CN103483449A - Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken - Google Patents
Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken Download PDFInfo
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Abstract
The invention discloses two types of ScFv (Single Chain Variable Fragment) antibodies, encoding genes of the antibodies and applications of the antibodies for preparing the preparations for treating or preventing infectious bursal disease of chicken. Two single-chain antibodies (namely, the ScFv antibodies) are provided; and the ScFv antibodies can be specially bound with the protein 2 (VP2) (Virus Protein 2) in an IBDV (Infectious Bursal Disease Virus) structure and a plurality of IBDV strains to block the cytopathic effect (CPE) of the chicken embryo fibroblast by the IBDV so as to protect the young chicken infected with IBDV. The immune serum and egg yolk antibody are tedious in preparation, high in production cost, unstable in effect, difficult to control industrialized production quality, capable of causing horizontal disease spread and the like in a use process; the ScFv antibodies disclosed by the invention can overcome the above disadvantages and have the advantages of strong specificity, good treatment effect, controllable industrialized production quality, capability of preventing the horizontal disease spread caused by the egg yolk antibody and the like; and therefore, by virtue of the ScFv antibodies, a new situation is opened up in the IBDV prevention and treatment history, and even in the entire prevention and treatment history for animal virus diseases.
Description
Technical field
The present invention relates to two kinds of scFv antibody, its encoding gene and the application in preparation treatment or prevention infectious bursal disease preparation thereof.
Background technology
Infectious bursal disease (Infectious Bursal Disease, IBD) be by chicken infectivity bursa of Fabricius virus (Infectious Bursal Disease Virus, IBDV) a kind of acute, the height contact chicken transmissible disease that cause, have that velocity of propagation is fast, infectivity strong, equal high characteristics of infection rate and mortality ratio.This disease causes heavy economic losses to aviculture.
IBDV can breed rapidly in lymphocyte, especially bone-marrow-derived lymphocyte in the chick fabricius bursa, impels the bone-marrow-derived lymphocyte apoptosis, and extremely atrophy of the fabricius bursa, finally cause immune deficiency and immunosuppression.Therefore very easily cause the secondary infection of bacterium (intestinal bacteria and Salmonellas), can also cause the immuning failure of other vaccines simultaneously.Polyclonal antibody (hyper-immune serum and yolk antibody) is at present effective treatment means, respond well at the morbidity early treatment, but due to the suitability for industrialized production poor controllability with have horizontal transmission disease (white dysentery, Mycoplasma, egg-decreasing syndrome and avian leukosis) and the anaphylaxis that caused by some non-specific antibodies etc. is former thereby be restricted.
Genetic engineering antibody is about to the gene of antibody and need to be processed, transforms and ressemble by difference, then imports the antibody molecule of being expressed in suitable recipient cell.In prokaryotic cell prokaryocyte, the current technology of expressing gene engineered antibody is relatively ripe, can realize industrialized production, the controlled homogeneous of quality product.There is not the danger of horizontal transmission communicable disease in prokaryotic expression system simultaneously.
Summary of the invention
The purpose of this invention is to provide two kinds of single-chain antibodies (scFv antibody), its encoding gene and the application in preparation treatment or prevention infectious bursal disease preparation thereof.
The invention provides two kinds of single-chain antibodies, called after IBDV-VP2 scFv29 antibody and IBDV-VP2 scFv30 antibody, comprise by variable region of heavy chain, variable region of light chain and the joining region between them and forming;
The variable region of heavy chain of described IBDV-VP2 scFv29 antibody is following (a) or (b): the protein that (a) sequence 1 forms from N-terminal 1-129 amino acids residue in sequence table; (b) by (a) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity;
Described IBDV-VP2 scFv29 antibody chain variable region is following (c) or (d): the protein that (c) sequence 1 forms from N-terminal 145-250 amino acids residue in sequence table; (d) by (c) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity.
Described single-chain antibody IBDV-VP2 scFv29 specifically can be as follows (e) or (f): (e) in sequence table
sequence 1shown protein; (f) by (e) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity.
The gene of described single-chain antibody IBDV-VP2 scFv29 of encoding also belongs to protection scope of the present invention.
In described single-chain antibody IBDV-VP2 scFv29 gene, the DNA molecular of the described variable region of heavy chain of encoding is following (1) or (2) or (3): the sequence 2 of (1) sequence table is from the DNA molecular shown in 5 ' end 1-387 position Nucleotide; (2) DNA molecular that the DNA sequence dna hybridization limited with (1) under stringent condition and coding have the albumen of identical activity; (3) DNA sequence dna limited with (1) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
In described single-chain antibody IBDV-VP2 scFv29 gene, the DNA molecular of the described variable region of light chain of encoding is following (4) or (5) or (6): the sequence 2 of (4) sequence table is from the DNA molecular shown in 5 ' end 433-753 position Nucleotide; (5) DNA molecular that the DNA sequence dna hybridization limited with (4) under stringent condition and coding have the albumen of identical activity; (6) DNA sequence dna limited with (4) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
Described single-chain antibody IBDV-VP2 scFv29 gene specifically can be (7) or (8) or (9) or (10) as follows: the sequence 2 of (7) sequence table is from the DNA molecular shown in 5 ' end 1-753 position Nucleotide; (8) DNA molecular shown in the sequence 2 of sequence table; (9) DNA molecular that the DNA sequence dna hybridization limited with (7) or (8) under stringent condition and coding have the albumen of identical activity; (10) DNA sequence dna limited with (7) or (8) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
The variable region of heavy chain of described IBDV-VP2 scFv30 antibody is following (a ') or (b '): (a ') protein that sequence 3 forms from N-terminal 1-135 amino acids residue in sequence table; (b ') will (a ') through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity;
Described IBDV-VP2 scFv30 antibody chain variable region is following (c ') or (d '): (c ') protein that sequence 3 forms from N-terminal 151-255 amino acids residue in sequence table; (d ') will (c ') through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity.
