CN106478816B - A kind of single-chain antibody of anti-avian influenza H9N2 virus - Google Patents
A kind of single-chain antibody of anti-avian influenza H9N2 virus Download PDFInfo
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- CN106478816B CN106478816B CN201611138384.9A CN201611138384A CN106478816B CN 106478816 B CN106478816 B CN 106478816B CN 201611138384 A CN201611138384 A CN 201611138384A CN 106478816 B CN106478816 B CN 106478816B
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Pulmonology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses single-chain antibody ScFv-AIV and its preparation method and application, and the gene, the carrier containing the gene and host cell that encode the single-chain antibody of a kind of anti-avian influenza H9N2 virus etc..The single-chain antibody of anti-avian influenza H9N2 virus is to be formed by connecting by antibody heavy chain variable region and light chain variable region by joint peptide, and can carry out high efficient expression by prokaryotic expression system.The molecular weight of single-chain antibody ScFv-AIV is about 28kD, can specific recognition bird flu H9N2 virus, the combination of blocking virus and natural sera, diagnosis, the exploitation for treating preparation and the epitope that can be further used for the virus study.
Description
Technical field
The invention belongs to bioengineering product technical fields, and in particular to a kind of single-chain antibody of anti-avian influenza H9N2 virus
And its preparation method and application.
Background technique
Bird flu (Avian Influenza, AI) is a kind of acute height of birds caused by influenza A (AIV)
Spend contagious disease.By its hemagglutinin (HA) and neuraminidase (NA) antigenic difference, AIV can be divided into 15 HA hypotypes
With 9 NA hypotypes.Wherein, Avian Influenza Virus H9N2 is Homme isolated for the first time in turkey equal to 1970,
It is widely distributed in birds, by inhibiting the immune system of host, the modes such as other pathogenic microorganisms being cooperateed with to endanger poultry cultivation,
Reduce production performance.Since nineteen ninety-eight, large area exists bird flu H9N2 virus in China, causes laying hen group's egg drop reduction
With other chicken group production performance declines, huge economic loss is brought to aviculture.Meanwhile bird flu H9N2 virus it is extensive,
Long-term existence to avian influenza virus it is quick variation, recombination etc. bring potential danger, such as in by the end of March, 2013 in Shanghai and Anhui
Two places take the lead in having found a kind of novel H7N9 subtype avian influenza virus, are exactly 6 internal genes with bird flu H9N2 hypotype
It is generated with other subtype avian influenza virus by gene rearrangement.Therefore, lasting epidemic disease is carried out to bird flu H9N2 virus
Hygienic monitoring on hands of childhood is of crucial importance to the sound development of poultry breeding industry and the publilc health of the maintenance mankind.
Antibody is the important tool of epidemic disease research, detection and treatment.Early stage people mainly use mouse, rabbit and goat etc.
Animal prepares polyclonal antibody and monoclonal antibody.With the parsing and various genetic engineering skills to immunoglobulin gene structure
Art is emerged in large numbers, and people start the means screening using genetic engineering, prepare antibody.Phage antibody library technique is one of weight
The preparation method for antibody wanted, principle are to connect antibody V domain genes with filobactivirus capsid protein gene, and express and biting
Phage surface carries out the affine absorption of more wheels to antibody library by purpose antigen, washes in a pan the specific antibody needed for sieve obtains.Bacteriophage
Antibody library avoids the complicated processes such as cell fusion, the subclone of conventional monoclonal antibody preparation, can develop lot of antibodies in a short time,
Important means as Antibody preparation of new generation.The single-chain antibody ScFv of phage antibody library technique exploitation is with complete antibody
The minimum antibody fragment of binding site is the 1/6 of complete antibody, has the advantages that a variety of: (1) maintaining the specificity of antibody, greatly
Reduce immunogenicity greatly；(2) without Fc sections, it can connect building bifunctional antibody with enzyme or cell factor and drug, be conducive to treatment
With diagnosis；(3) it is easy to the mass production of genetic manipulation and genetic engineering, it is such as thin in Escherichia coli, yeast, insect, mammal
The expression systems mass production such as born of the same parents.
In view of the significant damage of bird flu H9N2 virus, anti-avian influenza H9N2 virus is developed by phage antibody library technique
Single-chain antibody ScFv undoubtedly weighed in viral diagnosis reagent exploitation, newtype drug research and having in terms of epitope
The theory significance and application value wanted.
