CN106117349A - A kind of single domain heavy chain antibody of the specific recognition immunoglobulin Fc section in immune library source - Google Patents
A kind of single domain heavy chain antibody of the specific recognition immunoglobulin Fc section in immune library source Download PDFInfo
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- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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Abstract
The invention belongs to genetic engineering field, be specially the single domain heavy chain antibody for immunoglobulin Fc section, it has the aminoacid sequence shown in SEQ ID NO.1, can be used for the field such as immune detection, antigen enrichment purification.Aminoacid sequence provided by the present invention can be as precursor, transformed by random or site-directed mutagenesis technique, it is obtained in that character (affinity, specificity, stability etc.) preferably mutant, is further used for medicine, industry, the protein of agricultural or polypeptide for developing.
Description
Technical field
The present invention relates to single domain heavy chain antibody technology (being also called nano antibody technology), and genetic engineering antibody technology,
Particularly relate to for immunoglobulin G (immunoglobulin G, IgG) Fc section (fragment crystalline, Fc)
Single domain heavy chain antibody or polypeptide.
Technical background
Heavy chain antibody (Heavy-chain antibody) is a kind of natural deletions light chain, the antibody being only made up of heavy chain,
It is present in the animal such as camel, shark.Single domain heavy chain antibody (is also called nano antibody, VHH antibody, variable domain
Of heavy chain of heavy-chain antibody, lower same) refer to only by antibody heavy chain variable region (Variable
Region) genetic engineering antibody formed.Compared with common antibody, it is little that single domain heavy chain antibody has molecular weight, and stability is high, water
The advantages such as dissolubility is good, are widely used to basic research, medical diagnosis and the field such as detection, medicament research and development at present.
Immunoglobulin G (immunoglobulin G, IgG) is by the identical heavy chain group of two identical light chains and two
Become.Every chain all contains variable region (variable region, V district) and constant region (constant region, C district), wherein
Variable region provides antigen binding site and specificity, and constant region constitutes the framework of immunoglobulin, has biological effect function.With
Papain digestion IgG, can be divided into 3 functional areas, i.e. 2 identical Fab (fragment
Antigen binding, Fab) and a FC (fragment crystalline, Fc).Fc section is positioned at constant region,
It is made up of the half of two heavy chain c-terminuses.Owing to Fc section is away from antigen-binding site, it is thus provided that one and antibodies
And do not affect the region of antigen antibody reaction, it is the epi-position district of maximally effective two anti-reagent combinations.
At present, the open report of the conventional antibodies such as the polyclonal antibody for IgG Fc section, monoclonal antibody is had.With list
The shortcomings such as territory heavy chain antibody is compared, and it is higher that conventional antibodies exists production cost, and preparation process is loaded down with trivial details.
Summary of the invention
The purpose of the present invention is to provide the single domain heavy chain antibody of the Fc section for immunoglobulin G and (includes containing described list
The protein of all or part of functional area of territory heavy chain antibody or polypeptide) and aminoacid sequence, can be used for preparation detection or
Purification, the reagent of enrichment IgG and instrument.
The present invention provides single domain heavy chain antibody (the one immune library the most of the present invention of a kind of Fc section for immunoglobulin G
The nano antibody of the specific recognition immunoglobulin Fc section in source), there is the aminoacid sequence shown in SEQ ID NO.:1.
The IMGT numbering of its aminoacid sequence and the division of domain include four framework regions (Framework region,
FR) and three complementary determining regions (Complementarity-determining region, CDR).Complementary determining region is mainly born
The identification of duty antigen, framework region structure is the most stable, mainly plays a part Protein requirement structure.
The present invention provides a nucleic acid molecules, it is characterized in that encoding SEQ ID NO.:1, can be with by genetic codon
Time obtain the particular sequence of this nucleic acid molecules.
The present invention also provides for a nucleic acid molecules, it is characterized in that encoding SEQ ID NO.:1 partial domain, by heredity
Codon can obtain the particular sequence of this nucleic acid molecules at any time.It can be SEQ ID NO.:2 nucleic acid molecules.
Nucleotide sequence provided by the present invention or at least partly sequence can carry out table by suitable expression system
Reach to obtain corresponding protein or polypeptide.These expression systems include antibacterial, yeast, filamentous fungi, zooblast, insecticide
Cell, plant cell, or Cell free expression system.