Described single-chain antibody IBDV-VP2 scFv30 specifically can be as follows (e ') or (f '): (e ') protein shown in sequence 3 in sequence table; (f ') will (e ') through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity.
The gene of described single-chain antibody IBDV-VP2 scFv30 of encoding also belongs to protection scope of the present invention.
In described single-chain antibody IBDV-VP2 scFv30 gene, the DNA molecular of the described variable region of heavy chain of encoding is following (1 ') or (2 ') or (3 '): the sequence 4 of (1 ') sequence table is from the DNA molecular shown in 5 ' end 1-405 position Nucleotide; The DNA molecular that the DNA sequence dna hybridization that (2 ') limits with (1 ') under stringent condition and coding have the albumen of identical activity; The DNA sequence dna that (3 ') and (1 ') limit at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
In described single-chain antibody IBDV-VP2 scFv30 gene, the DNA molecular of the described variable region of light chain of encoding is following (4 ') or (5 ') or (6 '): the sequence 4 of (4 ') sequence table is from the DNA molecular shown in 5 ' end 451-768 position Nucleotide; The DNA molecular that the DNA sequence dna hybridization that (5 ') limits with (4 ') under stringent condition and coding have the albumen of identical activity; The DNA sequence dna that (6 ') and (4 ') limit at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
Described single-chain antibody IBDV-VP2 scFv30 gene specifically can be following (7 ') or (8 ') or (9 ') or (10 '): the sequence 4 of (7 ') sequence table is from the DNA molecular shown in 5 ' end 1-768 position Nucleotide; DNA molecular shown in the sequence 2 of (8 ') sequence table; The DNA molecular that the DNA sequence dna hybridization that (9 ') limits with (7 ') or (8 ') under stringent condition and coding have the albumen of identical activity; The DNA sequence dna that (10 ') and (7 ') or (8 ') limit at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5% SDS, 65
ohybridize under C, then use 2 * SSC, 0.1% SDS and 1 * SSC, 0.1% SDS respectively to wash film once.
The expression cassette that contains above arbitrary described gene, recombinant vectors, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.
The antibody of other form based on described two kinds of single-chain antibodies also belongs to protection scope of the present invention.The antibody of described other form can be the antibody of Fab form, the antibody of IgG form etc.
The present invention also protects the application of antibody in preparing product of described single-chain antibody or described other form; The function of described product is following (I), (II) or (III) or (IV): (I) detects chicken infectivity bursa of Fabricius virus; (II) assistant identification chicken infectivity bursa of Fabricius virus; (III) prevents and/or treats infectious bursal disease; (IV) prevents and/or treats the Other diseases brought out by chicken infectivity bursa of Fabricius virus.
The product of the antibody that contains described single-chain antibody or described other form also belongs to protection scope of the present invention; The function of described product is following (I), (II) or (III) or (IV): (I) detects infectious bursal disease virus; (II) assistant identification infectious bursal disease virus; (III) prevents and/or treats the disease that infectious bursal disease virus causes; (IV) prevents and/or treats infectious bursal disease.
The present invention also protects the application of antibody in the assistant identification infectious bursal disease virus of described single-chain antibody or described other form.Describedly be applied as non-methods for the diagnosis of diseases.
The invention provides two kinds of single-chain antibodies (being scFv antibody); scFv antibody has and IBDV structural protein 2(VP2) albumen and the multiple IBDV strain specific ability of being combined; IBDV capable of blocking produces cytopathy (CPE) to chick embryo fibroblast, the young chicken that can protect IBDV to infect.Immune serum and yolk antibody in use exist that preparation is loaded down with trivial details, production cost is high, effect is unstable controls and causes the drawback such as horizontal transmission disease with, suitability for industrialized production difficult quality.ScFv antibody provided by the invention can overcome above-mentioned drawback, there is high specificity, result for the treatment of is good, suitability for industrialized production is quality controllable, the advantages such as horizontal transmission disease of having avoided yolk antibody to cause, will be on the history anti-processed of IBDV, so preventing and treating on history of whole animal virosis opened up a new situation.
The accompanying drawing explanation
The SDS-PAGE collection of illustrative plates that Fig. 1 is IBDV-VP2 scFv29 and IBDV-VP2 scFv30 antibody-solutions.
The SEC-HPLC collection of illustrative plates that Fig. 2 is two kinds of scFv antibody-solutions.
