CN102276719B - 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 after transformation, is applied to the method for biosensor of avian influenza virus diagnosis.
Background technology
Influenza has become one of principal disease threatening human health gradually.H1N1virus in 2009 outburst in the world and the fear that since 2003, high pathogenic avian influenza H5N1 brings impel people to improve constantly to answer the Diagnosis and Treat ability of infected by influenza.Carry out diagnosis in early days at virus infection to be conducive to improving curative ratio, reduce virus disseminating.For the detection of influenza virus, except traditional detection method is as virus purification, RT-PCR etc., specific antibody is also widely used in the detection methods such as various ELISA and immunofluorescence.In addition, also there is a lot of trial improving detection specificity and sensitivity, such as, develop the stronger antibody of specificity, introduce microsphere etc. in the detection system.However, the highly sensitive influenza virus detection method developed based on antibody is still faced with huge challenge, also has very large space to go to explore.
Summary of the invention
Characteristic of the present invention be it be utilize engineered method to obtain for the high five affine poly-antibody of avian influenza virus and the detection carrying out avian influenza virus in being applied in based on magnetic nanoparticle biosensor.Rationale of the present invention is the special property based on camel antibodies: be made up of heavy chain antibody the IgG of nearly half in camel serum, and can functional conjugated antigen; The antibody of same disappearance light chain---neoantigen acceptor (NARs) finds in shark body.What clone built thus is only called single domain antibody, i.e. VHH antibody containing the antibody of variable region of heavy chain, and molecular weight is only 15kD, in conjunction with some less epitopes, can have higher binding activities.This small molecular antibody is screened by display technique of bacteriophage and obtains from the single domain antibody storehouse established in advance, and is subcloned in expression vector, by transformation of E. coli great expression.Single domain antibody only just can be played the function of conjugated antigen by variable region of heavy chain, and demonstrates analogous avidity.In addition, due to the heavy chain antibody of existence natural in camel antibodies not containing light chain, several key amino acids of its variable region sport hydrophilic amino acid by hydrophobic in conventional antibodies, and the single domain antibody therefore coming from camel native heavy antibody has following three distinguishing features: 1, have the avidity comparable with conventional antibodies; 2, during prokaryotic expression, solubility is very strong, and expression amount is higher; 3, molecular weight is less, is easier to carry out genetically engineered operation.This just compensate for the deadly defect of traditional small molecular antibody, and wide space is expanded out in the development for genetic engineering antibody.Therefore, compared with traditional mouse resource monoclonal antibody, utilize display technique of bacteriophage to prepare the single domain antibody required time cycle shorter, workload is less, and owing to using escherichia coli expression, output also improves greatly.
Based on above-mentioned theory basis, first, we utilize influenza virus immunization alpaca, establish influenza virus immunization single domain antibody phage library, and utilize the inactivating influenza virus of purifying from antibody library, filter out the affine single domain antibody VHH3B of a plant height.In order to improve the avidity of this antibody to avian influenza virus, make it to be more suitable for high-sensitivity detection, single domain antibody VHH3B and shiga toxin B subunit are carried out amalgamation and expression again by us, make expression product five dimerization by five dimerizations of shiga toxin B subunit self, form five poly-antibody.Finally utilize the virus in five of magnetic nanoparticle coupling poly-antibody capture samples, again with the mouse source avian influenza virus specific antibody 3C8 of colloid gold particle coupling for detect antibody, by such combination, set up high-sensitive method for biosensor, carry out the high-sensitivity detection of virus.
The innovation of the present invention in technological line is: 1, utilize deactivation H 5 N 1 avian influenza and H5N2 virus co-immunization to grow up alpaca, after three booster immunizations, be separated 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 dimerization, by bird flu single domain antibody amalgamation and expression with it, form five poly-antibody of avian influenza virus specific.Five poly-after antibody exhibits go out higher avidity, be more suitable for the needs of the high-sensitivity detection of avian influenza virus.3, five of magnetic nanoparticle coupling poly-antibody are utilized to carry out the detection of avian influenza virus as the mouse source anti-avian influenza monoclonal antibody of capture antibody and colloid gold particle coupling.4, the hydroquinone oxidation of golden catalysis is utilized to generate the reflection of reaction as detected result of benzoquinones.