Summary of the invention
The object of the present invention is to provide a kind of single-chain antibodies of anti-avian influenza H9N2 virus, to make up the prior art not
Foot.By constructing phage display single-chain antibody library, the circulate operation of " enrichment-elution-enrichment ", therefrom screening obtain one it is anti-
The single-chain antibody ScFv-IV of bird flu H9N2 virus, and single-chain antibody ScFv-AIV is obtained through further transformation, and construct table
Up to the genetic engineering bacterium of single-chain antibody, so as to largely prepare the single-chain antibody using the method for bioengineering.
Present invention firstly provides a kind of single-chain antibodies of anti-avian influenza H9N2 virus, and the single-chain antibody includes:
1) single-chain antibody for the SEQ ID NO:1 that amino acid sequence is；
2) 1) sequence in replaced, lacked, add one or several amino acid, the single-chain antibody as derived from 1).
A kind of gene of above-mentioned albumen is encoded, one kind nucleotide sequence is SEQ ID NO:2；
Preferably, the amino acid sequence of single-chain antibody is SEQ ID NO:3；
A kind of gene of above-mentioned albumen is encoded, one kind nucleotide sequence is SEQ ID NO:4；
Another aspect of the present invention provides a kind of for expressing the recombination table of the single-chain antibody of above-mentioned bird flu H9N2 virus
Up to plasmid, inserted with above-mentioned encoding gene；
Another aspect of the invention provides a kind of recombinant host bacterium, carries above-mentioned recombinant expression plasmid；
The present invention also provides a kind of preparation method of anti-avian influenza H9N2 virus single-chain antibody, preparation step is as follows:
1) single-chain antibody gene of anti-avian influenza H9N2 virus is connected into expression vector, is built into recombinant expression；
2) recombinant expression of building is converted into host strain, constructs the recombination base that can express anti-avian influenza H9N2 virus
Because of engineering bacteria；Go out the single-chain antibody with the recombination engineering bacterium expression；
3) single-chain antibody of recombinant expression is carried out after purification, can be used to develop diagnosis, the treatment of bird flu H9N2 virus
Preparation.
The present invention is constructed using pET30a (+) Expressing vector can express the big of anti-avian influenza H9N2 virus single-chain antibody
Enterobacteria BL21 (DE3) host strain.It is analyzed through SDS-PAGE, has given expression to the restructuring destination protein of 28kD or so.Egg will be recombinated
The white diagnosis that can be used for bird flu H9N2 virus after purification prevents and treats.
Specific embodiment
Further describe the present invention With reference to embodiment, but it will be understood by those skilled in the art that
Without departing from technical solution of the present invention can details to technical solution of the present invention and form modify or
Replacement, these modifications and replacement are each fallen in the scope of the present invention.
The building of 1 anti-avian influenza H9N2 viral phage single-chain antibody library of embodiment
1, it is small that BALB/c is immunized after bird flu H9N2 virus and Freund's complete adjuvant the 1:1 emulsification obtained ultracentrifugation
Mouse, booster immunization 2 times to antibody titer reach 1:100000 or more.Mouse spleen is taken, liquid nitrogen grinding is added by 50-100mg spleen
1ml Trizol reagent is stored at room temperature 5min, and 0.2ml chloroform is added, and acutely vibrates 15s repeatedly, is stored at room temperature 2-3min.4 DEG C,
12000g/min is centrifuged 15min, draws water phase in the centrifuge tube of another clean 1.5ml, and it is reverse mixed that 0.5ml isopropanol is added
It is even, 10min is centrifuged in -20 DEG C of precipitatings 30-60min, 12000g/min.Retain precipitating, 70% ethanol washing 1 time, in air
It dries, DEPC handles water dissolution precipitating RNA.
2, removal and reverse transcription the synthesis cDNA of genomic DNA are (using FastQuant cDNA first of Tiangeng company
Chain synthetic agent box):
The removal of 2.1 genomic DNAs: genomic DNA is prepared by following systems and removes system, is thoroughly mixed.Brief centrifugation,
42 DEG C are placed in, 3min is incubated for, is subsequently placed in and places on ice.
5×gDNA Buffer 2μl
1 μ g of RNA template
RNase-Free ddH2O is supplied to 10 μ l
The synthesis of 2.2cDNA: mixed liquor is prepared by following systems, is mixed well.
The removal system of said gene group DNA and reverse transcription system are mixed, mixed well, 42 DEG C of incubation 15min.95℃
Being put in the cDNA on ice, obtained after incubation 3min can be used for follow-on test.