Fig. 3 is that ELISA detects two kinds of scFv antibody to the specificity of VP2 albumen and the result of avidity.
Fig. 4 is that ELISA detects specificity and the avidity result of two kinds of scFv antibody to different I BDV virus.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop.Quantitative test in following examples, all arrange repeated experiments three times, results averaged.
PET-27b (+) carrier: purchased from Novagen company, Cat. No.69337-3.Intestinal bacteria Rosetta: purchased from Novagen company, Cat. No.71403-4.DF1 cell (chick fibroblast): purchased from Shanghai Inst. of Life Science, CAS cell resource center, Cat. No.3131C0001000400030.SPF chick: purchased from the Harbin veterinary institute.Newcastle disease inactivated vaccine (La Sota strain), buy from breathing out beast and grind Wei Ke biotech firm, numbers 080012008.
Infectious bursal disease live-vaccine (Gt strain): breathe out beast and grind Wei Ke biotech firm, number 080012122.Infectious bursal disease mesogenic living vaccine (NF8 strain): Yangzhou Brunswick biotechnology company limited, number 101042056.Infectious bursal disease live-vaccine (1-65 strain): Shafit InterGumboro, numbering S20651010A.Infectious bursal disease live-vaccine (BJ836 strain): sea, Shanghai sharp biologics company limited, number 090202026.Infectious bursal disease live-vaccine (MB strain): ABIC, numbering 20621150B.Infectious bursal disease live-vaccine (B87 strain): Hunan Province Zhong An Bioceuticals Inc., number 180022026.
Plasmid pHisSUMO: reference: Jiang Yuanyuan, Yin Chengkai, Li Jinnan, Ren Guiping, Zhang Wei, Li Deshan. utilize the research of the high efficient expression soluble recombinant protein of SUMO emerging system. Northeast Agricultural University's journal, 2008,39 (10): 57-62; Li Lu, Yin Chengkai, Li Deshan. high efficient expression soluble recombinant protein expression vector---pHisSUMO. biotechnology, 2009,19 (3): 11-14..
Coating buffer (pH9.6): get Na
2cO
30.15g, NaHCO
30.293g water-soluble and water is settled to 100mL.
PBS damping fluid: get NaCl 8g, KCl 0.2g, Na
2hPO
4.12H
2o 3.58g, KH
2pO
40.24g, be dissolved in 1L water.
The discovery of embodiment 1:scFv antibody (single-chain antibody) and encoding gene thereof
1, the structure of antibody library
Get the chicken spleen after the IBDV immunity, extract RNA after reverse transcription become cDNA.According to the antibody sequence on GenBank, design the gene primer of clonal antibody variable region, take cDNA as template, use the method clonal antibody variable region of PCR.VH and VL fragment are inserted into respectively to the upstream and downstream of the Linker of pTlinker carrier, construct the scFv antibody library.The scFv antibody library carried out to enzyme is cut and be connected with bacterium display carrier pBSD, building the bacterium surface displaying library of anti-IBDV antibody.The about 380bp of VH, the about 320bp of VL, the about 740bp of VH-Tlinker-VL;
2, the screening of antibody library
Collect and transform rear all clones, through IPTG(0.25mmol/ml) induce 4h, through EDTA-MgCl
2process, with the VP2 albumen of FITC mark, hatch 1h, after the PBS washing, utilize flow cytometer to be screened it;
After the three-wheel screening, obtain two single-chain antibodies that there is binding ability with the VP2 albumen of FITC mark, by its called after IBDV-VP2 scFv29 antibody and IBDV-VP2 scFv30 antibody;
IBDV-VP2 scFv29 antibody (be single-chain antibody, claim again scFv albumen) is as shown in the sequence 1 of sequence table, and its encoding gene is as shown in the sequence 2 of sequence table;
IBDV-VP2 scFv30 antibody (be single-chain antibody, claim again scFv albumen) is as shown in the sequence 3 of sequence table, and its encoding gene is as shown in the sequence 4 of sequence table.
