CN106914229A

CN106914229A - Affine sorbing material based on specific recognition c Myc label nano antibodies

Info

Publication number: CN106914229A
Application number: CN201710100783.4A
Authority: CN
Inventors: 吴红静; 付金衡; 涂追
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2017-02-23
Filing date: 2017-02-23
Publication date: 2017-07-04

Abstract

The invention belongs to genetic engineering field, specially for the single domain heavy chain antibody (also known as nano antibody) of c Myc labels, it has SEQ ID NO.:Amino acid sequence shown in 1.Amino acid sequence provided by the present invention can be as precursor, transformed by random or site-directed mutagenesis technique, it is obtained in that property (compatibility, specificity, stability etc.) preferably mutant, the nano antibody of the high specific can be purified as the affine in immunity sorbing material for c Myc to the recombinant protein containing c Myc labels.

Description

Affine sorbing material based on specific recognition c-Myc label nano antibodies

Technical field

It is special the present invention relates to affine in immunity sorbing material of the one kind based on single domain heavy chain antibody (also known as nano antibody technology) It is not directed to the affine in immunity sorbing material of c-Myc labels.

Technical background

The discovery of c-Myc label proteins comes from Evan in 1985 and has prepared one plant for people's Oncoprotein Myc The monoclonal antibody 9E10 of albumen, hereafter research finds that the epitope of the antibody recognition is made up of 10 amino acid residues, its sequence It is Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and this 10 amino acid and other protein fusion expressions Very strong antigen active can be still kept afterwards, can be recognized by corresponding antibodies, and do not influenceed by albumen framework.Cause This, c-Myc tag systems are widely used in the fields such as immunology detection, cell imaging, affinity purification and protein engineering.

The technologies such as immunoaffinity chromatography, affine in immunity magnetic bead and co-immunoprecipitation are usually used in isolating and purifying and contain c-Myc marks Sign fusion protein.This kind of affinity purification technology is based on the principle of aglucon and the specific binding of c-Myc labels, usually, will be special Property combination c-Myc labels aglucon and carrier conjugation or absorption, be subsequently used for the specific adsorption c-Myc tag fusions from solution Albumen.

In the market is directed to the affinity column and affine magnetic bead of c-Myc tag fusion protein purifying, and the aglucon of coupling is mostly Monoclonal antibody or polyclonal antibody.Research and development of monoclonal antibody and production process is relatively complicated and complexity, polyclonal antibody comes Source is limited.Existing affinity column and affine magnetic bead price for the purifying of c-Myc tag fusion proteins is generally higher.And nanometer is anti- Body can Escherichia coli, yeast etc. biology in great expression, advantageously reduce the production cost of correlated product.

Need to use acid in protein purification procedures or alkaline solution is eluted to destination protein.Due to monoclonal antibody or Polyclonal antibody is made up of heavy chain and light chain, and soda acid wash-out can inevitably cause the antibody activity to reduce, thus can not repeat Use.By contrast, single domain heavy chain antibody is only made up of a domain, with acid and alkali-resistance, high temperature resistant.There is provided in the present invention Affine sorbing material can be repeated several times and use.

The content of the invention

It is an object of the invention to provide a kind of for the affine in immunity sorbing material of c-Myc labels and its application.

To achieve the above object, the present invention is adopted the following technical scheme that：

Affine in immunity sorbing material for c-Myc labels includes carrier and aglucon, and the aglucon is single domain heavy chain antibody, With SEQ ID NO.:Amino acid sequence shown in 1.The aglucon can specific recognition c-Myc labels.

The aglucon can also be to be changed by random or site-directed mutagenesis technique on the basis of foregoing single domain heavy chain antibody Make the antibody that can be specifically bound with c-Myc labels for being obtained.

The carrier is magnetic bead, agarose gel microsphere, silica gel microball or porous material.