3, the mouse antibodies light and heavy chain of design and synthesis can be changed zone amplication primer:
Expand the primer of single-chain antibody heavy chain VH:
VH is upper:
5-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSAGGTSMARCTGCAGSAGTC-3；
Under VH:
5-GCCAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGT-3。
Expand the primer of single-chain antibody light chain VL:
VL is upper:
5-TCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATYGAGCTCACYCAGTCTCC-3
Under VL:
5-GAGTCATTCTGCGGCCGCCCGTTTBAKYTCCARCTTKGTSCC-3。
Remarks: B=T, C, G；K=T, G；M=A, C；R=A, G；S=G, C；W=A, T；Y=C, T.
Above-mentioned cDNA template is expanded respectively with above-mentioned primer, and glue recycling obtains its light and heavy chain variable region gene segment.
4, by above-mentioned amplification to VH and VL gene segment separated, recycled with 1% agarose gel electrophoresis respectively,
Then Overlap extension PCR (SOE-PCR) is carried out using VH and VL mixed in equal amounts as template to assemble ScFv segment, condition is 94 DEG C
1min denaturation, 55 DEG C of 1min annealing, 72 DEG C of 1min extend, totally 30 circulations.PCR product is single-chain antibody gene segment, VH
It is the linker sequence of 15 amino acid: GGGGSGGGGSGGGGS between VL gene.
5, the ScFv gene of glue recycling and pCANTAB5E carrier difference double digestion (Sfi I and Not I) are connected afterwards, it will
Connection product is transformed into 100 μ l TG1 competent cells, and 2 × YT culture medium of 37 DEG C of 900 μ l preheatings, 37 DEG C of vibrations are then added
Swing culture 1h.The 100 μ l of bacterium solution cultivated after taking step to convert does gradient doubling dilution with 2 × YT culture solution, and coating SOB-AG is flat
Plate, 30 DEG C of overnight incubations.The single colonie number on plate is counted, calculates that antibody library storage capacity is 1 × 108.It is trained after remaining conversion
Feeding bacterium solution, is added 2 × YT of 10ml (containing glucose: 2%, Amp:100 μ g/ml), and 37 DEG C of cultures to logarithmic growth phase are added 2
×1010Bacteriophage M13K07,37 DEG C of culture 1h, 4000g/min are centrifuged 10min, resuspension be deposited in 2 × YT of 200ml (containing Amp:
100 μ g/ml, kanamycins: 50 μ g/ml), 37 DEG C of shaken cultivations are stayed overnight.After 4000g/min is centrifuged 10min, 1/5 volume is added
The PEG/NaCl (NaCl of 20% PEG8000,2.5mol/L) of amount is in 4 DEG C of precipitating phage 30min, 9000g/min centrifugations
Precipitating phage is resuspended in the PBS of 2ml BSA containing 10g/L by 20min, and 12000g/min is centrifuged 5min, and supernatant is through 0.45 μm
Membrane filtration, i.e. acquisition phage antibody library.
The multifarious analysis of 2 single-chain antibody of embodiment
10 clones of random picking, extract plasmid after shaking bacterium amplification, and with Sfi I and Not I double digestion, Preliminary Identification is positive
Clone.It is with S1 (S1:5-CAACGTGAAAAAATTATTATT-3), S6 (S6:5-GTAAATGAATTTTCTGTATGAGG-3)
Primer carry out sequencing analysis, as a result 10 clone sequencing results show sequence with mouse immune globulin variable region sequences one
It causes, meets mouse weight chain variable region gene structure, arrangement mode VH-Linker-VL.Wherein the part VH is 357-367bp
Left and right, VL are 320-330bp or so, and the linker base sequence between heavy chain and light chain is all correct.Sequence alignment shows it
Homology is up to 80% or more.
The enrichment of 3 anti-avian influenza H9N2 virus single-chain antibody library of embodiment
1, bird flu H9N2 virus liquid buffer is coated with by 1: 5 (volume ratio) dilution carbonate to be coated with to immune pipe,
2ml/ pipe, 4 DEG C overnight.
2, coating finishes, and is washed pipe 3 times, is patted dry with PBS；It is closed with closing (2% skimmed milk PBS, MPBS) and pipe is immunized, 37 DEG C
Closing 2 hours.