The present invention also provides for a kind of carrier, comprises described nucleotide sequence.Owing to genetic codon has degeneracy, this core
Acid sequence can be different according to different application purposes.
The present invention also provides for a kind of host cell, including described protein or expression vector.
Present invention also offers a kind of purification or the method for detection immunoglobulin G, it is characterized in that containing above-mentioned protein
Or polypeptide.The protein provided based on the present invention or the polypeptide ability specific binding with immunoglobulin G, set up immune globulin
The purification of white G or detection method.Wherein, preferred method includes enzyme linked immunosorbent assay (Enzyme-linked
Immunosorbent assay, ELISA), fluorescent immune method (Fluoroimmunoassay, FIA), immuno-chip method and affine
Chromatography.
Aminoacid sequence provided by the present invention can be transformed by random or site-directed mutagenesis technique as precursor,
It is obtained in that character (water solublity, stability, affinity and specificity etc.) preferably mutant, is used for developing being further used for
Medical, industrial, agriculture protein or polypeptide.
The invention still further relates to the aforementioned nano antibody for immunoglobulin G Fc section at immune detection, enrichment and purification
In application.These immune detection refer to the immune detection of non-diseases diagnoses and treatment purpose.
The invention still further relates to the affine adsorbing material of immunity of the Fc section for immunoglobulin G, including carrier, be mounted in load
Aglucon on body, it is characterised in that this material using the nano antibody of the Fc section for immunoglobulin G as aglucon, described for
The nano antibody of the Fc section of immunoglobulin G has the aminoacid sequence shown in SEQ ID NO.:1.Carrier material is not limited to fine jade
Sepharose, it is also possible to select silicon ball, nanometer magnetic bead etc..
Some terms described in the present invention have a following implication:
Homology: describe the similarity degree of two or more aminoacid sequences, first aminoacid sequence and second ammonia
Between base acid sequence, the percentage ratio of homology can be by [with relevant position in the second aminoacid sequence in the first aminoacid sequence
The quantity of the identical amino acid residue of amino acid residue at place] take advantage of again divided by [aminoacid sum in first aminoacid sequence]
Calculate with [100%], wherein certain the amino acid whose disappearance in the second aminoacid sequence, insert, replace or add (with first
Aminoacid is compared) it is considered as to have difference.Alternatively, percent homology can also utilize known based on sequences match
Calculation machine operation program such as NCBI Blast obtains.
Domain: the fundamental structural unit of tertiary protein structure, is generally of certain function.
IMGT numbers: in IMGT data base (The International ImMunoGeneTics Database)
Plant the most standardized antibody amino acids sequence method for numbering serial.Concrete method for numbering serial be referred to document (Ehrenmann, F.,
Q.Kaas,et al.(2010)."IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database
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 Ig superfamily V-like domains."Dev Comp Immunol 27(1):
Description in 55-77.).
Codon (codon): be also called triplet code (triplet code), refer to corresponding to certain amino acid whose nucleoside
Acid triplet.The position of polypeptide chain in this kind of aminoacid insertion growth is determined during translation.
The invention provides the single domain heavy chain antibody of the identification IgG Fc section obtained from non-immune libraries, this single domain heavy chain resists
Body have passed through affinity maturation, has higher affinity, and recognition site is the dominant antigen epi-position of Fc.
Detailed description of the invention
Below by the preparation of chain antibody in single domain, analyzing and apply, the invention will be further described, and these are concrete
Embodiment is not construed in any way as limiting the range of application of the present invention.
Embodiment 1:
The structure of anti-Fc single domain heavy chain antibody non-immune libraries
After taking 300 μ g Fc recombiant proteins (this albumen can be by being either commercially available) and Freund's complete adjuvant emulsifying, to sheep
Camel (Lama pacos) carries out the immunity of subcutaneous multi-point injection.Booster immunization uses 150 μ g Fc recombiant proteins not exclusively to help with Freund
Agent emulsifying, is spaced 2 weeks and carries out, and 7 days posterior veins of immunity take blood every time, uses indirect elisa method to measure serum titer, selects serum
The sample separation lymphocyte that titer is the highest, extracts RNA.
The extraction of RNA is carried out with reference to TAKARA company RNAiso reagent description.With RNA as template, oligo dT is for drawing
Thing, with reference to TAKARA company reverse transcription description synthesis cDNA the first chain.
