CN107042092A

CN107042092A - Affine sorbing material based on specific recognition c Myc label single domain heavy chain antibodies

Info

Publication number: CN107042092A
Application number: CN201710100769.4A
Authority: CN
Inventors: 付金衡; 涂追; 许杨; 吴青松
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2017-02-23
Filing date: 2017-02-23
Publication date: 2017-08-15

Abstract

The invention belongs to genetic engineering field, specially the single domain heavy chain antibody (also known as nano antibody) for c Myc labels, it has the amino acid sequence shown in SEQ ID NO.1.Amino acid sequence provided by the present invention can be used as precursor, transformed by random or site-directed mutagenesis technique, result in 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 single domain heavy chain antibodies

Technical field

It is special the present invention relates to a kind of affine in immunity sorbing material based on single domain heavy chain antibody (also known as nano antibody technology) It is not the affine in immunity sorbing material for 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 antibody identification is made up of 10 amino acid residues, its sequence For 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 containing c-Myc marks Sign fusion protein.The principle that this kind of affinity purification technology is specifically bound based on aglucon and 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.The existing affinity column purified for c-Myc tag fusion proteins and affine magnetic bead price are generally higher.And nanometer is anti- Body can in Escherichia coli, yeast etc. are biological 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 elution inevitably can cause 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, which can be repeated several times, to be used.

The content of the invention

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

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

Include carrier and aglucon for the affine in immunity sorbing material of c-Myc labels, 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 on the basis of foregoing single domain heavy chain antibody by random or site-directed mutagenesis technique Make the antibody that can be specifically bound with c-Myc labels 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 in that：

When the carrier is magnetic bead, preparation method is：1mg carboxyl magnetic beads are taken in centrifuge tube, 600~1000 μ l is added and lives 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.It is separately added into after 1~5mg carbodiimides (EDC) and n-hydroxysuccinimide (NHS), vortex mixed, stands 35min.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, added with coupling buffer (10mM, Na2HPO4, pH 7.2) Anti- c-Myc labels single domain heavy chain antibody, reacts at room temperature 3~10h, 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 and resist Body, is mixed, and adds 1~10mg/ml of final concentration carbodiimide (EDC), 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 after chromatographic column, PBS (10 mM, the pH 7.2) washings for adding 8~10 times of bed volumes, 4 DEG C, 20% ethanol solution is stored in.

The invention further relates to be mounted with the affinity column of 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 of the label containing c-Myc 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, then uses pure water Elution, then with glycine hydrochloride (pH 2.2) elute specific adsorption the recombinant protein of label containing c-Myc, the eluent of collection, Sample extracting solution after purification, available for subsequent analysis detection.When carrier is magnetic bead, method is：Immunomagnetic beads is added pure In water, magnetic frame reclaims magnetic bead, and then immunomagnetic beads is added in sample extracting solution, mixed, magnetic frame by repeated washing 3~5 times Magnetic bead is reclaimed, after pure water is cleaned 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, is sample extracting solution after purification, available 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 aglucon can be produced 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.

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 the application of the present 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, takes 300 μ g c-Myc-BSA with after Freund's complete adjuvant emulsification, entering 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.

RNA extraction is carried out with reference to TAKARA company RNAiso reagents specification.Using 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, 600bp~750bp DNA fragmentation is reclaimed, is made For second wheel PCR template, 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.Reclaim, quantify through DNA fragmentation QIAquick Gel Extraction Kit, saved backup in -20 DEG C.It will bite Bacterium grain pHEN1 and pcr amplification product use Sfi I, Not I double digestions respectively, reclaimed through Ago-Gel, it is quantitative after, with 1: 3 Mol ratio, at 16 DEG C, is connected overnight.

The library construction of table 1 and identification primer used

Note：Underscore represents restriction endonuclease recognition sequence

Connection product is dissolved in 10 μ L sterilized waters after ethanol precipitation, and 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 24cm × 24cm ampicillin 2 × YT culture plates are all coated on, 37 DEG C, 13~16h of culture are inverted.Trained with 10mL, 2 × YT After foster base scrapes the lawn on culture plate, the glycerine of final concentration 20~30% is added, is dispensed, -80 DEG C save backup.

According to the storage capacity result of calculating, the living cells for being inoculated with 10 times of storage capacities (contains 2% grape in 20mL 2 × YT Sugar, 100 μ g/mL ampicillins), 37 DEG C, 200r/min is cultivated to OD₆₀₀Up to 0.5, add auxiliary by infection multiplicity 20: 1 and bite Thalline, 37 DEG C, 200r/min, 60 min.Culture is centrifuged, (contains 100 μ g/mL ampicillins and 50 with 50mL 2 × YT μ g/mL kanamycins) precipitation is resuspended, 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 centrifugation 30min, resuspension is deposited in the phosphoric acid containing 10% glycerine and delayed Fliud flushing (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 to determine titre, Remaining is sub-packed in -80 DEG C and saved backup.