3, incline deblocking liquid, is washed pipe 3 times, is patted dry with PBS；
4, by above-mentioned acquired primary phage antibody library supernatant, according to MPBS: supernatant=2:3 volume ratio will
MPBS and bacteriophage supernatant are mixed, and carry out interferenceization processing 20min at room temperature.
5, liquid will be handled in 4 well, the immune pipe closed is added, 2ml/ pipe after jog incubates 30min, then stands incubation
1.5 hour.
6, bacteriophage supernatant in immune manage is abandoned, is washed 3 times, then washed 3 times with PBS, is patted dry with PBST.
7, glycine-HCI (pH2.2) eluent is added, 2ml/ is managed, and after 37 DEG C of effect 6min, is rapidly added Tris buffering
Liquid (2mol/L pH 7.4), 200 μ l/ pipes neutralize the phage solution eluted, add the fresh TG1 bacterium solution of 2ml
(OD600Value is about 0.5) 37 DEG C of effect 1h.The first round naughty sieve is completed, Primary antibodies library is obtained.
8, after taking part bacterium solution to do 10 times of gradient dilutions, the bacteriophage that coating SOBAG plate calculates output washes in a pan sieve amount, remaining bacterium
Final concentration of 100 μ g/ml Amp and 2% glucose is added in liquid, while being added about 4 × 1010M13 K07 bacteriophage stands and incubates
After 30min, then 250rpm shaken cultivation 30min；1000g is centrifuged 10min, removes supernatant, is gently hanged with 2 × YT-AK of 100ml thin
Born of the same parents, 37 DEG C, 250rpm, overnight incubation starts next round and washes in a pan sieve.
9,3 wheels are eventually passed through and wash in a pan sieve, the bacterium solution of acquisition is taken, does gradient doubling dilution with 2 × YT culture medium, take dilution
100 μ l are coated with SOBAG plate, 30 DEG C of overnight incubations；
The plate for there are about 100-200 or so bacterium colonies is selected, colony count is calculated, multiplied by extension rate to get corresponding library
Hold.Sieve is washed in a pan by 3 wheel specific enrichments, obtaining titre is about 1.0 × 106The bacteriophage recombinant antibodies library of pfu/ml.It is remaining
Sterile glycerol is added to final concentration 20% in bacterium solution, and after mixing, -70 DEG C are frozen, and are labeled as three-level antibody library.
The screening of detection and the strong positive strain of 5 single-chain antibody ScFv-IV of embodiment
The centrifuge tube of 72 1.5ml is taken to set as culture plate on culturing rack, 2 × YT-AG culture medium, 400 μ l is added in every hole；
The single bacterium colony grown on the SOBAG plate of picking among the above, is seeded in each hole above, this plate is labeled as Master
Plate；Master Plate is placed in shaking table, 30 DEG C, 250rpm/min, shaken cultivation is overnight；Next day separately takes 72 holes to cultivate
Plate takes 400 μ l to contain 2.5 × 10102 × YT-AG of pfu/ml M13K07 is designated as P1Plate to every hole, this plate；From overnight training
Every hole takes 40 μ l culture solutions to P1Plate on feeding Master Plate；P1Plate is placed in shaking table, 37 DEG C, 150rpm/
Min, shaken cultivation 2 hours, 1500g was centrifuged 20min, carefully removed supernatant；400 2 × YT-AK of μ l training is added to the every hole P1Plate
Nutrient solution, 37 DEG C, 250rpm/min, shaken cultivation is overnight；1500g is centrifuged 20min, carefully takes supernatant stand-by.
4 DEG C of coated elisa plates of bird flu H9N2 virus liquid that ultracentrifugation obtains are stayed overnight, 2%MPBS (contains 2% skimmed milk
PBS) closing, after washing, using previously obtained phagocytosis body fluid as primary antibody, the anti-M13 monoclonal antibody of 37 DEG C of incubations 2h, HRP label is two
Anti-, TMB developing solution is added in 37 DEG C of reaction 1h after washing, 2M sulfuric acid color development stopping, microplate reader is added in 37 DEG C of reaction 30min
OD value is surveyed under 450nm wavelength, if M13K07 coating control wells (positive control) and blank are coated with control wells, finds out positive colony
(value >=0.3 OD and OD value/blank well >=3 OD can be determined as positive colony).ELISA measures each hole supernatant and bird flu H9N2
The combination situation of virus primarily determines that this 12 plants of recombinant clones are the positive as a result, it has been found that 12 strain clones and antigen have positive reaction,
Wherein one plant of phage antibody is strong positive, is named as IV plants.
