of anti-c-Myc-labeled nano antibody

The thermal stability of the nano-antibody is determined by ELISA experiments, which are as follows:

taking Myc-GST protein with the concentration of 5 mu g/mL, adding 100 mu L of Myc-GST protein into a 96-hole enzyme label plate per hole, and coating overnight at 4 ℃; wash plate 3 times with 0.05% PBST; sealing 3% skimmed milk at 37 deg.C l h; adding diluted c-Myc nano antibody, incubating for 1h at 37 ℃ and 100 mu L per well; adding 1: adding 100 mu L of HRP-labeled anti-His tag secondary antibody with the working concentration of 2000 into each hole, and incubating for 1h at 37 ℃; adding TMB color development liquid, and adding 100 mu L of TMB color development liquid into each hole; incubating at different temperatures for 30 min; after the reaction is finished, 2M sulfuric acid is added to stop the reaction, 50 mu L of sulfuric acid is added into each hole, and after the reaction is stopped by mixing, the light absorption value of 450nm is measured.

The results show that even if the temperature is as high as 70 ℃, 80 ℃ and 90 ℃, the protein activity still has biological activity and OD₄₅₀The values are 1.5, 1.3 and 1.1 respectively, and the heat resistance is better.

SEQUENCE LISTING

<110> university of Nanchang

<120> Nanobody against c-Myc tag

<130>2017

<160>9

<170>PatentIn version 3.3

<210>1

<211>127

<212>PRT

<213> Artificial sequence

<400>1

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

1 5 10 15

Ser Leu Thr LeuSer Cys Ser Ala Ser Gly Phe Asn Ile Ser Gln Tyr

20 25 30

Ser Val Gly Trp Phe Arg Glu Ala Pro Gly Glu Glu Arg Glu Gly Ile

35 40 45

Ser Cys Leu Asp Ile Asp Gly Lys Ile Thr Thr Phe Ser Asp Ala Ile

50 55 60

Gln Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Ala Asn Met Ile Tyr

65 70 75 80

Leu His Met Asp Ala Leu Asn Pro Leu Asp Thr Ala Val Tyr Arg Cys

85 90 95

Val Ala Arg Trp Asp Cys Ser Arg His Asp Phe Ile Thr Gln Lys Thr

100 105 110

Ala Thr Gly Ile Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ala

115 120 125

<210>2

<211>381

<212>DNA

<213>Lama pacos

<400>2

cagttgcagc tcgtggagtc agggggaggc tcggtgcagc ctggggggtc tctaacactc 60

tcctgctcag cctctggatt caatatatca caatattccg tggggtggtt ccgcgaggcc 120

ccaggggaag agcgtgaggg gatctcatgt ctggacattg atggcaaaat taccaccttc 180

tcagacgcca tacagggccg attcaccatc gcccgagaca atgctgcaaa tatgatatac 240

ttacacatgg acgccctgaa ccctctggat acggccgttt atcggtgtgt cgccagatgg 300

gactgttcgc gacacgattt tatcacccag aaaaccgcta ccggcatctg gggcccgggg 360

acccaggtca ccgtgtcagc a 381

<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

<213> Artificial sequence

<400>5

cgagtgcggc cgcggggtct tcgctgtggt gcg 33

<210>6

<211>34

<212>DNA

<213> Artificial sequence

<400>6

cgagtgcggc cgcttgtggt tttggtgtct tggg 34

<210>7

<211>23

<212>DNA

<213> Artificial sequence

<400>7

ggtacgtgct gttgaactgt tcc 23

<210>8

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<212>DNA

<213> Artificial sequence

<400>8

agcggataac aatttcacac agga 24

<210>9

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gccccattca gatcctcttc 20

Claims

1. A nano antibody aiming at a c-Myc label has an amino acid sequence shown in SEQ ID No. 1.

2. A nucleic acid molecule encoding the amino acid sequence of claim 1.

3. The nucleic acid molecule of claim 2, wherein the sequence is as set forth in 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 use of the nanobody of claim 1 against the c-Myc tag in immunodetection, enrichment and purification of the c-Myc tag.

7. The use of the nanobody of claim 1 against the c-Myc tag in the preparation of reagents or materials for immunodetection, enrichment and purification of c-Myc tags.

8. An immunoaffinity adsorption material aiming at a c-Myc label comprises a carrier and a ligand carried on the carrier, and is characterized in that the material takes a nano antibody aiming at the c-Myc label as the ligand, and the nano antibody aiming at the c-Myc label has an amino acid sequence shown in SEQ ID NO. 1.