CN102276719A - Avian influenza virus single domain antibody, pentameric antibody and preparation and application thereof - Google Patents
Avian influenza virus single domain antibody, pentameric antibody and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a single domain antibody having specific combination capability with avian influenza virus and a preparation method thereof. The invention also relates to a pentameric antibody having specific combination capability with avian influenza virus, and a preparation method and application thereof. The invention also relates to a magnetic pentameric antibody having specific combination capability with avian influenza virus, and an avian influenza virus detection method utilizing the same.
Description
Technical field
The invention belongs to molecular biology and antibody engineering technical field.Specifically, the present invention is that a class comes from the novel antibody of alpaca and is applied to the method for biosensor that avian influenza virus is diagnosed through after transforming.
Background technology
Influenza has become one of principal disease that threatens human health gradually.H1N1virus in 2009 in the world outburst and the fear that high pathogenic avian influenza H5N1 brings to people since 2003 impel people to improve constantly diagnosis and the treatment ability of reply influenza virus.Diagnose in early days at virus infection to help improving curative ratio, reduce virus disseminating.For the detection of influenza virus, except traditional detection method such as virus separation, RT-PCR etc., specific antibody also is widely used in the detection methods such as various ELISA and immunofluorescence.In addition, the trial of a lot of raising detection specificity and sensitivity is arranged also, for example develop the stronger antibody of specificity, in detection system, introduce microsphere etc.However, exploitation still is faced with great challenge based on the highly sensitive influenza virus detection method of antibody, also has very big space to go to explore.
Summary of the invention
Characteristic of the present invention is that it is to utilize being applied in based on the detection of carrying out avian influenza virus in the biosensor of magnetic nanoparticle for the high five affine poly-antibody of avian influenza virus and with it of engineered method acquisition.Rationale of the present invention is based on the special property of camel antibody: forms by heavy chain antibody by nearly half IgG in the camel serum, and can functional conjugated antigen; Same disappearance light chain antibody---neoantigen acceptor (NARs) is found in the shark body.The antibody that only contains variable region of heavy chain that makes up of clone is called single domain antibody thus, i.e. VHH antibody, and molecular weight only be 15kD, can be in conjunction with some littler epitope, have higher in conjunction with activity.This small molecular antibody screens from set up good single domain antibody storehouse in advance by display technique of bacteriophage and obtains, and is subcloned in the expression vector, by the transformed into escherichia coli great expression.Single domain antibody only just can be brought into play the function of conjugated antigen by variable region of heavy chain, and demonstrates analogous avidity.In addition, because natural existence does not contain the heavy chain antibody of light chain in the camel antibody, several key amino acids of its variable region are by the hydrophobic hydrophilic amino acid that sports in traditional antibody, and the single domain antibody that therefore comes from the natural heavy chain antibody of camel has following three distinguishing features: 1, have and avidity that traditional antibody is comparable; 2, solubility is very strong during prokaryotic expression, and expression amount is higher; 3, molecular weight is littler, is easier to carry out the genetically engineered operation.This has just remedied the deadly defect of traditional small molecular antibody, for wide space is expanded out in the development of genetic engineering antibody.Therefore, compare with traditional mouse resource monoclonal antibody, it is shorter to utilize display technique of bacteriophage to prepare the single domain antibody required time cycle, and workload is littler, and owing to can use escherichia coli expression, output is improve greatly also.
Based on the above-mentioned theory basis, at first, we utilize influenza virus immunity alpaca, have set up influenza virus immunity single domain antibody phage library, and have utilized the inactivating influenza virus of purifying to filter out the affine single domain antibody VHH3B of a plant height from antibody library.In order to improve the avidity of this antibody to avian influenza virus, make it more to be applicable to high-sensitivity detection, we carry out amalgamation and expression with single domain antibody VHH3B and shiga toxin B subunit again, and five dimerizations by shiga toxin B subunit self make and expression product five dimerizations form five poly-antibody.Utilize the virus in the magnetic nanoparticle link coupled five poly-antibody capture samples at last, serve as to detect antibody with colloid gold particle link coupled mouse source avian influenza virus specific antibody 3C8 again, by such combination, set up high-sensitive method for biosensor, carry out the high-sensitivity detection of virus.