The recombinant plasmid for extracting IV strain clone, does PCR identification, target fragment plastic recovery kit by primer of S1, S6
The sequencing of Hou Song company is recycled, sequencing result shows that purpose ScFv sequence arrangement is VH-Linker-VL, sequence alignment discovery
Meet mouse weight chain variable region gene structure.Blast tetraploid rice is carried out in Genebank, finds maximum homology
Only it is 93%, shows the variation that nucleosides has occurred, be a new mouse single-chain antibody ScFv.In order to improve IV plants of antibody effect
IV plants of 15 lysines (K) are become arginine (R), 100 third according to the protein tertiary structure information of biosoftware by valence
Propylhomoserin (A) becomes arginine (R), 192 serines (S) become aspartic acid (D), 228 serines (S) become tryptophan
(W), 229 serines (S) become glycine (G), 230 tyrosine (Y) become threonine (T), and are named as single-chain antibody
ScFv-AIV, the amino acid sequence of coding albumen are SEQ ID NO:3, and nucleotides sequence is classified as SEQ ID NO:4, and carries out complete
Gene chemical synthesis, both ends add BamHI and Hind III restriction enzyme site respectively.
The prokaryotic expression of embodiment 6 improved single-chain antibody ScFv-AIV and original single-chain antibody ScFv-IV
1, the amplification of single-chain antibody ScFv-IV genetic fragment and the identification of transformation front and back recombinant plasmid
Pair of primers is designed according to the gene order of ScFv-IV, both ends add BamHI and Hind III restriction endonuclease respectively:
IV-P1:5-CCAGGATCCATGGCCCAGGTG-3
IV-P2:5-CCGAAGCTTTTACTTCAGCTCCAG-3
Following reagent is added in a 0.2mlPCR pipe to be expanded:
Purified pcr product connects after distinguishing digestion with protein expression vector pET-30a, and is transferred to TOP10 strain inoculated and arrives
In LB culture medium containing kanamycins, 37 DEG C of concussions overnight, extract recombinant plasmid digestion identification.
By full genome synthesis improved ScFv-AIV gene BamHI and Hind III endonuclease digestion after with albumen
Expression vector pET-30a is connected after distinguishing digestion, and is transferred to TOP10 strain inoculated into the LB culture medium containing kanamycins, and 37
DEG C concussion overnight, extract recombinant plasmid digestion identification.
Company is sent to be sequenced pET30a-ScFv-IV, pET30a-ScFv-AIV for having identified, as a result correctly.
2, the prokaryotic expression of single-chain antibody ScFv-IV, ScFv-AIV and purifying
Recombinant plasmid pET30a-ScFv-IV, pET30a-ScFv-AIV for having identified are transferred to BL21 (DE3) large intestine respectively
In bacillus, at 37 DEG C of culture to bacterium solution absorbance about 0.6, add IPTG inducing expression destination protein, 25 DEG C of induction 6h, not add
IPTG bacterium solution compares, and carries out SDS-PAGE and detects protein expression situation.Destination protein is mainly expressed with soluble form, using nickel
Column purification, the specific steps are as follows:
(1) for bacterium solution product through 4 DEG C, 8000g is centrifuged 15min after inducing expression, removes supernatant.
(2) it is resuspended with the Wash Buffer of 0.1 times of culture volume (20mM Tris-HCl, pH7.5,10mM EDTA)
Precipitating, mixes well.
(3) it operates on ice, ultrasound cracking bacterium.
(4) 10000g is centrifuged 10min, collects ultrasonication supernatant.
(5) ni-sepharose purification, imidazoles elution.The recombinant protein eluent of collection is put into bag filter, PBS liquid is outer as dialysis
Liquid, up to single-chain antibody ScFv-IV, ScFv-AIV after dialysis desalting.
(6) Coomassie Brilliant Blue measures protein concentration respectively, is adjusted to 1mg/ml, -20 DEG C of preservations with PBS liquid.