Particular content is as follows:
1. obtain the method for single domain antibody avian influenza virus to specific combination ability, it is characterized in that described method comprises the following steps:
1) avian influenza virus immunity single domain antibody phage library is set up, and
2) utilize display technique of bacteriophage from described single domain antibody phage library, screen the single domain antibody clone high to avian influenza virus avidity,
Wherein, preferably, described avian influenza virus is influenza vaccines H5N1 hypotype Re1 strain and H5N2 hypotype N28 strain.
2. avian influenza virus is had to a single domain antibody for specific combination ability, it is characterized in that described single domain antibody is obtained by the method for above 1.
3. avian influenza virus is had to a single domain antibody for specific combination ability, it is characterized in that the aminoacid sequence of described single domain antibody is as shown in SEQ ID No:1.
4. one kind has five poly-antibody of specific combination ability to avian influenza virus, it is characterized in that described five poly-antibody are the five poly-antibody formed by pentamer core by the single domain antibody described in above 2 or 3, wherein said pentamer core can be the pentamer carrier containing shiga toxin B subunit gene fragment, preferably pVT2.
5. antibody is gathered in five more than described in 4, it is characterized in that described five poly-antibody have the monomer of 5 aminoacid sequences as shown in SEQ ID No:2.
6. build the method for the poly-antibody in above five described in 4 or 5, it is characterized in that described method comprises the following steps:
1) gene fragment of the single domain antibody described in above 2 or 3 is subcloned into obtain recombinant vectors in the pentamer carrier containing shiga toxin B subunit gene fragment, and
2) described recombinant vectors is expressed,
The wherein said pentamer carrier containing shiga toxin B subunit gene fragment is preferably pVT2.
7. described in single domain antibody more than described in 2 or 3 or more 4 or 5 five poly-antibody are for catching the application of avian influenza virus particle.
8. the magnetic five that pair avian influenza virus has a specific combination ability gathers antibody, it is characterized in that described magnetic five is gathered antibody and formed by making five of above 4 or 5 poly-antibody and magnetic nanoparticle coupling.
9. detect the test kit of avian influenza virus, it comprises
1) magnetic five more than described in 8 gathers antibody;
2) with the anti-avian influenza antibody of gold nano grain coupling; With
3) Resorcinol.
10. detect the method for avian influenza virus, it is characterized in that said method comprising the steps of:
1) magnetic five adding above 8 in sample and blank is respectively gathered antibody and utilizes magnetic to be separated, to obtain the magnetic separated object of tool,
2) to step 1) in add with the anti-avian influenza antibody of gold nano grain coupling and utilize magnetic to be separated, to obtain the magnetic separated object of tool in the separated object that obtains
3) to step 2) in obtain separated object in add Resorcinol and 390nm place measurement its absorbance value, and
4) be greater than the absorbance value to the 390nm place that described blank obtains by the absorbance value at the 390nm place obtained described sample, determine, in described sample, there is avian influenza virus.
Magnetic five described in more than 11. 8 gathers antibody and the test kit described in above 9 for detecting the application of avian influenza virus.
accompanying drawing is sketched
Fig. 1. avian influenza virus antibody storehouse calculates through four-wheel screening enrichment number;
Fig. 2 .SDS-PAGE analyzes the purity of anti-avian influenza virus single domain antibody, wherein left side swimming lane: protein molecular weight standard; Right lanes: 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 left side swimming lane: protein molecular weight standard; Right lanes: 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 analyzes the binding ability of pentamer antibody;
Fig. 8. the biosensor based on magnetic nanoparticle detects avian influenza virus;
Fig. 9. double crush syndrome detects avian influenza virus;
Figure 10. phagemid vector pCANTAB5E collection of illustrative plates;
Figure 11. expression vector pET28a collection of illustrative plates; With
Figure 12. pentamer carrier pVT2 sequence map.
sequence explanation
The aminoacid sequence of SEQ ID No:1 single domain antibody VHH3B; With
SEQ ID No:2 five gathers the aminoacid sequence of the monomer of antibody pVHH3B.
Embodiment
In order to comprehend and application the present invention, provide the following example.