The innovation part of the present invention aspect technological line is: 1, utilize the adult alpaca of the common immunity of deactivation H 5 N 1 avian influenza and H5N2 virus, through behind three booster immunizations, separate the B cell and set up immune alpaca single domain antibody storehouse, and filter out the special high-affinity single domain antibody of bird flu; 2, utilize the character of shiga toxin B subunit self five dimerizations,, form five poly-antibody of avian influenza virus specific bird flu single domain antibody amalgamation and expression with it.Five antibody after poly-show higher avidity, more are applicable to the needs of the high-sensitivity detection of avian influenza virus.3, utilize magnetic nanoparticle link coupled five poly-antibody to carry out the detection of avian influenza virus as capture antibody and colloid gold particle link coupled mouse source anti-avian influenza monoclonal antibody.4, utilize golden catalytic hydroquinone oxidation to generate the reflection of the reaction of benzoquinones as detected result.
Particular content is as follows:
1. obtain that avian influenza virus is had the method for the single domain antibody of specific combination ability, it is characterized in that described method comprises the following steps:
1) set up avian influenza virus immunity single domain antibody phage library and
2) utilize display technique of bacteriophage from described single domain antibody phage library, to screen the single domain antibody clone high to avian influenza virus avidity,
Wherein, preferably, described avian influenza virus is Re1 strain of influenza vaccines H5N1 hypotype and H5N2 hypotype N28 strain.
2. single domain antibody that avian influenza virus is had the specific combination ability is characterized in that described single domain antibody is to obtain by above 1 method.
3. single domain antibody that avian influenza virus is had the specific combination ability, the aminoacid sequence that it is characterized in that described single domain antibody is shown in SEQ ID No:1.
4. five poly-antibody that avian influenza virus had the specific combination ability, it is characterized in that described five poly-antibody are the five poly-antibody that formed by the pentamer core by above 2 or 3 described single domain antibodies, wherein said pentamer core can be to contain the segmental pentamer carrier of shiga toxin B subunit gene, preferably pVT2.
5. above 4 described five poly-antibody is characterized in that described five poly-antibody have the monomer of 5 aminoacid sequences shown in SEQ ID No:2.
6. make up the method for above 4 or 5 described five poly-antibody, it is characterized in that described method comprises the following steps:
1) with the gene fragment subclone of above 2 or 3 described single domain antibodies to contain in the segmental pentamer carrier of shiga toxin B subunit gene with obtain recombinant vectors and
2) express described recombinant vectors,
The wherein said preferably pVT2 of the segmental pentamer carrier of shiga toxin B subunit gene that contains.
7. above 2 or 3 described single domain antibodies or above 4 or 5 described five poly-antibody are used to catch the avian influenza virus particulate and use.
8. the poly-antibody of magnetic five that avian influenza virus had the specific combination ability is characterized in that described magnetic five poly-antibody are by making above 4 or 5 five poly-antibody and magnetic nanoparticle coupling formation.
9. detect the test kit of avian influenza virus, it comprises
1) above 8 described magnetic five poly-antibody;
2) with gold nano grain link coupled anti-avian influenza antibody; With
3) Resorcinol.
10. detect the method for avian influenza virus, it is characterized in that said method comprising the steps of:
1) in sample and blank, add above 8 magnetic five poly-antibody and utilize magnetic to separate respectively, the separated object that has magnetic with acquisition,
2) add with gold nano grain link coupled anti-avian influenza antibody in the separated object that in step 1), obtains and utilize magnetic to separate, to obtain separated object with magnetic
3) to step 2) in add in the separated object that obtains Resorcinol and 390nm place its absorbance value of measurement and
4) absorbance value by 390nm place that described sample is obtained determines to have avian influenza virus in the described sample greater than the absorbance value to the 390nm place of described blank acquisition.