Using PrimeSTAR high-fidelity DNA polymerase, the variable region encoding gene obtaining heavy chain antibody through nest-type PRC (is adopted
Primer be shown in Table 1).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 extend 10min.
By first round PCR primer with the agarose gel electrophoresis of 1.2%, reclaim the DNA fragmentation of 600bp～750bp, as
Second template taking turns PCR, respectively with primer AlpVh-SfiI and AlpVHHR1-NotI, AlpVh-SfiI and AlpVHHR2-
NotI, expands, 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 extend 10min.Through DNA fragmentation recovery test kit recovery, quantitatively, save backup in-20 DEG C.To bite
Bacterium grain pHEN1 and pcr amplification product respectively with Sfi I, Not I double digestion, reclaim through agarose gel, quantitatively after, rub with 1: 3
That ratio, at 16 DEG C, overnight connects.
Primer used by table 1 library construction and qualification
Note: underscore represents restriction endonuclease recognition sequence
Connection product, after ethanol precipitates, is dissolved in 10 μ L sterilized water, carries out Electroporation Transformation e. coli tg1 in ten times.
Take 10 μ L electric shock, cultivate after bacterium solution doubling dilution, be coated with ampicillin 2 × YT culture plate, 37 DEG C, be inverted cultivate 12～
16h, uses primer M13-R and pHEN-R to carry out bacterium colony PCR, calculates storage capacity；Remainder all coats 24cm × 24cm ammonia
Benzylpcnicillin 2 × YT culture plate, is inverted and cultivates 12～16h by 37 DEG C.By 10mL, 2 × YT culture medium, the lawn on culture plate is scraped
After washing, adding final concentration 15～30% glycerol, subpackage ,-80 DEG C save backup.
According to calculate storage capacity result, inoculation 10 times of storage capacities living cells in 20mL 2 × YT (containing 2% glucose,
100 μ g/mL ampicillin), 30 DEG C, 220r/min cultivates and reaches 0.5 to OD600, adds helper phage by infection multiplicity 20: 1
Body, 37 DEG C, 220r/min, 60min.Culture is centrifuged, with the 2 × YT of 50mL (containing 100 μ g/mL ampicillin and 50 μ g/
ML kanamycin) resuspended precipitation, 30 DEG C, after 220r/min incubated overnight, 3000g centrifuging and taking supernatant, add 5 × PEG/NaCl molten
Liquid, places 1h or 4 DEG C overnight on ice, and 12000rpm is centrifuged 30min, the resuspended phosphate buffer being deposited in containing 10% glycerol
(PBS, 0.01M, pH 7.4), i.e. obtains the single domain heavy chain antibody non-immune libraries of anti-Fc, takes 10 μ L and measures titre, remaining subpackage
Save backup in-80 DEG C.
Embodiment 2:
The elutriation of anti-igg Fc section single domain heavy chain antibody and qualification
Use the affine elutriation of solid phase method from the single domain heavy chain antibody non-immune libraries of anti-Fc elutriation for IgG Fc section
Single domain heavy chain antibody.Use affinity chromatography purified mouse serum, obtain IgG solution.With PBS dilution IgG solution to 50～
100 μ g/mL, every hole adds 100 μ L, 4 DEG C, is coated overnight；Sucking-off is coated liquid, and PBS washes plate 3 times, and every hole adds 300 μ L 3%
BSA-PBS, closes 2h by 37 DEG C；PBS washes plate 6 times, adds 100 μ L phage antibody libraries (containing about 2 × 1011CFU), 37 DEG C, hatch
1.5h；The unconjugated phage of sucking-off, washes plate 5 times (increasing by 1 time by wheel) with PBST (containing 0.5%Tween-20), then washes with PBS
Plate 10 times (washing plate number of times to increase by 5 times by wheel)；Adsorb in enzyme mark hole with 100 μ L eluents (glycine-HCI, pH 2.2) eluting
In phage, neutralize eluates with 50 μ L Tris-HCl (1mol/L, pH 8.0), take 10 μ L for titer determination, remaining is washed
For next round elutriation after de-thing amplification.