Embodiment one: the Preparation and identification of single domain antibody
Application 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 from Beijing animal doctor master station) and H5N2 hypotype N28 strain (being purchased from Beijing animal doctor master station) to two adult alpaca (Alpaca, Camelidae, yamma belongs to, purchased from alpaca cultivation base, Vicugna pacos) (code name is respectively B007 and B008) carry out the subcutaneous multiple spot immunization of neck both sides lymphoglandula, every immunity in 10 days once, three times are divided into.Within after last immunization 10 days, get the peripheral blood of two alpacas respectively, with erythrocyte cracked liquid (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.) cracking hemocyte, collected by centrifugation white corpuscle.Add 1mlTrizol (being purchased from Invitrogen), room temperature leaves standstill 5min.Add 200 μ l chloroforms and leave standstill 2-3min.12000rpm, 4 DEG C of centrifugal 15min.Transfer upper strata aqueous phase, in another new centrifuge tube, adds 500 μ l Virahols and fully mixes afterwards after-20 DEG C of standing 10min, 12000rpm 4 DEG C of centrifugal 10min, abandon supernatant.Precipitated rna 1ml 75% ethanol rinse 2-3 time, 4 DEG C of centrifugal 5min of 7500rpm, abandon supernatant.RNA precipitates drying at room temperature 5-10min, and the ultrapure water 50 μ l processed with diethylpyrocarbonate (DEPC) dissolves RNA completely in 55 DEG C of incubation 10min.
Be that (20 μMs of oligo dT 3 μ l, 10 μMs of dNTP 1 μ l, in PCR pipe, add water to 13 μ l to template reverse transcription synthesis cDNA for Invitrogen test kit, reaction conditions: add RNA 2 μ g with RNA.Hatch 5min for 65 DEG C, put rapidly 2-3min on ice.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 cDNA product for template, successively use two couples of primer VHBACKA6 in table 1, CH2FORTA4 and F-PRCCAMEL, R-PCRCAMEL carry out nest-type PRC (reaction conditions: 94 DEG C, 5min denaturation; 94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 40s, 30 circulations; 72 DEG C extend 7min), amplify special VHH segment.
Table 1 utilizes nested PCR method amplification VHH fragment two groups of primers used
Then the VHH fragment amplified and phagemid vector pCANTAB5E (are purchased from Amersham Pharmacia, cut with restriction enzyme SfiI enzyme in 50 DEG C of water-baths respectively as shown in Figure 10), after reclaiming the VHH fragment after cutting and carrier segments, connect with T4DNA ligase enzyme (being purchased from TaKaRa) 16 DEG C and spend the night.Afterwards, electricity transforms connection product and enters intestinal bacteria (E.Coli), concrete grammar is as follows: will connect product PCR purification kit (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.) and remove ion, join in previously prepd competent cell e. coli tg1 (being purchased from Stratagene), mixing, places 30min on ice.Add in the electric revolving cup (1mm, BioRad) of precooling, at voltage 2.5kV, under the condition of resistance 2000 Ω, electric capacity 25 μ F, carry out Electroporation Transformation.Join rapidly 2 × YT-AG substratum (1.6% Tryptones, 1% yeast extract, 0.5% sodium-chlor afterwards, penbritin to final concentration is 100 μ g/ml, kantlex to final concentration 50 μ g/ml, 2% glucose) in, 180rpm, cultivates 1h by 37 DEG C.5000g, 10 minutes centrifugal, the resuspended precipitation of appropriate 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 is 100 μ g/ml, kantlex is to final concentration 50 μ g/ml, 2% glucose) on, 37 DEG C, be inverted overnight incubation.Transformed bacteria is stored in-70 DEG C with 15% glycerine after flat board scrapes.This is influenza virus nano antibody gene pool, and its size is 2 × 10
6, refrigerate at-70 DEG C.