The preparation method of above-mentioned c-Myc labels affine in immunity sorbing material, it is characterized by：

When the carrier is magnetic bead, preparation method is：1mg carboxyl magnetic beads are taken in centrifuge tube, adds 600~1000 μ l to live Change buffer solution (10mM, NaH₂PO₄, pH 6.0), vortex mixed is uniform, and magnetic frame reclaims magnetic bead, then washs 2 with activation buffer Time.1~5mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) are separately added into, after vortex mixed, 35min is stood.With Coupling buffer (10mM, Na₂HPO₄, pH 7.4) and wash magnetic bead 5 times, anti-c-Myc labels single domain heavy chain antibody is added, room temperature is anti- 3~5h is answered, the immunomagnetic beads of the anti-c-Myc labels single domain heavy chain antibody of covalent coupling is obtained；

When the carrier is agarose gel microsphere, preparation method is：The dry glue that CNBr is activated is washed with 0.1M HCl 10~20 times, 6~10min is balanced every time.Washed 5~20 times with coupling buffer (10mM, Na2HPO4, pH 7.2), added anti- C-Myc label single domain heavy chain antibodies, 3~10h of room temperature reaction obtains the anti-c-Myc labels single domain heavy chain antibody of covalent coupling Affine in immunity sorbing material；

When the carrier is silica gel microball, preparation method is：By silica gel microball pure water and phosphate buffer (PBS, 10mM, pH 6.5) alternately washing 5~15 times, with PBS suspension silica gel microball, add anti-c-Myc labels single domain heavy chain to resist Body, is mixed, and adds the carbodiimide (EDC) of 1~10mg/ml of final concentration, and rapid to mix, 4 DEG C of 12~20h of stirring reaction are obtained The covalent coupling affine in immunity sorbing material of anti-c-Myc labels single domain heavy chain antibody.

Above-mentioned affine in immunity sorbing material (carrier is agarose gel microsphere and silica gel microball) is filled to chromatographic column, i.e., C-Myc label immune affinity chromatographic columns are obtained, method is：According to chromatography column capacity, appropriate above-mentioned affine in immunity sorbing material is taken In chromatographic column, after adding 8~10 times of PBS of bed volume (10mM, pH 7.2) washings, 4 DEG C, 20% ethanol solution is stored in.

Affinity column the invention further relates to be mounted with c-Myc labels affine in immunity sorbing material described in claim 1.

The application of above-mentioned c-Myc labels affine in immunity sorbing material, with the c-Myc labels affine in immunity sorbing material pair The albumen containing c-Myc labels is purified in sample.When the carrier of immune absorption material is that agarose gel microsphere and silica gel are micro- During ball, method is：Immune absorption material is washed with PBS (10mM, pH 7.2) first, sample extracting solution is added, pure water is then used Drip washing, then the recombinant protein of label containing c-Myc of specific adsorption is eluted with glycine hydrochloride (pH 2.2), the eluent of collection, Sample extracting solution after purification, can be used for subsequent analysis detection.When carrier is magnetic bead, method is：Immunomagnetic beads is added pure In water, magnetic frame reclaims magnetic bead, and repeated washing 3~5 times, then by immunomagnetic beads addition sample extracting solution, is mixed, magnetic frame Magnetic bead is reclaimed, after pure water cleans 3~5 times, the c-Myc labels for eluting specific adsorption with glycine hydrochloride (pH 2.2) recombinate egg In vain, the eluent of collection, sample extracting solution as after purification can be used for subsequent analysis detection.

The present invention is single domain heavy chain antibody for the affine in immunity sorbing material aglucon of c-Myc labels, with SEQ ID NO.:Amino acid sequence shown in 1, the aglucon can specific recognition c-Myc labels.The single domain heavy chain antibody is readily available, resistance to Heat, it is single domain heavy chain antibody that can produce aglucon by biological method mass propgation, it is to avoid the cumbersome production such as artificial antibody Method, greatly reduces production cost, and reusable, has a extensive future.

Specific embodiment

Below by the preparation and application of c-Myc label affine in immunity sorbing materials, the present invention will be further described, this A little specific embodiments are not construed in any way as limiting range of application of the invention.

Embodiment 1：

The structure of anti-c-Myc labels single domain heavy chain antibody (i.e. for the single domain heavy chain antibody of c-Myc labels) non-immune libraries

By c-Myc labels and bovine serum albumin(BSA) (Bovine serum albumin, BSA) covalent coupling, c-Myc is obtained Artificial antigen c-Myc-BSA, after taking 300 μ g c-Myc-BSA and Freund's complete adjuvant emulsification, enters to alpaca (Lama pacos) The subcutaneous multi-point injection of row is immunized.Booster immunization is emulsified using 150 μ g c-Myc-BSA with incomplete Freund's adjuvant, and interval is entered for 2 weeks OK, venous blood sampling after being immunized 7 days every time, serum titer, selection serum titer highest sample point are determined using indirect elisa method From lymphocyte, RNA is extracted.