The preparation of embodiment 2, scFv antibody
1, the double chain DNA molecule shown in the sequence 2 of composition sequence table;
2, take the synthetic double chain DNA molecule of step 1 is template, and the primer formed with F1 and R1 be take streaming screening plasmid that positive bacteria is extracted and carried out pcr amplification as template, obtains pcr amplification product;
F1：5'–CATG
CCATGGGCCGTGACGTTGGACGAG-3'；
R1：5'?–CCC
AAGCTTTTAACCTAGGACGGTCAGGG-3；
3, use restriction enzyme
ncoi and
hindthe pcr amplification product of III double digestion step 2, reclaim enzyme and cut product;
4, use restriction enzyme
ncoi and
hindiII double digestion pET-27b (+) carrier, reclaim the carrier framework of about 5367bp;
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid.According to sequencing result, recombinant plasmid is carried out to structrual description as follows: at pET-27b (+) carrier
ncoi and
hindinserted the double chain DNA molecule shown in the sequence 2 of sequence table between the III restriction enzyme site;
6, recombinant plasmid step 5 obtained imports intestinal bacteria Rosetta, obtains recombinant bacterium;
7, recombinant bacterium step 6 obtained is seeded to the LB liquid nutrient medium containing 50 μ g/ml kantlex, and 37 ℃, 100r/min shaking culture are to OD
600nm=0.35; Adding IPTG and making its concentration is 0.25mmol/L, 37 ℃, 100r/min shaking culture 4h;
8, get the culture system of 25L completing steps 7,4 ℃, the centrifugal 30min of 4000r/min are also collected bacterial sediment;
9, get the bacterial sediment that step 8 obtains, resuspended with the PBS damping fluid, adding lysozyme soln (purchased from Amresco) and making the concentration of N,O-Diacetylmuramidase is 1mg/ml, place 1h for 4 ℃, then carry out ultrasonication (power of 25 watts, 3min), 4 ℃, the centrifugal 30min of 10000g, collecting precipitation;
10, use AKTA Purifier 100 protein chromatography systems (purchased from GE company) purifying target protein
Get the precipitation that step 9 obtains, dissolve damping fluid (8mol/L aqueous solution of urea with 100 milliliters, pH8.0) fully dissolve, then be splined on HiLoad 16/60 Superdex75 pg pillar (purchased from GE company), then use 500 milliliters of renaturation buffer (2mol/L aqueous solution of urea, pH8.0) wash-out and collected post after elutriant, then dialysed overnight in the PBS damping fluid, obtain solution and be the scFv antibody-solutions.Institute is in steps all under 4 ℃ of environment.Fig. 1 is shown in by the SDS-PAGE collection of illustrative plates of scFv antibody-solutions, shows the band that is about 28KD, with expection, conforms to;
11, get the scFv antibody-solutions that step 10 obtains, carry out the SEC-HPLC analysis
Silica gel filler, model is G-3000swxl; By after scFv antibody-solutions loading, (pH8.0, solvent is water to the elutriant that is 0.5ml/min with flow velocity, containing 50mM Na
3pO
4with 150mM NaCl) carry out wash-out.Fig. 2 is shown in by the SEC-HPLC collection of illustrative plates, the purity 90% of target protein.
The preparation of embodiment 3, VP2 albumen
One, the structure of recombinant plasmid
1, the IBDV vp2 double chain DNA molecule shown in the sequence 6 of composition sequence table;
2, take the synthetic double chain DNA molecule of step is template, and the primer pair vp2 formed with F2 and R2 carries out pcr amplification, obtains pcr amplification product;
F2：5’-
GAAGACTTAGGT?ACAAACCTGCAAGATCAA-3’；
R2：5’-
GGATCCTTATGCTCCTGCAATCTTCAG-3’；
3, use restriction enzyme
bbsi and
bamHthe pcr amplification product of I double digestion step 3, reclaim enzyme and cut product;
4, use restriction enzyme
bbsi and
bamHi double digestion plasmid pHisSUMO, reclaim the carrier framework of about 5700bp;
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid.In recombinant plasmid, the encoding sequence of the encoding sequence of the encoding gene of VP2 albumen and the molecular chaperones SUMO on carrier framework and the His label on carrier framework (is positioned at the upstream of the encoding sequence of SUMO, by 6 histidine residues, formed) merge, form fusion gene, expressed fusion protein (fusion rotein is held to C and held and be followed successively by His label, molecular chaperones SUMO and VP2 albumen from N).
Two, the preparation of VP2 albumen and purifying
1, recombinant plasmid step 1 obtained imports intestinal bacteria Rosetta, obtains recombinant bacterium;
2, recombinant bacterium step 1 obtained is seeded to the LB liquid nutrient medium containing 100 μ g/ml penbritins, and 37 ℃, 100r/min shaking culture are to OD
600nm=0.35; Adding IPTG and making its concentration is 0.4mmol/L, 25 ℃, 65r/min shaking culture 10h;
3, take into the culture system of step 2,4 ℃, the centrifugal 30min of 4000r/min, and collect bacterial sediment;
4, get the bacterial sediment that step 3 obtains, resuspended with Binding buffer, adding lysozyme soln (purchased from Amresco) and making the concentration of N,O-Diacetylmuramidase is 1mg/ml, place 1h for 4 ℃, then carry out ultrasonication (power of 25 watts, 3min), 4 ℃, the centrifugal 30min of 10000g, collect supernatant liquor;
5, get the supernatant liquor that step 4 obtains, carry out HisTrapTM FF crude colum affinity chromatography;
The pillar model is: column length 0.7cm, the high 2.5cm of post;
Applied sample amount is 10ml;
Elution process: (solvent is water, each solute that contains following concentration: 40mmol/L imidazoles, 500mmol/L NaCl and 50mmol/L Na first to use the foreign protein elutriant of 5 times of column volumes
3pO
4; PH7.4) wash-out is to remove foreign protein, and flow velocity is 1ml/min; Then (solvent is water, each solute that contains following concentration: 500mmol/L imidazoles, 500mmol/L NaCl and 50mmol/L Na to use the target protein elutriant of 3 times of column volumes
3pO
4; PH7.4) wash-out, flow velocity is 1ml/min, the 280nm wavelength monitoring is collected target peak (being the peak of peak value higher than 80mAU), is fusion rotein solution;
6, the fusion rotein solution that adopts HiPrepTM 26/10 Desalting that step 5 is obtained carries out desalination;
7, getting the solution that step 6 obtains, is 1:50 by the mol ratio of SUMO proteolytic enzyme I(SUMO proteolytic enzyme I and fusion rotein) and final concentration be 2mmol/L 4 ℃ of cuttings of DTT are spent the night;
8, get the solution that step 7 obtains, carry out HisTrapTM FF crude colum affinity chromatography.