11. above 8 described magnetic five poly-antibody and above 9 described test kits are used to detect the application of avian influenza virus.
The accompanying drawing summary
Fig. 1. calculate through four-wheel screening enrichment number in the avian influenza virus antibody storehouse;
Fig. 2 .SDS-PAGE analyzes the purity of anti-avian influenza virus single domain antibody, wherein the left side swimming lane: protein molecular weight standard; Right side swimming lane: the anti-avian influenza virus single domain antibody of purifying;
Fig. 3. anti-avian influenza virus single domain antibody binding specificity;
Fig. 4. the binding ability of surface plasma resonance technology analysis list domain antibodies;
Fig. 5 .SDS-PAGE analyzes the purity of anti-avian influenza virus pentamer antibody, wherein the left side swimming lane: protein molecular weight standard; Right side swimming lane: the anti-avian influenza virus pentamer antibody of purifying;
Fig. 6. anti-avian influenza virus pentamer antibody binding specificity;
Fig. 7. surface plasma resonance technology is analyzed the binding ability of pentamer antibody;
Fig. 8. the biosensor based on magnetic nanoparticle detects avian influenza virus;
Fig. 9. double antibodies sandwich ELISA detects avian influenza virus;
Figure 10. phagemid carrier pCANTAB5E collection of illustrative plates;
Figure 11. expression vector pET28a collection of illustrative plates; With
Figure 12. pentamer carrier pVT2 sequence map.
The sequence explanation
The aminoacid sequence of SEQ ID No:1 single domain antibody VHH3B; With
The monomeric aminoacid sequence of SEQ ID No:2 five poly-antibody pVHH3B.
Embodiment
In order more fully to understand and apply the invention, provide the following example.
Embodiment one: the preparation of single domain antibody and evaluation
Use display technique of bacteriophage manufacture order domain antibodies VHH3B (Arbabi Ghahroudi and Muyldermans, Selection and identification of single domain antibody fragments from camel heavy-chain antibodies, FEBS Letters, 1997).Be summarized as follows: utilize commercial avian influenza vaccine H5N1 hypotype Re1 strain (being purchased master station) and H5N2 hypotype N28 strain (being purchased master station) two adult alpaca (Alpaca from Beijing animal doctor from Beijing animal doctor, Camelidae, yamma belongs to, available from Chinese alpaca cultivation base, Vicugna pacos) (code name is respectively B007 and B008) carries out the subcutaneous multiple spot immunization of neck both sides lymphoglandula, every immunity in 10 days once, be divided into three times.Got the peripheral blood of two alpacas after the last immunization in 10 days respectively, with erythrocyte cracked liquid (being purchased) cracking hemocyte, centrifugal collection white corpuscle from TIANGEN Biotech (Beijing) Co., Ltd..Add 1mlTrizol (being purchased from Invitrogen), room temperature leaves standstill 5min.Add 200 μ l chloroforms and leave standstill 2-3min.12000rpm, 4 ℃ of centrifugal 15min.Shift the upper strata water in another new centrifuge tube, add behind the abundant mixing of 500 μ l Virahols-20 ℃ and leave standstill 10min, behind 4 ℃ of centrifugal 10min of 12000rpm, abandon supernatant.Precipitated rna 1ml 75% ethanol rinsing 2-3 time, 4 ℃ of centrifugal 5min of 7500rpm abandon supernatant.RNA precipitation drying at room temperature 5-10min, the ultrapure water 50 μ l that handle with diethylpyrocarbonate (DEPC) dissolve RNA fully in 55 ℃ of incubation 10min.