The extraction of RNA is carried out with reference to TAKARA companies RNAiso reagents specification.With RNA as template, oligo dT are to draw Thing, with reference to the TAKARA companies reverse transcriptase specification synthesis chains of cDNA first.

Using PrimeSTAR high-fidelity DNA polymerases, the variable region encoding gene for obtaining heavy chain antibody through nest-type PRC (is adopted 1) primer is shown in Table.First round PCR expands cDNA with primer AlpVh-LD and CH2-R respectively, and reaction condition is, 98 DEG C, 10s, 55 DEG C, 20s, 72 DEG C, 1min, 20 circulations, 98 DEG C, 10s, 68 DEG C, 1min, 72 DEG C of extension 10min.

By first round PCR primer with 1.2% agarose gel electrophoresis, the DNA fragmentation of 600bp~750bp is reclaimed, as The template of the second wheel PCR, respectively with primer AlpVh-SfiI and AlpVHHR1-NotI, AlpVh-SfiI and AlpVHHR2- NotI, is expanded, and reaction condition is, 98 DEG C, 10s, 50 DEG C, 20s, 72 DEG C, 40s, 5 circulations, 98 DEG C, 10s, 68 DEG C, 40s, 30 circulations, 72 DEG C of extension 10min.Reclaimed through DNA fragmentation QIAquick Gel Extraction Kit, quantified, saved backup in -20 DEG C.To bite Bacterium grain pHEN1 and pcr amplification product use Sfi I, Not I double digestions respectively, after Ago-Gel recovery, quantifying, are rubbed with 1: 3 You compare, and at 16 DEG C, overnight connect.

The library construction of table 1 and identification primer used

Note：Underscore represents restriction endonuclease recognition sequence

After connection product is through ethanol precipitation, 10 μ L sterilized waters are dissolved in, Electroporation Transformation e. coli tg1 is carried out in ten times. The bacterium solution doubling dilution after 10 μ L electric shocks, culture is taken, ampicillin 2 × YT culture plates are coated with, storage capacity is calculated.Remainder is complete Portion coats 24cm × 24cm ampicillin 2 × YT culture plates, 37 DEG C, is inverted 13~16h of culture.With 10mL, 2 × YT cultures After base scrapes the lawn on culture plate, the glycerine of final concentration 20~30% is added, dispensed, -80 DEG C save backup.

According to calculate storage capacity result, inoculation 10 times of living cells of storage capacity in 20mL 2 × YT (contain 2% glucose, 100 μ g/mL ampicillins), 37 DEG C, 200r/min is cultivated to OD₆₀₀Up to 0.5, helper phage is added by infection multiplicity 20: 1 Body, 37 DEG C, 200r/min, 60min.Culture is centrifuged, (100 μ g/mL ampicillins and 50 μ g/ are contained with the 2 × YT of 50mL ML kanamycins) resuspended precipitation, 37 DEG C, after 200r/min incubated overnights, 8000rpm centrifuging and taking supernatants add 5 × PEG/NaCl Solution, places 1.5h or 4 DEG C overnight on ice, and 8000rpm is centrifuged 30min, resuspended to be deposited in the phosphate buffer containing 10% glycerine (PBS, 0.01M, pH 7.4), that is, obtain anti-c-Myc labels single domain heavy chain antibody non-immune libraries, takes 10 μ L and determines titre, remaining - 80 DEG C are sub-packed in save backup.

Embodiment 2：

The elutriation of anti-c-Myc labels single domain heavy chain antibody and identification

Using the method for the affine elutriation of solid phase from the anti-c-Myc labels single domain heavy chain antibody non-immune libraries of gained of embodiment 1 Single domain heavy chain antibody of the elutriation for c-Myc labels.Merged to the Myc-GST for adding 120 μ L PBS to dilute in each enzyme mark hole Albumen (albumen that Myc labels are merged with glutathione), 4 DEG C, overnight, the coating concentration for often taking turns elutriation is respectively 100 to coating, 75,50 μ g/mL；Coating buffer is suctioned out, PBS board-washings 5 times add 300 μ L 3%BSA-PBS per hole, 37 DEG C, close 2h；PBS board-washings 5 times, 100 μ L phage antibody libraries are added (containing about 1 × 10¹¹CFU), 37 DEG C, it is incubated 2.0h；Uncombined bacteriophage is suctioned out, With PBST (contain 0.5%Tween-20) board-washing 3-5 time (by wheel increase by 5 times), then with PBS board-washings 15-25 times；Eluted with 100 μ L Bacteriophage of liquid (glycine-HCI, pH 2.2) the wash-out absorption in enzyme mark hole, with 35 μ L Tris-HCl (1mol/L, pH 8.0) eluate is neutralized, 10 μ L is taken for titer determination, next round elutriation is used for after the amplification of remaining 125 μ L eluate.