Embodiment 2：

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

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 the gained of embodiment 1 Elutriation is directed to the single domain heavy chain antibody of c-Myc labels.The Myc-GST that 120 μ L PBS dilute is added into each enzyme mark hole to melt Hop protein (albumen that Myc labels are merged with glutathione), 4 DEG C, coating is stayed overnight, and the coating concentration for often taking turns elutriation is respectively 100,75,50 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-washing 5 times, adds 100 μ L phage antibody libraries (containing about 1 × 10¹¹CFU), 37 DEG C, it is incubated 2.0h；Suction out uncombined phagocytosis Body, with PBST (contain 0.5%Tween-20) board-washing 3-5 time (increasing by 5 times by wheel), then with PBS board-washings 15-25 times；With 100 μ L Bacteriophage of eluent (glycine-HCI, pH 2.2) the elution absorption in enzyme mark hole, with 35 μ L Tris-HCl (1 mol/L, PH 8.0) eluate is neutralized, take 10 μ L to be used for titer determination, be used for next round elutriation after the amplification of remaining 125 μ L eluate.

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

The indirect phage-ELISA of table 2 is loaded table

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

DNA sequence dna (SEQ ID NO.:2)：

CAGTTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTG ACGCTCTCCTGTCTAGCCTCTGGATTCACTCTTAGTAACTACTACATGAGCTGGGTCCG CCAGGCTCCAGGAAAGGGGCCCGAGTGGGTCGCGGAGGTTAATGATAGTGGCTTTGG CAGAAACTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACGCCAAG AGCACGGTATACTTGGAAATGAATGACCTGAAACCTGAGGACACGGCCCTATATTACTG TGCGCGCGATCGGTATCTTGACCTCAGAGAGTATGATTACTGGGGCCAGGGGACCCAG GTCACCGTCTCCTCA

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

QLQLVESGGGLVQPGGSLTLSCLASGFTLSNYYMSWVRQAPGKGPEWVAEVNDSGF GRNYGDSVKGRFTISRDNAKSTVYLEMNDLKPEDTALYYCARDRYLDLREYDYWGQGT QVTVSS。

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. directly send biotechnology service company by anti-c-Myc labels single domain heavy chain antibody coded sequence Carry out chemical synthesis；3. expand in designing specific primer, the cDNA storehouses originated by round pcr from alpaca (Lama pacos) Increase.

Obtained anti-c-Myc tags single domain heavy chain antibody genes fragment 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 to the inoculum concentration of 1% culture volume in 50mL LB-A fluid nutrient mediums, 37 DEG C, 220r/min shaken cultivations are to OD₆₀₀0.5 (about needing 3~3.5h) is reached, final concentration 0.1mM IPTG, 30 DEG C, 200r/ is added Min Fiber differentiations.

Induced cultures 8000r/min is centrifuged, and 25mL phosphate buffers (pH 7.4) are added in cell precipitation and are mixed, 8000r/min is centrifuged, and removes supernatant, retains cell precipitation；15mL same buffers are added in cell precipitation, mixes, surpasses on ice Sound wave clasmatosis is handled, and ultrasonication condition is 200W, crushes 2s, interval 5s, totally 250 circulations, broken to cell at 4 DEG C The 8000r/min that minces centrifuges 15min, takes supernatant to carry out affinitive layer purification and SDS-PAGE electrophoretic analysis, or add 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, 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 (containing 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 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

Nano antibody heat endurance is measured by ELISA experiments, experimental method is as follows：

It is 5 μ g/mL Myc-GST albumen to take concentration, is added with every μ L of hole 100 in 96 hole elisa Plates, 4 DEG C of coatings are stayed overnight； 0.05%PBST board-washings 3 times；3% 37 DEG C of closing lh of skim milk；The c-Myc nano antibodies added after dilution, 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, mix and 450nm light absorption values are determined after terminating reaction.

Even if as a result display temperature is up to 70 DEG C, 80 DEG C, 90 DEG C, protein active still has bioactivity, OD₄₅₀Value point Not Wei 1.4,1.3,1.2, with preferable heat resistance.