With RNA for the synthetic cDNA of template reverse transcription (the Invitrogen test kit, reaction conditions: add RNA 2 μ g, 20 μ M oligo dT, 3 μ l, 10 μ M dNTP, 1 μ l adds water to 13 μ l in the PCR pipe.Hatch 5min for 65 ℃, put 2-3min on ice rapidly.Add 5 * buffer, 4 μ l, 100mM DTT1 μ l, ThermoScript II SSIII RT 1 μ l, RNase inhibitor HRPI 1 μ l mixes.50℃?1h，75℃?15min)。With the cDNA product is template, successively uses two couples of primer VHBACKA6 in the table 1, CH2FORTA4 and F-PRCCAMEL, R-PCRCAMEL carry out nest-type PRC (reaction conditions: 94 ℃, the pre-sex change of 5min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 40s, 30 circulations; 72 ℃ are extended 7min), amplify special VHH segment.
Table 1 utilizes the used two groups of primers of nested PCR method amplification VHH fragment
Then VHH fragment and the phagemid carrier pCANTAB5E that amplifies (is purchased the Pharmacia from Amersham, cut with restriction enzyme SfiI enzyme in 50 ℃ of water-baths respectively as shown in figure 10), after reclaiming the VHH fragment and carrier segments after cutting, spend the night with 16 ℃ of connections of T4DNA ligase enzyme (being purchased) from TaKaRa.Afterwards, electricity transforms the connection product and goes into intestinal bacteria (E.Coli), concrete grammar is as follows: will connect product and remove ion with PCR purification kit (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.), join in the previously prepd competent cell e. coli tg1 (being purchased) from Stratagene, mixing is placed 30min on ice.Add to precooling electric revolving cup (1mm, BioRad) in, at voltage 2.5kV, resistance 2000 Ω carry out electroporation and transform under the condition of electric capacity 25 μ F.Join rapidly afterwards in 2 * YT-AG substratum (0.5% sodium-chlor, penbritin to final concentration are 100 μ g/ml for 1.6% Tryptones, 1% yeast extract, and kantlex is to final concentration 50 μ g/ml, 2% glucose), 180rpm, cultivates 1h by 37 ℃.5000g, 10 minutes centrifugal, and the resuspended precipitation of an amount of 2 * YT is applied to SOB-AG solid medium (2% Tryptones, 0.5% yeast extract, 0.05% sodium-chlor, 1% agarose, penbritin to final concentration are 100 μ g/ml, kantlex is to final concentration 50 μ g/ml, 2% glucose) on, 37 ℃, be inverted overnight incubation.Transformed bacteria is scraped the back from flat board be stored in-70 ℃ with 15% glycerine.This is influenza virus nano antibody gene pool, and its size is 2 * 10
6, refrigeration is at-70 ℃.
The screening process of the special single domain antibody of avian influenza virus is as follows: take out 500 μ l and be used for screening from the single domain antibody phage antibody library, 2 * YT substratum (1.6% Tryptones that at first it is added 9.5ml, 1% yeast extract, 0.5% sodium-chlor, 2% glucose) in, 37 ℃, 220rpm activates 1h.Add 10 μ l M13 helper phages (being purchased from Stratagene) again, add penbritin, 37 ℃, 220rpm cultivates 2h.5000g, the centrifugal receipts of 10min bacterium.Be resuspended in (A, G represent respectively and contain penbritin and kantlex) in 10ml2 * YT-AG substratum, 37 ℃, 220rpm, incubated overnight.Centrifugal overnight culture precipitates 1h with supernatant with PEG/NaCl, 10000g, and 4 ℃ of centrifugal 20min, precipitation is phage antibody.The resuspended precipitation of 2%BSA adds in advance and wraps quilt with inactivated avian influenza virus (avian influenza virus A/Chicken/Henan/16/2004 (H5N1) is available from Wuhan virus institute of the Chinese Academy of Sciences), and in the immune pipe with the 2%BSA adequate closure, hatches 2h for 37 ℃.Again successively with PBST and PBS purge to remove non-special absorption, add 2.5ml elutriant (0.1M Gly-HCl, pH 2.2) incubated at room 10min with the interaction force between the destruction antigen-antibody.Be drawn in another clean pipe after the piping and druming for several times, use 60 μ l 2M Tris (pH7.4) neutralization immediately.Elutriant is added in e. coli tg1 (being purchased from Stratagene) the bacterium liquid of 10ml OD600=0.3, room temperature leaves standstill 15min, makes phage fully infect intestinal bacteria.Finish first round screening like this, and therefrom draw a small amount of bacterium liquid coating SOB-AG flat board (2% Tryptones, 0.5% yeast extract, 0.05% sodium-chlor, penbritin to final concentration is 100 μ g/ml, and kantlex is to final concentration 50 μ g/ml, 2% glucose) counting.Repeat above step, carried out 3 screening of taking turns and enrichments again, number of times and the dynamics with PBST and PBS purge continues to increase (Fig. 1) at every turn.