After three-wheel elutriation, use helper phage KM13 that the monoclonal of random picking is rescued, respectively obtain exhibition
Show the phage particle of antibody variable region, then it is right to set the positive with the combination activity experiment of indirect ELISA mensuration phage particle
According to, negative control and ground control, concrete 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 colony X, obtain the DNA sequence of Insert Fragment,
It encodes the single domain heavy chain antibody for immunoglobulin Fc section, (SEQ ID NO.:2) specific as follows:
CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGCA
GCCTCTGGATTCACCTTCAGTAGCTATGTTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTC
AGATATTAATAGTGGTGGTGGTAGCACAGACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACG
CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCCCTAGGGCGT
AACCGGGTAGCGACTAGGAGGTCGAATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCAGCA
The amino of the single domain heavy chain antibody of immunoglobulin Fc section it is available for according to DNA sequencing result and password sublist
Acid sequence (SEQ ID NO.:1):
QLQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSDINSGGGSTDYADSVKGRFT
ISRDNAKNTLYLQMNSLKPEDTAVYYCALGRNRVATRRSNDYWGQGTQVTVSA
The single domain heavy chain antibody of the identification IgG Fc section obtained from non-immune libraries that the present invention provides, this single domain heavy chain resists
Body have passed through affinity maturation, and through controlled trial, the single domain heavy chain separating acquisition from natural antibody library comparing routine resists
Body has higher affinity, and recognition site is the dominant antigen epi-position of Fc.
Embodiment 3:
Anti-igg Fc section single domain heavy chain antibody is at expression in escherichia coli and purification.
The acquisition of the DNA fragmentation of coding anti-igg Fc section single domain heavy chain antibody: 1. use restricted enzyme SfiI/
NotI, double digestion phasmid pHEN-X, agarose gel electrophoresis reclaims anti-igg Fc section single domain heavy chain antibody genes；The most directly will
Anti-igg Fc section single domain heavy chain antibody coded sequence send biotechnology service company to carry out chemosynthesis；3. design specificity draws
Thing, is expanded from the cDNA storehouse that alpaca (Lama pacos) is originated by round pcr.
The anti-igg Fc section single domain heavy chain antibody genes fragment obtained is cloned into expression vector pRXS, through PCR and enzyme action
Identify, built the colibacillus expression plasmid of anti-igg Fc section single domain heavy chain antibody, named pRXS-X.
Converting expression plasmid pRXS-X to e. coli bl21, picking list bacterium colony carries out abduction delivering.List bacterium colony is connect
Enter in 4mL LBA (Luria-Bertani broth with 100 μ g/mL ampicillin) fluid medium, 37 DEG C,
250r/min shaken cultivation 12h；It is transferred in 50mL LBA fluid medium with the inoculum concentration of 1% culture volume, 37
DEG C, 250r/min shaken cultivation to OD600 reach 0.5 (about needing 2.5～3h), add the IPTG of final concentration 0.1mM, 30 DEG C,
200r/min inducing culture.
Induced cultures 8000r/min is centrifuged, and adds 20mL phosphate buffer (pH 7.4) mixing in cell precipitates,
8000r/min is centrifuged, and removes supernatant, retains cell precipitation；In cell precipitates, add 10mL same buffer, mixing, surpass on ice
Sound wave cell breakage processes, and ultrasonication condition is 200W, broken 2s, intermittently 3s, and cell is broken at 4 DEG C by totally 240 circulations
The 12000r/min that minces is centrifuged 20min, takes supernatant and carries out affinitive layer purification and SDS-PAGE electrophoretic analysis, or adds in supernatant
Enter the glycerol of final concentration 30%, mixing, it is stored in-20 DEG C of refrigerator-freezers stand-by.
By optimizing abduction delivering condition (such as Host Strains, expression vector, inducing culture time, temperature and IPTG concentration
Deng), destination protein (single domain antibody) expression can be improved further, provide approach for a large amount of preparation anti-igg single domain antibodies.
Embodiment 4:
The amalgamation and expression of anti-igg Fc section single domain heavy chain antibody.
Anti-igg Fc section single domain heavy chain antibody genes is cloned into fusion expression vector pAP, identifies through PCR and enzyme action, structure
The alkaline phosphatase fusion expression plasmid of anti-igg Fc section single domain heavy chain antibody, named pAP-X are built.
Alkali phosphatase can generate inorganic phosphate and corresponding alcohol, phenol or saccharide with the hydrolysis of non-specific catalytic phosphatase monoesters
Compound.This enzyme is used for the detection methods such as ELISA, immunoblotting, histochemistry frequently as signal label.Fusion expression plasmid
Anti-igg Fc section single domain heavy chain antibody is blended in the N end of alkali phosphatase by pAP-X, and the expression in reference example 3 can
With at expression in escherichia coli, be purified into fusion protein AP-X.