After through four-wheel elutriation, the monoclonal of random picking is rescued using helper phage KM13, respectively obtain exhibition Show the phage particle of antibody variable region, then the binding activity and specificity of phage particle determined with indirect phage-ELISA, Experiment setting control, specific load procedure is shown in Table 2.

The indirect phage-ELISA of table 2 is loaded table

Send the biotechnology service company to carry out sequencing ELISA positive colonies, obtain the DNA sequence dna of Insert Fragment, Single domain heavy chain antibody of its coding for c-Myc labels.

DNA sequence dna (SEQ ID NO.:2)：

CAGTTGCAGCTCGTGGAGTCAGGGGGAGGCTCGGTGCAGCCTGGGGGGTCTCTAACACTCTCCTGCTCA GCCTCTGGATTCAATATATCACAATATTCCGTGGGGTGGTTCCGCGAGGCCCCAGGGGAAGAGCGTGAGGGGATCTC ATGTCTGGACATTGATGGCAAAATTACCACCTTCTCAGACGCCATACAGGGCCGATTCACCATCGCCCGAGACAATG CTGCAAATATGATATACTTACACATGGACGCCCTGAACCCTCTGGATACGGCCGTTTATCGGTGTGTCGCCAGATGG GACTGTTCGCGACACGATTTTATCACCCAGAAAACCGCTACCGGCATCTGGGGCCCGGGGACCCAGGTCACCGTGTC AGCA

Coding has SEQ ID NO.:Amino acid sequence shown in 1：

QLQLVESGGGSVQPGGSLTLSCSASGFNISQYSVGWFREAPGEEREGISCLDIDGKITTFSDAIQGRFTIARDNAAN MIYLHMDALNPLDTAVYRCVARWDCSRHDFITQKTATGIWGPGTQVTVSA。

Embodiment 3：

It is prepared by the scale of anti-c-Myc labels single domain heavy chain antibody

Encode the acquisition of the DNA fragmentation of anti-c-Myc labels single domain heavy chain antibody：1. using restriction enzyme SfiI/ NotI, double digestion phasmid pHEN-anti-c-Myc single domain heavy chain antibody genes, agarose gel electrophoresis reclaims anti-c-Myc marks Sign a bill domain heavy chain antibody genes；2. biotechnology service company directly is sent by anti-c-Myc labels single domain heavy chain antibody coded sequence Carry out chemical synthesis；3. specific primer is designed, is expanded by the cDNA storehouses that round pcr is originated from alpaca (Lama pacos) Increase.

The anti-c-Myc tags single domain heavy chain antibody genes fragment that will be obtained is cloned into expression vector pET25-flag (by carrier, institute band c-Myc tag replacements are Flag labels in itself：DYKDDDDK), identified through PCR and digestion, build and complete anti- The colibacillus expression plasmid of c-Myc label single domain heavy chain antibodies.

Expression plasmid is converted to Escherichia coli rosetta, picking single bacterium colony carries out induced expression.Single bacterium colony is accessed In 5mL LB-A (the μ g/mL ampicillin of Luria-Bertani broth with 100) fluid nutrient medium, 37 DEG C, 220r/ Min shaken cultivations 12h；It is transferred in 50mL LB-A fluid nutrient mediums with the inoculum concentration of 1% culture volume, 37 DEG C, 220r/min shaken cultivations are to OD₆₀₀0.5 (about needing 3~3.5h) is reached, the IPTG, 30 DEG C, 200r/ of final concentration 0.1mM is added Min Fiber differentiations.

Induced cultures 8000r/min is centrifuged, and adds 25mL phosphate buffers (pH 7.4) to mix in cell precipitation, 8000r/min is centrifuged, and removes supernatant, retains cell precipitation；15mL same buffers are added in cell precipitation, is mixed, surpassed on ice Sound wave clasmatosis is processed, and ultrasonication condition is 200W, crushes 2s, interval 5s, totally 250 circulations, broken to cell at 4 DEG C The 8000r/min that minces is centrifuged 15min, and taking supernatant carries out affinitive layer purification and SDS-PAGE electrophoretic analysis, or adds in supernatant Enter the glycerine of final concentration 20%, mix, be stored in -20 DEG C of refrigerator-freezers stand-by.