In the end one take turns screening and finish after, 46 clones of picking at random from the flat board that screens back coating prepare phage antibody according to the method described above respectively.Utilize direct ELISA to carry out the screening of phage antibody again, concrete grammar is as follows: by elisa plate, 4 ℃ of bags are spent the night with H 5 N 1 avian influenza subtype virus bag; Add 2%BSA room temperature sealing 2h; Anti-with the phage antibody that 2%BSA is resuspended as one, hatch 1h for 37 ℃; Two is anti-for Anti-M13-HRP (being purchased from Promega), hatches 40min for 37 ℃.Last O-Phenylene Diamine (OPD) substrate colour developing (being purchased from Shanghai life worker bio-engineering corporation) is used 2M H behind the colour developing 5min
2SO
4Termination reaction, microplate reader is at the OD/490nm reading.Filter out the clone of a strain by this method, called after VHH3B to H 5 N 1 avian influenza subtype virus high-affinity.VHH3B is made up of 120 amino acid, and its sequence is shown in SEQ ID No:1.
For a large amount of special single domain antibody VHH3B of avian influenza virus that obtain, we with its gene fragment by the primer in the table 2, subclone is gone into expression vector pET28a and (is purchased from Novagen, as shown in figure 11), and in e. coli bl21 (DE3) (being purchased), obtain great expression (Fig. 2) from Novagen, compare with other genetic engineering antibody, its solubility is very strong, and expression amount also can reach every liter of bacterium 10mg.Through direct elisa assay, the single domain antibody VHH3B of prokaryotic expression shows the strong bonding force to avian influenza virus, and other irrelevant antigen is not had bonding force (Fig. 3).VHH3B is carried out dynamic analysis (the Jianbing Zhang of antibody through SPR (SurfacePlasmon Resonance), et al., Pentamerization of Single-domain Antibodies from Phage Libraries:A Novel Strategy for the Rapid Generation of High-avidity Antibody Reagents, J.Mol.Biol. (2004) 335,49-56), learn that its affinity constant KD is 2.66 * 10
-7Nm (Fig. 4), as a strain gene engineering antibody, its avidity can be comparable with a strain mouse resource monoclonal antibody, is very beneficial for its further application.
Table 2VHH3B subclone is gone into the pET28a the primer
Embodiment two: the preparation of avian influenza virus pentamer antibody pVHH3B
The antibody of a high-affinity of our ultimate demand acquisitions is used for the detection and the treatment of avian influenza virus, therefore we go into pentamer carrier pVT2 with the VHH3B gene fragment by the primer subclone in the table 3 (is provided by Canadian National Research Council doctor Zhang Jianbing, as shown in figure 12, reference: Jianbing Zhang, et al., Pentamerization of Single-domainAntibodies from Phage Libraries:A Novel Strategy for the Rapid Generationof High-avidity Antibody Reagents, J.Mol.Biol. (2004) 335,49-56) in.Because the shiga toxin B subunit in this carrier can self five dimerization, thus with shiga toxin B subunit amalgamation and expression single domain antibody VHH3B after, form five poly-antibody structures, we name it is pVHH3B.The monomer of pVHH3B is made up of 195 amino acid, and its sequence is shown in SEQ ID No:2.We are its great expression (Fig. 5) in e. coli tg1 subsequently, and identify itself and avian influenza virus bonded specificity (Fig. 6) by ELISA, analyze by SPR, and pVHH3B demonstrates the avidity stronger than VHH3B (Fig. 7).