Embodiment 5:
IgG detection method based on anti-igg Fc section single domain heavy chain antibody.
Principle based on direct elisa, the method setting up detection IgG.Use affinitive layer purification serum
Or ascites obtains IgG solution, or buy commercialization IgG product；The preparation of fusion protein AP-X is with reference to application example 3, colour developing
Reagent is analytical pure 4-NPP (pNPP 2Na).
Detecting step includes, (1) is antigen coated: with 10mM phosphate buffer (pH 7.4), IgG is diluted to 5 μ g/mL,
100 μ L/ holes, are coated in ELISA Plate, and 4 DEG C overnight, and the phosphate buffer containing 0.5%Tween-20 (W/V) washes plate 5 times, pats dry plate
Bar, adds 3% skim milk (W/V), 300 μ L/ holes, closes 2h for 37 DEG C.(2) combine: after phosphate buffer washes plate 3 times, add
The fusion protein AP-X of doubling dilution, 100 μ L/ holes, horizontal direction mixes gently, 37 DEG C of incubation 30min.(3) after taking out incubation
Lath, phosphate buffer is washed plate 5 times, is patted dry, and adds 100 μ L/ hole pNPP nitrite ions, 37 DEG C of lucifuge colour developing 5min.(4) 50 are added
μ L/ hole stop buffer (2M H2SO4), microplate reader reading.
Embodiment 6
Anti-Fc single domain heavy chain antibody is for the preparation of affinity purification material
By anti-igg Fc section single domain heavy chain antibody recombinant expressed for the present invention and solid phase carrier agarose coupling, concrete grammar
As follows:
The agarose dry glue 0.1M HCl activated by CNBr washs 10 times, balances 5min every time.Use coupling buffer
(10mM, Na2HPO4, pH 7.4) wash 10 times, add anti-igg Fc section single domain heavy chain antibody (2mg/ every gram agarose microbeads),
Room temperature reaction 4h, makes the agarose gel microsphere covalent coupling of anti-igg Fc section single domain heavy chain antibody and CNBr activation.Use coupling
Buffer (10mM, Na2HPO4, pH 7.4) washing 2 times after, add confining liquid room temperature reaction 2h to close unreacted active group
Group.Alternately washing 3 times of the phosphate buffer (10mM, pH 7.4) amassed by colloid with 5 and acetate buffer solution (0.1M, pH 4.0),
Obtain the affine adsorbing material of immunity of covalent coupling anti-igg Fc section single domain heavy chain antibody.
Solid support material is not limited to agarose gel, it is also possible to select silicon ball, nanometer magnetic bead etc..
Claims (9)
1. a single domain heavy chain antibody for the specific recognition immunoglobulin Fc section in immune library source, has SEQ ID NO.:1
Shown aminoacid sequence.
2. a nucleic acid molecules, is characterized in that encoding aminoacid sequence described in claim 1.
Nucleic acid molecules the most according to claim 2, it is characterised in that sequence such as SEQ ID NO.:2.
4. the carrier of the nucleotide sequence comprised described in claim 2.
5. the host cell of the carrier comprised described in claim 4.
6. the single domain heavy chain antibody of the specific recognition immunoglobulin Fc section described in claim 1 immune detection, be enriched with pure
Change the application in specific recognition immunoglobulin.
7. the single domain heavy chain antibody of the specific recognition immunoglobulin Fc section described in claim 1 is exempted from preparing immunoglobulin
Application in epidemic disease detection, enrichment and purified reagent or material.
8. the single domain heavy chain antibody of the specific recognition immunoglobulin Fc section described in claim 1 is by random or rite-directed mutagenesis
Technology carries out the antibody being combined of house of correction acquisition with specific for immunoglobulin.
9. for the affine adsorbing material of immunity of immunoglobulin, including carrier, the aglucon being mounted on carrier, its feature
Be this material single domain heavy chain antibody using specific recognition immunoglobulin Fc section as aglucon, described specific recognition immunity
The single domain heavy chain antibody of globulin Fc section has the aminoacid sequence shown in SEQ ID NO.:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109336979A (en) * | 2018-11-16 | 2019-02-15 | 宁夏医科大学 | Nano antibody, coded sequence and its screening technique of a kind of clostridium difficile glutamte dehydrogenase and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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