The screening process of the special single domain antibody of avian influenza virus is as follows: from single domain antibody phage antibody library, take out 500 μ l for screening, first 2 × YT substratum (1.6% Tryptones of 9.5ml is added, 1% yeast extract, 0.5% sodium-chlor, 2% glucose) in, 37 DEG C, 220rpm activates 1h.Add 10 μ l M13 helper phage (being purchased from Stratagene) again, add penbritin, 37 DEG C, 220rpm cultivates 2h.5000g, 10min centrifugal receipts bacterium.Be resuspended in (A, G represent respectively containing penbritin and kantlex) in 10ml2 × YT-AG substratum, 37 DEG C, 220rpm, incubated overnight.Centrifuged overnight culture, precipitates 1h by supernatant PEG/NaCl, 10000g, 4 DEG C of centrifugal 20min, and precipitation is phage antibody.The resuspended precipitation of 2%BSA, add prior inactivated avian influenza virus (avian influenza virus A/Chicken/Henan/16/2004 (H5N1), purchased from Wuhan virus institute of the Chinese Academy of Sciences) wrap quilt, and with in the immune pipe of 2%BSA adequate closure, hatch 2h for 37 DEG C.Priority PBST and PBS purge are to remove non-specific adsorption again, add 2.5ml elutriant (0.1M Gly-HCl, pH 2.2) incubated at room 10min to destroy the interaction force between antigen-antibody.Be drawn in another clean pipe after piping and druming for several times, use 60 μ l 2M Tris (pH7.4) neutralizations immediately.Be added to by elutriant 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.Complete 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, kantlex to final concentration 50 μ g/ml, 2% glucose) counting.Repeat above step, carried out again 3 screening taken turns and enrichments, number of times and the dynamics of each PBST and PBS purge continue to increase (Fig. 1).
In the end one take turns after screening completes, random picking 46 clone the flat board of coating after screening, prepares phage antibody according to the method described above respectively.Recycling Salmonella carries out the screening of phage antibody, and concrete grammar is as follows: with H 5 N 1 avian influenza subtype virus bag by elisa plate, 4 DEG C of bags are spent the night; Add 2%BSA room temperature and close 2h; With the resuspended phage antibody of 2%BSA as primary antibodie, hatch 1h for 37 DEG C; Two resist for Anti-M13-HRP (being purchased from Promega), hatch 40min for 37 DEG C.Last O-Phenylene Diamine (OPD) substrate colour developing (being purchased from Shanghai Sheng Gong bio-engineering corporation), uses 2M H after colour developing 5min
2sO
4termination reaction, microplate reader is at OD/490nm reading.The clone of a strain to H 5 N 1 avian influenza subtype virus high-affinity is filtered out, called after VHH3B by this method.VHH3B is made up of 120 amino acid, and its sequence is as shown in SEQ ID No:1.
In order to obtain the special single domain antibody VHH3B of avian influenza virus in a large number, its gene fragment is passed through the primer in table 2 by us, subclone enters expression vector pET28a and (is purchased from Novagen, as shown in figure 11), and great expression (Fig. 2) is obtained in e. coli bl21 (DE3) (being purchased from Novagen), compared with other genetic engineering antibody, its solubility is very strong, and expression amount also can reach often liter of bacterium 10mg.Through Salmonella analysis, the single domain antibody VHH3B of prokaryotic expression shows the strong bonding force to avian influenza virus, and does not have bonding force (Fig. 3) to other irrelevant antigen.Through SPR (SurfacePlasmon Resonance), dynamic analysis (the Jianbing Zhang of antibody is carried out to VHH3B, 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 it and further applies.
Table 2VHH3B subclone enters pET28a the primer
Embodiment two: the preparation of avian influenza virus pentamer antibody pVHH3B
We finally need the test-and-treat of antibody for avian influenza virus of an acquisition high-affinity, therefore VHH3B gene fragment is entered pentamer carrier pVT2 by the primer subclone in table 3 by us (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).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 to be pVHH3B.The monomer of pVHH3B is made up of 195 amino acid, and its sequence is as shown in SEQ ID No:2.We are by its great expression (Fig. 5) in e. coli tg1 subsequently, and identify its specificity be combined with avian influenza virus (Fig. 6) by ELISA, analyzed by SPR, pVHH3B demonstrates the avidity (Fig. 7) stronger than VHH3B.
Table 3VHH3B subclone enters pVT2 the primer
Embodiment three: set up method for biosensor detection avian influenza virus with the mouse source special monoclonal antibody of avian influenza virus of the poly-antibody of the avian influenza virus of magnetic nanoparticle coupling special five and gold nano grain coupling
Magnetic nanoparticle (MNPs) (be purchased and doubly think happy chromatographic technique development centre from the Tianjin) coupling five of 1 μm of coated with silica is gathered antibody pVHH3B by us, forms MNP-pVHH3B.