The activity of the detection of 7 indirect elisa method of embodiment single-chain antibody ScFv-IV, ScFv-AIV
According to best coating condition, 4 DEG C of coated elisa plates of bird flu H9N2 virus liquid are stayed overnight；Coating buffer is abandoned, PBST is used
Washing 2 times, pats dry ELISA Plate；2% skimmed milk PBS (MPBS) is added to full hole, 37 DEG C of closing 1.5h；Confining liquid is abandoned, PBST is used
Washing 3 times, pats dry ELISA Plate；Recombinant single chain antibody ScFv-IV, the ScFv-AIV for adding different dilutions respectively, set up simultaneously
PBS liquid is as negative control, 37 DEG C of reaction 1h；Recombinant antibodies liquid is abandoned, is washed 3 times with PBST, pats dry ELISA Plate；HRP is marked
The antibody of anti-His is 1:4000 dilution, 100 holes μ l/, 37 DEG C of incubation reaction 1h with PBS；Enzyme labelled antibody liquid is abandoned, washs 3 with PBST
It is secondary, pat dry ELISA Plate；Colour developing: 100 hole μ l/ TMB developing solutions, 37 DEG C of incubations colour developing 15min are added；It terminates: with 100 holes μ l/ end
Only liquid, color development stopping, the readings at 450nm.As a result it see the table below.Result of indirect ELISA shows single-chain antibody ScFv-IV and changes
Single-chain antibody ScFv-AIV after making has stronger binding ability, improved single-chain antibody with bird flu H9N2 virus
The binding ability of ScFv-AIV and virus is stronger (table 1).
Table 1: the activity of indirect ELISA detection transformation front and back recombinant single chain antibody
The activity of the blocking of embodiment 8 ELISA method detection recombinant single chain antibody ScFv-AIV
According to best coating condition, the bird flu H9N2 virus of purifying is coated with 96 orifice plates, 4 DEG C of coatings are overnight.2%MPBS
Closing.4 gradients are arranged by 8 times, 4 times, 2 times, 1 times of peridium concentration in bird flu H9N2 virus liquid, each gradient sets up blocking
Hole and non-blacked hole respectively repeat for three.Bird flu H9N2 virus liquid and each 50 μ l room temperature outside hole of single-chain antibody ScFv-AIV liquid
30min is reacted, moves to blocking aperture, PBS liquid is same with recombinant single chain antibody ScFv-AIV liquid to react and moves back to non-blacked hole, and 37 DEG C
React 1h；Reaction solution is abandoned, is washed 3 times with PBST, pats dry ELISA Plate；The antibody of anti-His is marked with PBS to be 1:4000 HRP dilute
It releases, 100 holes μ l/, 37 DEG C of incubation reaction 1h；Enzyme labelled antibody liquid is abandoned, is washed 3 times with PBST, pats dry ELISA Plate；Colour developing: 100 are added
The hole μ l/ TMB developing solution, 37 DEG C of incubations colour developing 15min；Terminate: with 100 hole μ l/ terminate liquids, color development stopping is read at 450nm
Value.The virus concentration for being used to block as the result is shown is higher, remaining viral must to react single-stranded with being coated on elisa plate
Amount of antibody is fewer, the OD of ELISA450It is worth smaller, illustrates recombinant single chain antibody ScFv-AIV and bird flu H9N2 virus association reaction
Specificity preferably, have blocking effect.
Table 2: the activity of ELISA detection recombinant single chain antibody ScFv-AIV is blocked
The activity of 9 double sandwich-ELISA method of embodiment detection single-chain antibody ScFv-AIV
Recombinant single chain antibody ScFv-AIV is coated with 96 orifice plates, 4 DEG C of coatings are overnight.2%MPBS closing, is added pure after washing
The bird flu H9N2 virus of change, respectively does three repetitions, the serum of rabbit-anti bird flu H9N2 virus is added in 37 DEG C of reaction 1h after washing
The goat anti-rabbit antibody of HRP label is added in (1:4000 dilution), 37 DEG C of reaction 1h after washing, 37 DEG C of reaction 1h ibid develop the color and survey
Determine OD value.Double sandwich-ELISA result following table illustrates that screened recombinant single chain antibody ScFv-AIV is able to suppress anti-avian influenza
The mostly anti-association reaction with virus of the rabbit of H9N2 virus.