Table 3VHH3B subclone is gone into the pVT2 the primer
Embodiment three: set up method for biosensor with magnetic nanoparticle link coupled avian influenza virus special five poly-antibody and the special monoclonal antibody of gold nano grain link coupled mouse source avian influenza virus and detect avian influenza virus
We form MNP-pVHH3B with the magnetic nanoparticle (MNPs) of 1 μ m coated with silica (be purchased doubly think from Tianjin happy chromatographic technique development centre) coupling five poly-antibody pVHH3B.
Concrete grammar is as follows: get the magnetic nanoparticle of 1ml 10mg/ml, add succinimide (NHS is purchased from Sigma) and the carbodiimide (EDC of 50mg/ml, be purchased from Sigma) each 50 μ l, incubated at room 30min uses washed with de-ionized water, removes unnecessary NHS/EDC.The sodium acetate that adds 1ml pH6.0, the pVHH3B of 100 μ g, mixing is hatched 2h for 4 ℃, and the PBS washing adds 50mM pH 7.4Tris-Cl sealing activatory carboxyl, and PBS is resuspended, 4 ℃ of preservations.Use mouse monoclonal antibody mAb 3C8 (being purchased pharmaceutical Co. Ltd) mark 14nm gold nano grain (GNPs) (being purchased pharmaceutical Co. Ltd) simultaneously from Beijing Wan Tai from Beijing Wan Tai.Concrete grammar is as follows: get the 1ml gold nano grain, use 25mM K
2CO
3Transfer pH to 8.5, add 10 μ l, the 3C8 of 1mg/ml, mixing, room temperature leaves standstill 10min.Add 100 μ l 10%BSA, mixing, room temperature leaves standstill 10min, and the centrifugal 20min of 10000rpm absorbs supernatant.With the resuspended gold nano grain precipitation of 50 μ l PBS, 4 ℃ of preservations.At first catch influenza virus particles with MNP-pVHH3B, the MNPs that will have a virion is with magnet after separating from solution, to wherein adding and gold nano grain link coupled mAb 3C8, what add forms complex body by combining influenza virus particles with magnetic nanoparticle with gold nano grain link coupled mAb 3C8, and this complex body is separated with magnet.To wherein adding Resorcinol, because the golden catalysis hydroquinone oxidation in the described mixture generates benzoquinones.So benzoquinones is made comparisons at the absorbance value at the 390nm place absorbance value when not adding virion by will add influenza virus particles the time, realizes the high-sensitivity detection to virus.By this method influenza virus is detected, can detect the influenza virus (Fig. 8) that is low to moderate the 10ng/ml level, compare with double antibodies sandwich ELISA, sensitivity has improved 10 times (Fig. 9).
IB101881.ST25.txt
SEQUENCE?LISTING
＜110〉Institute of Biophysics, Academia Sinica
＜120〉avian influenza virus single domain antibody, five poly-antibody and preparation and application
<130>IB101881
<160>2
<170>PatentIn?version?3.1
<210>1
<211>118
<212>PRT
<213>Vicugna?pacos
<400>1
Glu?Val?Gln?Leu?Gln?Ala?Ser?Gly?Gly?Gly?Leu?Val?Gln?Ala?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Arg?Thr?Phe?Ser?Ser?Gly
20 25 30
Ala?Met?Gly?Trp?Phe?Arg?Gln?Ala?Pro?Gly?Lys?Glu?Arg?Glu?Phe?Leu
35 40 45
Ser?Ala?Ile?Arg?Trp?Asp?Gly?Lys?Ile?Ile?Arg?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Met?Asn?Arg?Val?Phe
65 70 75 80
Leu?Gln?Met?Asp?Ser?Leu?Lys?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Pro?Gly?Pro?Asp?Ile?Ile?Thr?Phe?Asp?Ser?Trp?Gly?Gln?Gly?Thr
100 105 110
Gln?Val?Thr?Val?Ser?Ser
115
<210>2
<211>195
<212>PRT
<213>Vicugna?pacos
<400>2
Thr?Pro?Asp?Cys?Val?Thr?Gly?Lys?Val?Glu?Tyr?Thr?Lys?Tyr?Asn?Asp
1 5 10 15