The pillar model is: column length 0.7cm, the high 2.5cm of post;
Applied sample amount is 15ml, and the 280nm wavelength monitoring is collected target peak (being the peak of peak value higher than 30mAU), is the VP2 protein solution.The VP2 protein solution is carried out to the polyacrylate hydrogel electrophoresis, shows that molecular weight is about the single protein band of 42KDa, reclaim protein band order-checking, N hold front 15 amino-acid residues as the sequence 5 of sequence table from as shown in N-terminal the 1st to 15 amino acids residues.
One, ELISA detection IBDV-VP2 scFv29 and IBDV-VP2 scFv30 antibody specificity and the avidity to VP2 albumen;
1, the scFv antibody-solutions that is 300 μ g/ml, 60 μ g/ml, 12 μ g/ml, 2.4 μ g/ml with protein concentration respectively (is the scFv antibody-solutions of embodiment 2 preparations, adjust protein concentration with coating buffer) coated elisa plate, 4 ℃ are spent the night, then with PBST damping fluid washing 3 times, and each 2min;
2, every hole adds the VP2 protein solution that 100 μ l protein concentrations are 40 μ g/ml (be the VP2 protein solution of embodiment 3 preparations, with the PBS damping fluid, adjust protein concentration), hatches 1h for 37 ℃, then with PBST damping fluid washing 3 times, and each 2min;
3, add yolk antibody (B87 strain Immune Laying Hens obtains, and working concentration is that 1:200 doubly dilutes), hatch 1h for 37 ℃, then with PBST damping fluid washing 3 times, each 2min;
4, add the rabbit anti-chicken antibody (Cat.No.11-7018 is purchased from eBioscience company, and working concentration is that 1:7500 doubly dilutes) of HRP mark, hatch 1h for 37 ℃, then with PBST damping fluid washing 3 times, each 2min;
5, add the tmb substrate nitrite ion, 37 ℃ of lucifuge colour developing 5min;
6, every hole adds the H of 50 μ l2mol/L
2sO
4the aqueous solution detects the OD value by microplate reader under wavelength 450nm;
Arrange with isopyknic PBS damping fluid and replace scFv antibody-solutions in step 1, VP2 protein solution in step 2 and the PBS group of the yolk antibody in step 3.During the scFv antibody-solutions coated elisa plate that is 300 μ g/ml with protein concentration in step 1: the control group 1 that does not add the VP2 protein solution in setting steps 2, the control group 2 that does not add yolk antibody in step 3, do not add the control group 3 that does not add yolk antibody in VP2 protein solution and step 3 in step 2, with equal-volume and wait the control group 4 of the BSA solution replacement VP2 protein solution of protein concentration;
Each processing arranges 3 multiple holes;
The results are shown in Figure 3.The ELISA result shows, IBDV-VP2 scFv29 and IBDV-VP2 scFv30 antibody can be combined with the VP2 protein-specific.
Two, ELISA detection sIBDV-VP2 scFv29 and IBDV-VP2 scFv30 antibody specificity and the avidity to different I BDV virus
1, the scFv antibody-solutions that is 300 μ g/ml with protein concentration (be the scFv antibody-solutions of embodiment 2 preparations, with coating buffer, adjust protein concentration) coated elisa plate, 4 ℃ are spent the night, then with PBST damping fluid washing 3 times, each 2min;
2, every hole adds 100 μ l IBDV virus liquids (virus titer is 100TCID
50, TCID
50=10
-6.8/ 0.1ml), hatch 1h for 37 ℃, then with PBST damping fluid washing 3 times, each 2min;
3, add yolk antibody (B87 strain Immune Laying Hens obtains, and working concentration is that 1:200 doubly dilutes), hatch 1h for 37 ℃, then with PBST damping fluid washing 3 times, each 2min;
4, add the rabbit anti-chicken antibody (Cat.No.11-7018 is purchased from eBioscience company, and working concentration is that 1:7500 doubly dilutes) of HRP mark, hatch 1h for 37 ℃, then with PBST damping fluid washing 3 times, each 2min;
5, add the tmb substrate nitrite ion, 37 ℃ of lucifuge colour developing 5min;
6, every hole adds the H of 50 μ l 2mol/L
2sO
4the aqueous solution detects the OD value by microplate reader under wavelength 450nm;
Adopt respectively the strain of following IBDV to carry out above-mentioned experiment: Gt strain, NF8 strain, 1-65 strain, BJ836 strain, MB strain, B87 strain;
Arrange with isopyknic PBS damping fluid and replace scFv antibody-solutions in step 1, IBDV virus liquid in step 2 and the PBS group of the yolk antibody in step 3.The control group 1 that does not add the IBDV virus liquid in setting steps 2, the control group 2 that does not add yolk antibody in step 3, do not add the control group 3 that does not add yolk antibody in IBDV virus liquid and step 3 in step 2, replace the control group 4 of IBDV virus liquid with the Newcastle Disease venom of the titres such as equal-volume;
Each processing arranges 3 multiple holes;
The results are shown in Figure 4.The ELISA result shows, IBDV-VP2 scFv29 and IBDV-VP2 scFv30 antibody can be combined with different I BDV strain specific, and different IBDV strains is had to different avidity.