By optimizing induced expression condition (such as Host Strains, expression vector, Fiber differentiation time, temperature and IPTG concentration Deng), destination protein (single domain heavy chain antibody) expression quantity can be further improved, it is largely to prepare anti-c-Myc labels single domain heavy chain Antibody provides approach.

The amalgamation and expression of anti-c-Myc labels single domain heavy chain antibody：

Anti- c-Myc labels single domain heavy chain antibody genes of the invention are cloned into fusion expression vector pAP and (contain alkaline phosphatase Enzyme gene), identified through PCR and digestion, build the alkaline phosphatase fusion expression matter for completing anti-c-Myc labels single domain heavy chain antibody Grain.

Alkaline phosphatase can be with non-specific catalytic phosphatase monoesters hydrolysis generation inorganic phosphate and corresponding alcohol, phenol or carbohydrate Compound.The enzyme is used for the detection methods such as ELISA, Western blotting, histochemistry frequently as signal element.Fusion expression plasmid will Anti- c-Myc labels single domain heavy chain antibody is blended in the N-terminal of alkaline phosphatase, with reference to the expression in application example 3, Ke Yi Expression in escherichia coli, it is purified into fusion protein AP-anti-c-Myc label single domain heavy chain antibodies.

The heat-resistant experiment of the nano antibody of anti-c-Myc labels

Tested by ELISA and nano antibody heat endurance is measured, experimental technique is as follows：

Concentration is taken for 5 μ g/mL Myc-GST albumen, in adding 96 hole elisa Plates with the μ L of every hole 100,4 DEG C of coatings are overnight； 0.05%PBST board-washings 3 times；3% 37 DEG C of closing l h of skim milk；The c-Myc nano antibodies after dilution are added, per the μ of hole 100 L, is incubated 1h by 37 DEG C；Add 1：The anti-His labels secondary antibody of 2000 working concentration HRP marks, 100 μ L are added per hole, 37 DEG C, are incubated Educate 1h；TMB nitrite ions are added, 100 μ L are added per hole；30min is incubated at a temperature of in multigroup difference；After reaction terminates, add 2M sulfuric acid terminating reactions, 50 μ L are added per hole, and 450nm light absorption values are determined after mixing terminating reaction.

Even if result shows that temperature is up to 70 DEG C, 80 DEG C, 90 DEG C, protein active still has bioactivity, OD₄₅₀Value point Not Wei 1.5,1.3,1.1, with preferable heat resistance.

Embodiment 4：

It is prepared by a small amount of of c-Myc label affine in immunity magnetic beads

Using nanometer magnetic bead as carrier, after being coupled anti-c-Myc labels single domain heavy chain antibody, obtain c-Myc labels and be immunized Magnetic bead, specific preparation method is as follows：

The magnetic bead of 1mg carboxyl modifieds is taken in centrifuge tube, 500 μ l activation buffers (10mM, NaH are added₂PO₄, pH 6.0), vortex mixed is uniform, and magnetic frame reclaims magnetic bead, then is washed with activation buffer 3 times.It is separately added into 2mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS), after vortex mixed, 25min is stood.With coupling buffer (10mM, Na₂HPO₄, pH 7.4) washing magnetic bead 3 times, addition is dissolved in anti-c-Myc labels single domain heavy chain antibody 1mg, the room temperature reaction 3.5h of coupling buffer, Magnetic bead is washed with coupling buffer 5 times, add 500 μ l to contain 1% (w/v) bovine serum albumin(BSA) (BSA) or 1% (w/v) egg white egg The coupling buffer of (OVA) closes unreacted active group, room temperature reaction 35min in vain.Magnetic bead 5 is washed with coupling buffer It is secondary, PBS solution (10mM, pH 7.2,0.02%w/v, Na₃N 4 DEG C are stored in after) resuspended.