Table 3: the activity of double sandwich-ELISA method detection recombinant single chain antibody ScFv-AIV
The specific test of 10 recombinant single chain antibody ScFv-AIV of embodiment
Respectively by the bird flu H9N2 virus of purifying, newcastle disease virus (NDV), avian infectious bronchus viral (IBV)
96 orifice plates are coated with, 4 DEG C of coatings are overnight.2%MPBS closing, is added recombinant single chain antibody ScFv-AIV as primary antibody after washing, and 37
DEG C reaction 1h, washing.The antibody of anti-His is marked to be 1:4000 dilution, 100 holes μ l/, 37 DEG C of incubation reaction 1h with PBS HRP；
Enzyme labelled antibody liquid is abandoned, is washed 3 times with PBST, pats dry ELISA Plate；Colour developing: 100 hole μ l/ TMB developing solutions, 37 DEG C of incubation colour developings are added
15min；It terminates: with 100 hole μ l/ terminate liquids, color development stopping, the readings at 450nm.As a result it see the table below, illustrate that recombinant single chain is anti-
Body ScFv-AIV can only have with bird flu H9N2 virus to react, and with other viral no signals, there is virus identification specificity.
Table 4: the specificity of recombinant single chain antibody ScFv-AIV
SEQUENCE LISTING
<110>to be determined
<120>a kind of single-chain antibody of anti-avian influenza H9N2 virus
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 242
<212> PRT
<213> 1
<400> 1
Met Ala Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro
1 5 10 15
Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Ser Tyr Trp Ile His Trp Ile Lys Gln Arg Pro Val His Gly Leu Glu
35 40 45
Trp Ile Gly Tyr Phe Asn Pro Ser Thr Gly Tyr Thr Ala Tyr Asn Gln
50 55 60
Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr
65 70 75 80
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
85 90 95
Tyr Cys Thr Ala Arg Ser Gly Gly Asp Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala
130 135 140
Ser Leu Ser Val Ser Pro Gly Glu Thr Val Thr Ile Thr Cys Arg Ala
145 150 155 160
Ser Glu Asn Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Lys Ser Pro Gln Leu Leu Val Tyr Ala Ala Thr Ser Asn Leu Ala Ser
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser
195 200 205
Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys
210 215 220
Gln His Phe Ser Ser Tyr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Leu Lys
<210> 2
<211> 726
<212> DNA
<213> 2
<400> 2
atggcccagg tgaagttgca gcagagcgga gccgagctgg ttaaacctgg cgcttccgtt 60
aagttgtcct gcaaggcctc cggctatacc ttcaccagct actggattca ctggatcaag 120
cagaggcctg tgcacggcct ggagtggatc ggctacttca accctagcac cggctacacc 180
gcatacaacc agaagttcaa ggacaaggcc accctgaccg ccgacaagag cagcagcacc 240
gcttacatgc agctgagctc cctgactagc gaggacagcg ccgtgtacta ctgcaccgcc 300
aggagcggtg gcgactattt cgactactgg ggccagggca ccacagtgac agtgagcagc 360
ggaggaggcg gaagtggagg tggtggatcc ggcggagggg gtagcgatat agagctcacc 420
cagagccctg ccagcctgag tgtctccccc ggagaaactg tgaccatcac ttgtagggcc 480
agcgagaaca tctacagcaa ccttgcctgg tatcaacaga agcctgggaa gtcaccccaa 540
ttgctggtgt acgccgccac ctcaaatctg gcaagcggcg tgccttccag gttcagcggc 600
agcgggagcg ggacccaata ttctctgaag atcaatagcc tgcagagtga ggacttcggc 660
agctattact gtcagcactt cagcagctac cctaccttcg gtggggggac taagctggag 720
ctgaag 726
<210> 3
<211> 242
<212> PRT
<213> 3
<400> 3
Met Ala Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
1 5 10 15
Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr
20 25 30
Ser Tyr Trp Ile His Trp Ile Lys Gln Arg Pro Val His Gly Leu Glu
35 40 45
Trp Ile Gly Tyr Phe Asn Pro Ser Thr Gly Tyr Thr Ala Tyr Asn Gln
50 55 60
Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr
65 70 75 80
Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Arg Ser Gly Gly Asp Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala
130 135 140
Ser Leu Ser Val Ser Pro Gly Glu Thr Val Thr Ile Thr Cys Arg Ala
145 150 155 160
Ser Glu Asn Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Lys Ser Pro Gln Leu Leu Val Tyr Ala Ala Thr Ser Asn Leu Ala Asp
180 185 190
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser
195 200 205
Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys
210 215 220
Gln His Phe Trp Gly Thr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu
225 230 235 240
Leu Lys
<210> 4
<211> 726
<212> DNA
<213> 4
<400> 4
atggcgcagg tcaaactcca gcagtctggt gcggaactgg ttcgtccggg tgcgtctgtt 60
aaactgtctt gcaaagcgtc tgactacacc ttcacctctt actggattca ctggatcaaa 120
cagcgtccag ttcacggtct ggaatggatc ggttacttca acccgtctac cggttacact 180
gcgtacaacc agaaattcaa agacaaagcg accctgaccg cggacaaatc ttcttccacc 240
gcgtacatgc agctgtcttc tctgacttct gaagactctg cggtttacta ctgcacccgt 300
cgttctggtg gtgattactt cgactactgg ggtcaaggta ccactgttac cgtttcttcc 360
ggcggtggtg gctccggtgg cggcggttct ggcggtggtg gtagcgatat tgaactgacc 420
cagtctccgg cgagcctgtc tgtttctccg ggcgagacgg tcactatcac ctgccgtgca 480
tctgagaaca tctactctaa cctggcctgg tatcagcaga aaccgggtaa atctccgcaa 540
ctgctcgttt acgcggcgac ctctaatctc gcggacggtg ttccatctcg tttcagcggt 600
agcggcagcg gcacccagta ctctctgaaa atcaacagcc tgcaatccga agacttcggt 660
tcttattact gccagcattt ttggggtacg ccgaccttcg gtggcggtac gaagctggaa 720
ctgaaa 726
Claims (7)
1. a kind of single-chain antibody of anti-avian influenza H9N2 virus, which is characterized in that the amino acid sequence of the single-chain antibody is SEQ
ID NO:3。
2. a kind of nucleotide fragments, which is characterized in that the nucleotide fragments encode single-chain antibody described in claim 1.
3. nucleotide fragments as claimed in claim 2, which is characterized in that the sequence of the nucleotide fragments is SEQ ID
NO:4。
4. a kind of recombinant expression plasmid, which is characterized in that the recombinant expression plasmid is inserted with nucleosides as claimed in claim 2
Acid fragment.
5. a kind of recombinant host bacterium, which is characterized in that the recombinant host bacterium carries recombinant expression as claimed in claim 4
Plasmid.
6. the preparation method of single-chain antibody described in claim 1, which is characterized in that the preparation method includes following
Step:
1) nucleotide fragments as claimed in claim 2 are connected into prokaryotic expression carrier, are built into recombinant expression plasmid；
2) recombinant expression plasmid of building is converted into host strain, building can express the recombination engineering bacteria of single-chain antibody, with this
Recombination engineering bacterium expression goes out single-chain antibody.
7. single-chain antibody described in claim 1 is in preparation bird flu H9N2 viral diagnosis, treatment preparation and epitope research
Application in product.
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| WO2003078600A2 (en) * | 2002-03-13 | 2003-09-25 | Kirin Beer Kabushiki Kaisha | Human monoclonal antibodies to influenza m2 protein and methods of making and using same |
| CN101993492A (en) * | 2010-11-25 | 2011-03-30 | 南京医科大学 | ScFv antibody for resisting H5N1 type highly-pathogenic avian influenza and application thereof |
| CN102399754A (en) * | 2011-05-27 | 2012-04-04 | 山东省农业科学院畜牧兽医研究所 | H9N2 avian influenza virus vaccine strain and application of H9N2 avian influenza virus vaccine strain in immune protection |
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| 抗H9N2亚型禽流感病毒单克隆抗体的制备及初步鉴定;王凤强 等;《现代畜牧兽医》;20050830(第08（2005）期);第32页摘要，第33页2.3小节，第33页第3小节 |
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Address after: No. 85 Keyun Road, High tech Zone, Qingdao, Shandong Province, 266000 Patentee after: Qingdao Blue Animal Health Group Co.,Ltd. Patentee after: QINGDAO ANIMAL PROTECTION NATIONAL ENGINEERING TECHNOLOGY RESEARCH CENTER CO.,LTD. Patentee after: SHANDONG DELINORE BIO-ENGINEERING Co.,Ltd. Patentee after: QINGDAO VLAND BIOTECH Inc. Address before: No. 16 Tianhai Road, Hongdao Street, Chengyang District, Qingdao City, Shandong Province, 266114 Patentee before: QINGDAO VLAND BIOTECH GROUP CO.,LTD. Patentee before: QINGDAO ANIMAL PROTECTION NATIONAL ENGINEERING TECHNOLOGY RESEARCH CENTER CO.,LTD. Patentee before: SHANDONG DELINORE BIO-ENGINEERING Co.,Ltd. Patentee before: QINGDAO VLAND BIOTECH Inc. |