The neutralization activity of embodiment 5, IBDV-VP2 scFv29 and IBDV-VP2 scFv30 antibody
One, the mensuration of IBDV titre
To be inoculated in 96 porocyte culture plates in the DF1 of logarithmic phase cell, will be with DMEM substratum 10
1to 10
11the IBDV virus liquid of gradient dilution (B87 strain) is inoculated into (every hole 100 μ l) in monolayer cell, 8 cell holes of each extent of dilution inoculation; The control group that does not add the IBDV virus liquid is set.Tissue Culture Plate is put in cell culture incubator to 37 ℃, 5% CO
2cultivate, Continuous Observation 7 days, record cell growth state every day.Calculate virus titer, within the 7th day, the results are shown in Table 1.
The result of the mensuration of table 1 IBDV titre
Calculate TCID according to Reed-Muench Liang Shi method
50=10
-6.8/ 0.1ml.
Two, the neutralization activity of scFv antibody
To be inoculated in 96 porocyte culture plates in the DF1 of logarithmic phase cell, will be with the scFv antibody-solutions after DMEM substratum gradient dilution (being the scFv antibody-solutions of embodiment 2 preparation) and 100TCID
50iBDV virus liquid (B87 strain) equal-volume mix and 37 ℃ hatch 1h, then be inoculated in monolayer cell 8 cell holes of each gradient inoculation; The control group that does not add the control group of virus liquid and do not add the scFv antibody-solutions is set.Tissue Culture Plate is put in thin incubator, 37 ℃, 5% CO
2cultivate Continuous Observation 7 days, record cell growth state every day.Within the 7th day, the results are shown in Table 2a and 2b.
The measurement result of table 2a IBDV-VP2 scFv29 antibody neutralization
Result shows, it is active that IBDV-VP2 scFv29 antibody has neutralization, and blocking-up or the minimum protein concentration that suppresses CPE are 9.375 μ g/ml.
The measurement result of table 2b IBDV-VP2 scFv30 antibody neutralization
Result shows, it is active that IBDV-VP2 scFv29 antibody has neutralization, and blocking-up or the minimum protein concentration that suppresses CPE are 4.688 μ g/ml.
Infectious bursal disease virus HB/11 strain: reference: yellow obvious, Zhang little Fei, Ding Meijuan, Yin Xiufeng, Xu Xiumei, Wang Wei, Jin Yutian, separation and the evaluation of infectious bursal disease virus virulent strain HB/11, " Chinese zoonosis journal " the 5th phase in 2012, the 8-11 page.
One, security detects
Get 10 13 age in days SPF chick, the scFv antibody-solutions that chest muscle injection protein concentration is 2mg/ml respectively (be the scFv antibody-solutions of embodiment 2 preparations, with the PBS damping fluid, adjust protein concentration), every 1ml, observe 14 days.Without injection site and systemic adverse reactions.
Two, the mensuration of IBDV semilethal rate
Get the chicken embryo of 9 ages in days, be divided into 9 groups, 10 every group, the 1st group to the 8th group, injecting respectively 200 μ L is 10 by the dilution gradient after the dilution of PBS damping fluid
1-10
8infectious bursal disease virus HB/11 strain virus liquid, the 9th group of control group (every embryo is injected 200 μ l physiological saline) that is physiological saline.Observe 3-6 days, record chicken embryo survival condition;
Semilethal rate after 6 days is 10
5.5eID
50.
Three, antibody potency test
13 age in days SPF chickens are divided into five groups, 10 every group;
First group: every chest muscle injection 0.2ml PBS damping fluid;
Second group: (the viral dosage that every 0.2ml contains is 10 to inoculation B87 strain virus liquid
4.0eID
50), every chest muscle injection 0.2ml;
The 3rd group: every chest muscle injection scFv antibody-solutions (1mg albumen/kg body weight), every injection 0.2ml;
The 4th group: every chest muscle injection scFv antibody-solutions (0.5mg albumen/kg body weight), every injection 0.2ml;
The 5th group: every chest muscle injection scFv antibody-solutions (0.1mg albumen/kg body weight), every injection 0.2ml.