Glu?Asp?Thr?Phe?Thr?Val?Lys?Val?Gly?Asp?Lys?Glu?Leu?Phe?Thr?Asn
20 25 30
Arg?Ala?Asn?Leu?Gln?Ser?Leu?Leu?Leu?Ser?Ala?Gln?Ile?Thr?Gly?Met
35 40 45
Thr?Val?Thr?Ile?Lys?Thr?Asn?Ala?Cys?His?Asn?Gly?Gly?Gly?Phe?Ser
50 55 60
Glu?Val?Ile?Phe?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Met?Ala?Glu?Val?Gln
65 70 75 80
Leu?Gln?Ala?Ser?Gly?Gly?Gly?Leu?Val?Gln?Ala?Gly?Gly?Ser?Leu?Arg
85 90 95
Leu?Ser?Cys?Ala?Ala?Ser?Gly?Arg?Thr?Phe?Ser?Ser?Gly?Ala?Met?Gly
100 105 110
Trp?Phe?Arg?Gln?Ala?Pro?Gly?Lys?Glu?Arg?Glu?Phe?Leu?Ser?Ala?Ile
115 120 125
Arg?Trp?Asp?Gly?Lys?Ile?Ile?Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg
130 135 140
Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Met?Asn?Arg?Val?Phe?Leu?Gln?Met
145 150 155 160
Asp?Ser?Leu?Lys?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Ala?Gly
165 170 175
Pro?Asp?Ile?Ile?Thr?Phe?Asp?Ser?Trp?Gly?Gln?Gly?Thr?Gln?Val?Thr
180 185 190
Val?Ser?Ser
195
Claims (11)
1. obtain that avian influenza virus is had the method for the single domain antibody of specific combination ability, it is characterized in that described method comprises the following steps:
1) set up avian influenza virus immunity single domain antibody phage library and
2) utilize display technique of bacteriophage from described single domain antibody phage library, to screen the single domain antibody clone high to avian influenza virus avidity,
Wherein, preferably, described avian influenza virus is Re1 strain of influenza vaccines H5N1 hypotype and H5N2 hypotype N28 strain.
2. single domain antibody that avian influenza virus is had the specific combination ability is characterized in that described single domain antibody is that method by claim 1 obtains.
3. single domain antibody that avian influenza virus is had the specific combination ability, the aminoacid sequence that it is characterized in that described single domain antibody is shown in SEQ ID No:1.
4. five poly-antibody that avian influenza virus had the specific combination ability, it is characterized in that described five poly-antibody are the five poly-antibody that formed by the pentamer core by claim 2 or 3 described single domain antibodies, wherein said pentamer core can be to contain the segmental pentamer carrier of shiga toxin B subunit gene, preferably pVT2.
5. claim 4 described five poly-antibody is characterized in that described five poly-antibody have the monomer of 5 aminoacid sequences shown in SEQ ID No:2.
6. make up the method for claim 4 or 5 described five poly-antibody, it is characterized in that described method comprises the following steps:
1) with the gene fragment subclone of claim 2 or 3 described single domain antibodies to contain in the segmental pentamer carrier of shiga toxin B subunit gene with obtain recombinant vectors and
2) express described recombinant vectors,
The wherein said preferably pVT2 of the segmental pentamer carrier of shiga toxin B subunit gene that contains.
7. claim 2 or 3 described single domain antibodies or claim 4 or 5 described five poly-antibody are used to catch the application of avian influenza virus particulate.
8. the magnetic five poly-antibody that avian influenza virus had the specific combination ability is characterized in that described magnetic five poly-antibody form by five poly-antibody and the magnetic nanoparticle coupling that makes claim 4 or 5.
9. detect the test kit of avian influenza virus, it comprises
1) the described magnetic five poly-antibody of claim 8;
2) with gold nano grain link coupled anti-avian influenza antibody; With
3) Resorcinol.