Embodiment 5：

The preparation of c-Myc label affine in immunity sorbing materials and affinity column

Using agarose microbeads as carrier, anti-c-Myc labels single domain heavy chain antibody is coupled, specific preparation method is as follows：

The dry glue that CNBr is activated is washed 10 times with 0.1M HCl, 5min is balanced every time.With coupling buffer (10mM, Na₂HPO₄, pH 7.2) wash 10 times, anti-c-Myc labels single domain heavy chain antibody (every gram of agarose microbeads of 2mg/) is added, room temperature is anti- 3.5h is answered, makes the agarose gel microsphere covalent coupling of anti-c-Myc labels single domain heavy chain antibody and CNBr activation.Buffered with coupling Liquid (10mM, Na₂HPO₄, pH 7.2) and after washing 3 times, confining liquid room temperature reaction 2.5h is added closing unreacted active group. The phosphate buffer (10mM, pH 7.2) and acetate buffer solution (0.1M, pH 4.5) accumulated with 6 times of colloids alternately washing 3 times, obtain The covalent coupling affine in immunity sorbing material of anti-c-Myc labels single domain heavy chain antibody.Take the above-mentioned affine in immunity adsorption materials of 0.2ml Expect the chromatographic column for 1ml in capacity, after 8~10 times of PBS of bed volume (10mM, pH 7.2) washings, add 20% ethanol molten Liquid, 4 DEG C of preservations.

Embodiment 6：

Using silica gel microball as carrier, anti-c-Myc labels single domain heavy chain antibody is coupled, specific preparation method is as follows：

Alternately washing 6~10 times of 2g silica gel microballs pure water and phosphate buffer (PBS, 10mM, pH 6.5) are taken, 10ml is used PBS suspension silica gel microball, adds the anti-c-Myc labels single domain heavy chain antibodies of 5mg, mixes, and adds final concentration 5mg/ml's Carbodiimide (EDC), rapid to mix, 4 DEG C of 12~20h of stirring reaction obtain the anti-c-Myc labels single domain heavy chain of covalent coupling and resist The affine in immunity sorbing material of body.The above-mentioned affine in immunity sorbing materials of 0.2ml are taken in the chromatographic column that capacity is 1ml, 58~10 times After PBS (10mM, the pH 6.5) washings of bed volume, add and contain 0.02% (w/v) Na₃The PBS (10mM, pH 6.5) of N, 4 DEG C Preserve.

Embodiment 7：

The adsorbance of c-Myc label affinity columns, reuse are determined

Pillars are cleaned with 6 times of bed volume PBS (10mM, pH 7.2), protein sample solution is added, efflux mistake again Post.Then with 3 times of bed volume pure water drip washing, then being marked containing c-Myc for specific adsorption is eluted with glycine hydrochloride (pH 2.2) Sign recombinant protein, the eluent of collection, protein solution as after purification.Test result indicate that, be filled with 1mL embodiments 5,6, 7 affinity column/the magnetic beads for preparing can be with specific adsorption destination protein.Reuse 10 times afterwards, the rate of recovery is still above 80%. It is single domain heavy chain antibody that aglucon can be produced by biological method mass propgation, it is to avoid the cumbersome producer such as artificial antibody Method, greatly reduces production cost, and reusable, has a extensive future.

SEQUENCE LISTING

<110>University Of Nanchang

<120>Affine sorbing material based on specific recognition c-Myc label nano antibodies

<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 Leu Ser 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

<211> 24

<212> DNA

<213>Artificial sequence

<400> 8

agcggataac aatttcacac agga 24

<210> 9

<211> 20

<212> DNA

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gccccattca gatcctcttc 20

Claims

1. a kind of c-Myc labels affine in immunity sorbing material, including carrier, is mounted in the aglucon on carrier, it is characterised in that should Material is using the single domain heavy chain antibody of specific recognition c-Myc labels as aglucon, the single domain of the specific recognition c-Myc labels Heavy chain antibody has SEQ ID NO.:Amino acid sequence shown in 1.

2. c-Myc labels affine in immunity sorbing material according to claim 1, it is characterised in that the aglucon is in SEQ ID NO.:The energy and c-Myc of house of correction acquisition are carried out on the basis of amino acid sequence shown in 1 by random or site-directed mutagenesis technique The antibody of label specific binding.

3. c-Myc labels affine in immunity sorbing material according to claim 1, it is characterised in that the carrier is magnetic bead.

4. c-Myc labels affine in immunity sorbing material according to claim 1, it is characterised in that the carrier is agar Sugared microballoon.

5. c-Myc labels affine in immunity sorbing material according to claim 1, it is characterised in that the carrier is silica gel Microballoon or porous material.