Embodiment 4：

A small amount of preparations of c-Myc label affine in immunity magnetic beads

Using nanometer magnetic bead as carrier, it is coupled after anti-c-Myc labels single domain heavy chain antibody, obtains 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 3 times with activation buffer.It is separately added into 2mg carbodiimides (EDC) and n-hydroxysuccinimide (NHS), after vortex mixed, 25min is stood.With coupling buffer (10mM, Na₂HPO₄, pH 7.4) washing magnetic bead 3 times, adds the anti-c-Myc labels single domain heavy chain antibody 1mg for being dissolved in coupling buffer, reacts at room temperature 3.5h, Magnetic bead is washed with coupling buffer 5 times, add 500 μ l and 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 in vain, reacts at room temperature 35min.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) being 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, and 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, the agarose gel microsphere covalent coupling for making anti-c-Myc labels single domain heavy chain antibody be activated with CNBr.With coupling buffering Liquid (10mM, Na₂HPO₄, pH 7.2) and after washing 3 times, confining liquid room temperature reaction 2.5h is added to close unreacted active group 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 affine in immunity sorbing material of the anti-c-Myc labels single domain heavy chain antibody of covalent coupling.The above-mentioned affine in immunities of 0.2ml are taken to inhale Enclosure material adds 20% second in after the chromatographic column that capacity is 1 ml, PBS (10mM, the pH 7.2) washings of 8~10 times of bed volumes Alcoholic solution, 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 The affine in immunity sorbing material of antibody.The above-mentioned affine in immunity sorbing materials of 0.2ml are taken in the chromatographic column that capacity is 1ml, 58~10 After PBS (10mM, the pH 6.5) washings of times bed volume, add and contain 0.02% (w/v) Na₃N PBS (10mM, pH 6.5), 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 eluted with 3 times of bed volume pure water, then being marked containing c-Myc for specific adsorption is eluted with glycine hydrochloride (pH 2.2) Recombinant protein is signed, the eluent of collection is protein solution after purification.Test result indicates that, be filled with 1mL embodiments 5,6, 7 affinity column/the magnetic beads prepared can be with specific adsorption destination protein.After reusing 10 times, 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 single domain heavy chain antibodies

<130> 2017

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 360

<212> PRT

<213>Artificial sequence

<400> 1

Cys Ala Gly Thr Thr Gly Cys Ala Gly Cys Thr Cys Gly Thr Gly Gly

1 5 10 15

Ala Gly Thr Cys Ala Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr

20 25 30

Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly

35 40 45

Thr Cys Thr Cys Thr Gly Ala Cys Gly Cys Thr Cys Thr Cys Cys Thr

50 55 60

Gly Thr Cys Thr Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr

65 70 75 80

Cys Ala Cys Thr Cys Thr Thr Ala Gly Thr Ala Ala Cys Thr Ala Cys

85 90 95

Thr Ala Cys Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys

100 105 110

Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Ala Ala Ala

115 120 125

Gly Gly Gly Gly Cys Cys Cys Gly Ala Gly Thr Gly Gly Gly Thr Cys

130 135 140

Gly Cys Gly Gly Ala Gly Gly Thr Thr Ala Ala Thr Gly Ala Thr Ala

145 150 155 160

Gly Thr Gly Gly Cys Thr Thr Thr Gly Gly Cys Ala Gly Ala Ala Ala

165 170 175

Cys Thr Ala Thr Gly Gly Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly

180 185 190

Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala

195 200 205

Thr Cys Thr Cys Thr Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys

210 215 220

Cys Ala Ala Gly Ala Gly Cys Ala Cys Gly Gly Thr Ala Thr Ala Cys

225 230 235 240

Thr Thr Gly Gly Ala Ala Ala Thr Gly Ala Ala Thr Gly Ala Cys Cys

245 250 255

Thr Gly Ala Ala Ala Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys

260 265 270

Gly Gly Cys Cys Cys Thr Ala Thr Ala Thr Thr Ala Cys Thr Gly Thr

275 280 285

Gly Cys Gly Cys Gly Cys Gly Ala Thr Cys Gly Gly Thr Ala Thr Cys

290 295 300

Thr Thr Gly Ala Cys Cys Thr Cys Ala Gly Ala Gly Ala Gly Thr Ala

305 310 315 320

Thr Gly Ala Thr Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly

325 330 335

Gly Gly Gly Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys Cys Gly

340 345 350

Thr Cys Thr Cys Cys Thr Cys Ala

355 360

<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

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<221> misc_feature

<222> (44)..(44)

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<210> 5

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<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

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gccccattca gatcctcttc 20

Claims

1. a kind of c-Myc labels affine in immunity sorbing material, including carrier, are 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.