Concrete grammar is as follows: the magnetic nanoparticle getting 1ml 10mg/ml, add the succinimide (NHS of 50mg/ml, be purchased from Sigma) and carbodiimide (EDC, be purchased from Sigma) each 50 μ l, incubated at room 30min, by washed with de-ionized water, remove unnecessary NHS/EDC.Add the sodium acetate of 1ml pH6.0, the pVHH3B of 100 μ g, mixing, hatches 2h for 4 DEG C, and PBS washs, and add the carboxyl that 50mM pH 7.4Tris-Cl closes activation, PBS is resuspended, 4 DEG C of preservations.Use mouse monoclonal antibody mAb 3C8 (being purchased from Beijing Wan Tai pharmaceutical Co. Ltd) to mark 14nm gold nano grain (GNPs) (being purchased from Beijing Wan Tai pharmaceutical Co. Ltd) simultaneously.Concrete grammar is as follows: get 1ml gold nano grain, uses 25mM K
2cO
3adjust 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, the centrifugal 20min of 10000rpm, absorbs supernatant.By the resuspended gold nano grain precipitation of 50 μ l PBS, 4 DEG C of preservations.First influenza virus particles is caught with MNP-pVHH3B, after MNPs magnet with virion is separated from solution, add the mAb 3C8 with gold nano grain coupling wherein, that add with mAb 3C8 that is gold nano grain coupling by forming complex body in conjunction with influenza virus particles with magnetic nanoparticle, this complex body magnet is separated.Add Resorcinol wherein, due to the golden catalysis hydroquinone oxidation in described mixture, generate benzoquinones.So made comparisons by the absorbance value by the absorbance value of benzoquinones at 390nm place when adding influenza virus particles and when not adding virion, realize the high-sensitivity detection to virus.Detected by this method infected by influenza, the influenza virus (Fig. 8) being low to moderate 10ng/ml level can be detected, compare with double crush syndrome, sensitivity improves 10 times (Fig. 9).
Claims (5)
1. avian influenza virus is had to five poly-antibody of specific combination ability, it is characterized in that described five poly-antibody are made up of the monomer of 5 aminoacid sequences as shown in SEQ ID No:2.
2. according to claim 1 five poly-antibody are for catching the application of the non-diagnostic therapeutic purpose of avian influenza virus particle.
3. the magnetic five that pair avian influenza virus has a specific combination ability gathers antibody, it is characterized in that described magnetic five is gathered antibody and formed by making five of claim 1 poly-antibody and magnetic nanoparticle coupling.
4. magnetic five according to claim 3 gathers the application of antibody in the test kit for the preparation of detection avian influenza virus, and wherein said detection is undertaken by following steps:
1) magnetic five adding claim 3 respectively in sample and blank is gathered antibody and utilizes magnetic to be separated, to obtain the magnetic separated object of tool,
2) to step 1) in add with the antibody anti AIV of gold nano grain coupling and utilize magnetic to be separated in the separated object that obtains, to obtain the magnetic separated object of tool,
3) to step 2) in obtain separated object in add Resorcinol and 390nm place measurement its absorbance value, and
4) be greater than the absorbance value to the 390nm place that described blank obtains by the absorbance value at the 390nm place obtained described sample, determine, in described sample, there is avian influenza virus.
5. detect the test kit of avian influenza virus, it comprises:
1) magnetic five according to claim 3 gathers antibody;
2) with the antibody anti AIV of gold nano grain coupling; With
3) Resorcinol,
Wherein said detection is undertaken by following steps:
1) magnetic five adding claim 3 respectively in sample and blank is gathered antibody and utilizes magnetic to be separated, to obtain the magnetic separated object of tool,
2) to step 1) in add with the antibody anti AIV of gold nano grain coupling and utilize magnetic to be separated in the separated object that obtains, to obtain the magnetic separated object of tool,
3) to step 2) in obtain separated object in add Resorcinol and 390nm place measurement its absorbance value, and
4) be greater than the absorbance value to the 390nm place that described blank obtains by the absorbance value at the 390nm place obtained described sample, determine, in described sample, there is avian influenza virus.
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| CN106146657B (en) * | 2016-07-12 | 2020-07-17 | 晋明(天津)生物医药技术开发有限公司 | Recombinant antibody fragment capable of combining influenza virus A in broad spectrum and preparation method and application thereof |
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| 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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