10. detect the method for avian influenza virus, it is characterized in that said method comprising the steps of:
1) in sample and blank, add the magnetic five poly-antibody of claim 8 and utilize magnetic to separate respectively, obtaining separated object with magnetic,
2) add with gold nano grain link coupled anti-avian influenza antibody in the separated object that in step 1), obtains and utilize magnetic to separate, obtaining separated object with magnetic,
3) to step 2) in add in the separated object that obtains Resorcinol and 390nm place its absorbance value of measurement and
4) absorbance value by 390nm place that described sample is obtained determines to have avian influenza virus in the described sample greater than the absorbance value to the 390nm place of described blank acquisition.
11. described magnetic five poly-antibody of claim 8 and the described test kit of claim 9 are used to detect the application of avian influenza virus.
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| CN105044009A (en) * | 2015-07-14 | 2015-11-11 | 上海拜豪生物科技有限公司 | Chromium-very low density lipoprotein chelate and preparation method and application thereof |
| CN105044007A (en) * | 2015-07-14 | 2015-11-11 | 上海拜豪生物科技有限公司 | cadmium-IgM chelate as well as preparation method and application thereof |
| CN106146657A (en) * | 2016-07-12 | 2016-11-23 | 晋明（天津）生物医药技术有限公司 | Wide spectrum recombinant antibody fragment combining influenza virus A and preparation method and application |
| CN107207611A (en) * | 2015-02-05 | 2017-09-26 | 扬森疫苗与预防公司 | Binding molecule for enza hemagglutinin and application thereof |
| JP2020012700A (en) * | 2018-07-17 | 2020-01-23 | パナソニックＩｐマネジメント株式会社 | Detector |
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| CN107207611A (en) * | 2015-02-05 | 2017-09-26 | 扬森疫苗与预防公司 | Binding molecule for enza hemagglutinin and application thereof |
| US11136379B2 (en) | 2015-02-05 | 2021-10-05 | Janssen Vaccines & Prevention B.V. | Binding molecules directed against influenza hemagglutinin and uses thereof |
| CN107207611B (en) * | 2015-02-05 | 2021-11-09 | 扬森疫苗与预防公司 | Binding molecules against influenza hemagglutinin and uses thereof |
| CN105044009A (en) * | 2015-07-14 | 2015-11-11 | 上海拜豪生物科技有限公司 | Chromium-very low density lipoprotein chelate and preparation method and application thereof |
| CN105044007A (en) * | 2015-07-14 | 2015-11-11 | 上海拜豪生物科技有限公司 | cadmium-IgM chelate as well as preparation method and application thereof |
| CN105044009B (en) * | 2015-07-14 | 2018-04-03 | 上海拜豪生物科技有限公司 | Chromium-very low density lipoprotein chelate and preparation method and application thereof |
| CN105044007B (en) * | 2015-07-14 | 2018-08-21 | 上海拜豪生物科技有限公司 | cadmium-IgM chelate as well as preparation method and application thereof |
| CN106146657A (en) * | 2016-07-12 | 2016-11-23 | 晋明（天津）生物医药技术有限公司 | Wide spectrum recombinant antibody fragment combining influenza virus A and preparation method and application |
| JP2020012700A (en) * | 2018-07-17 | 2020-01-23 | パナソニックＩｐマネジメント株式会社 | Detector |
| JP7178623B2 (en) | 2018-07-17 | 2022-11-28 | パナソニックＩｐマネジメント株式会社 | detector |
| CN111116752A (en) * | 2019-12-24 | 2020-05-08 | 北京纽安博生物技术有限公司 | Immunoglobulin-binding single domain antibody, anti-avian influenza single domain antibody, bifunctional antibody and application thereof |
| CN111116752B (en) * | 2019-12-24 | 2021-09-03 | 北京纽安博生物技术有限公司 | Immunoglobulin-binding single domain antibody, anti-avian influenza single domain antibody, bifunctional antibody and application thereof |
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