(every chest muscle injection 0.2ml, the viral dosage contained is 10 to detect NAT inoculative infection bursa of Fabricius virus HB/11 strain virus liquid after immune 21 days
5.5eID
50) attacked poison;
The measuring method of NAT (microtitrimetry): (1) gets serum, 56 ℃ of water-bath deactivation 30min, naturally cooling, use Hank ' s serum to make 2 times of serial dilutions (from 1:2, being diluted to 1:4096), and every kind of diluent adds equal-volume viral suspension (100TCID
50/ ml) fully concussion mixes, and cultivates 1h for 37 ℃; (2) chick fibroblast is inoculated in to Tissue Culture Plate, every hole 100 microlitres, the mixed solution that every hole adds 100 microlitre steps (1) to obtain; The control group that does not add the control group of viral suspension and do not add serum is set; 37 ℃, 5% CO
2cultivate, Continuous Observation 3-5 days, if antibody has neutralization active, the DF1 cell not there will be pathology, calculates the antibody titer (greatest dilution that can suppress DF1 cell generation pathology) of the 7th day.The results are shown in Table 3a and 3b;
After attacking malicious 72h, add up the number of elements of dead chicken in every group.The results are shown in Table 3a and 3b;
The dead chicken number of table 3a IBDV-VP2 scFv29 antibody titer result and every group
The dead chicken number of table 3b IBDV-VP2 scFv30 antibody titer result and every group
sequence table
AVTLDEPGGGLQTPGGALSLVCKASGFTFSDRGMGWMRQAPGRGLEWVASINGAGSYTYYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYFCAKSAYGTTTWSGCIAKDVDTWGHGTEVIVSSAS
GGGGSGGGGSGGGGSALTQPSSVSANLGGTVEITCSGSSGSYGWYQQKSPGSAPVTLIYYNNKRPSDVPSRFSGSRSGSTATLTITGVQAEDEAVYFCGGYDSSAAYVGIFGAGTTLTVLG
The part of double underline is linker
Sequence 2:IBDV-VP2 scFv29 antibody VH-linker-VL gene nucleotide series
GCCGTGACGTTGGACGAGCCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGCCTCGTCTGCAAGGCCTCCGGGTTCACCTTCAGTGACCGTGGCATGGGATGGATGCGCCAGGCGCCTGGCAGGGGGCTGGAGTGGGTCGCGAGTATTAATGGTGCTGGTAGTTACACATACTACGCGCCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACAGTGAGGCTGCAGCTGAACAACCTCAGGGCTGAGGACACCGGCACCTACTTCTGCGCCAAAAGTGCTTATGGTACTACTACTTGGAGTGGTTGTATTGCTAAAGATGTCGACACATGGGGCCACGGGACCGAAGTCATCGTCTCCTCCGCTAGCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCCGGTGGTGGTGGATCCGCGCTGACTCAGCCGTCCTCGGTGTCAGCAAACCTGGGAGGAACCGTCGAGATCACCTGCTCCGGGAGTAGTGGCAGCTATGGCTGGTATCAGCAGAAGTCTCCTGGCAGTGCCCCTGTCACTCTGATCTATTACAACAACAAGAGACCCTCGGACGTCCCTTCACGATTCTCCGGTTCCAGATCCGGCTCCACAGCCACATTAACCATCACTGGGGTCCAAGCCGAGGACGAGGCTGTCTATTTCTGTGGTGGCTACGACAGCAGTGCTGCTTATGTTGGTATATTTGGGGCCGGGACAACCCTGACCGTCCTAGGTTAA
AVTLDEPGGGLQTPGGALSLVCKASGFTFSSYAMHWVRQAPGKGLEWVAGIYSSGSSTYYAPAVKGRATITRDNGQSTVRLQLNNLRAEDTGTYYCAKAGSGYCGWSAYGYGCAAYPGNIDAWGHGTEVIVSSAS
GGGGSGGGGSGGGGSALTQPSPVSANPGETVKITCSGGYSNYYGWYQQKSPASAPVTVIYDNNKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSRDSNYIGIFGAGTTLTVLG
The part of double underline is linker
Sequence 4:IBDV-VP2 scFv30 antibody VH-linker-VL gene nucleotide series
GCCGTGACGTTGGACGAGCCCGGGGGCGGCCTCCAGACGCCCGGAGGAGCGCTCAGTCTCGTCTGCAAGGCCTCCGGGTTCACCTTCAGCAGTTACGCCATGCACTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAGTGGGTCGCGGGTATTTACAGCAGTGGTAGTAGCACATACTACGCGCCGGCGGTGAAGGGCCGTGCCACCATCACGAGGGACAACGGGCAGAGCACAGTGAGGCTGCAGCTGAACAACCTCAGGGCTGAGGACACCGGCACCTACTACTGCGCCAAAGCTGGTAGTGGTTACTGTGGTTGGAGTGCTTACGGTTACGGTTGTGCTGCTTATCCTGGTAACATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCCGCTAGCGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGATCCGCGCTGACTCAGCCGTCCCCGGTGTCAGCAAACCCAGGAGAAACCGTCAAGATCACCTGCTCCGGGGGTTACAGCAACTACTATGGCTGGTATCAGCAGAAGTCTCCTGCCAGTGCCCCTGTCACTGTGATCTATGACAACAACAAGAGACCCTCGGACATCCCTTCTCGATTCTCCGGTTCCAAATCCGGCTCCACGGGCACATTAACCATCACTGGGGTCCAAGCCGAGGACGAGGCCGTCTATTACTGTGGGAGCAGGGACAGCAACTATATTGGTATATTTGGGGCCGGGACAACCCTGACCGTCCTAGGTTAA
TNLQDQTQQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSIVGAHYTLQSNGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRSSTLPGGVYALNGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAIGLDPKMVATCDSSDRPRVYTITAANDYQFSSQYQAGGVTITLFSANIDAITSLSIGGELVFQTSVQGLILGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNIVIPTSEITQPITSIKLEIVTSKSGGQAGDQMSWSASGSLAVTIHGGNYPGALRPVTLVAYERVATGSVVTVAGVSNFELIPNPELAKNLITEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGA
ATGACAAACCTGCAAGATCAAACCCAACAGATTGTTCCGTTCATACGGAGCCTTCTGATGCCAACAACCGGACCGGCGTCCATTCCGGACGACACCCTAGAGAAGCACACTCTCAGGTCAGAGACCTCGACCTACAATTTGACTGTGGGGGACACAGGGTCAGGGCTAATTGTCTTTTTCCCTGGTTTCCCTGGCTCAATTGTGGGTGCTCACTACACACTGCAGAGCAATGGGAACTACAAGTTCGATCAGATGCTCCTGACTGCCCAGAACCTACCGGCCAGCTACAACTACTGCAGGCTAGTGAGTCGGAGTCTCACAGTGAGGTCAAGCACACTCCCTGGTGGCGTTTATGCATTAAACGGAACCATAAACGCCGTGACCTTCCAAGGAAGCCTGAGTGAACTGACAGATGTTAGCTACAATGGGTTGATGTCTGCGACGGCCAACATCAACGACAAAATCGGGAACGTCCTAGTAGGGGAAGGGGTAACTGTCCTCAGCTTACCCACATCATATGATCTGGGGTATGTGAGACTCGGTGACCCCATTCCCGCTATAGGGCTTGACCCAAAGATGGTAGCAACATGTGACAGCAGTGATAGGCCCAGAGTCTACACCATAACTGCAGCCAATGACTACCAATTCTCATCACAGTACCAAGCAGGTGGAGTGACAATCACACTGTTCTCAGCAAACATCGATGCCATCACAAGCCTCAGCATCGGGGGAGAACTTGTGTTTCAAACAAGCGTCCAAGGCCTTATACTGGGCGCTACCATCTACCTTATAGGCTTCGATGGGACTGCGGTAATCACCAGAGCTGTGGCCGCAGACAATGGGCTAACGGCCGGCACTGACAACCTTATGCCATTCAATATTGTGATTCCAACCAGCGAGATAACCCAGCCAATCACATCCATCAAACTGGAGATAGTTACCTCCAAAAGTGGTGGTCAGGCGGGGGATCAGATGTCATGGTCAGCAAGTGGGAGCCTAGCAGTGACGATCCACGGTGGCAACTATCCAGGGGCCCTCCGTCCCGTCACACTAGTAGCCTACGAAAGAGTGGCTACAGGATCTGTCGTTACGGTCGCCGGGGTGAGCAACTTCGAGCTGATCCCAAATCCTGAACTAGCAAAGAACCTGATCACAGAATACGGCCGATTTGACCCAGGGGCCATGAACTACACAAAACTGATACTGAGTGAGAGGGACCGTCTTGGCATCAAGACCGTGTGGCCAACAAGGGAGTACACCGACTTTCGCGAGTACTTTATGGAGGTGGCCGACCTCAACTCTCCCCTGAAGATTGCAGGAGCA
Claims (10)
1. two kinds of single-chain antibodies, comprise by variable region of heavy chain, variable region of light chain and the joining region between them and forming;
Described IBDV-VP2 scFv29 antibody heavy chain variable region is following (a) or (b): the protein that (a) sequence 1 forms from N-terminal 1-129 amino acids residue in sequence table; (b) by (a) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity;
Described IBDV-VP2 scFv29 antibody chain variable region is following (c) or (d): the protein that (c) sequence 1 forms from N-terminal 145-250 amino acids residue in sequence table; (d) by (c) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity;
Described IBDV-VP2 scFv30 antibody heavy chain variable region is following (a ') or (b '): (a ') protein that sequence 1 forms from N-terminal 1-135 amino acids residue in sequence table; (b ') will (a ') through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity;
Described IBDV-VP2 scFv30 antibody chain variable region is following (c ') or (d '): (c ') protein that sequence 1 forms from N-terminal 151-255 amino acids residue in sequence table; (d ') will (c ') through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity.
2. two kinds of single-chain antibodies as claimed in claim 1 is characterized in that:
Described IBDV-VP2 scFv29 single-chain antibody is following (e) or (f): (e) protein shown in sequence 1 in sequence table; (f) by (e) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity;
Described IBDV-VP2 scFv30 single-chain antibody is following (e ') or (f '): (e ') protein shown in sequence 3 in sequence table; (f ') will (e ') through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the derivative protein by it with identical activity.
3. the gene of coding claim 1 or 2 described single-chain antibody.
4. gene as claimed in claim 3 is characterized in that:
In described IBDV-VP2 scFv29 single-chain antibody gene, the DNA molecular of the described variable region of heavy chain of encoding is following (1) or (2) or (3): the sequence 2 of (1) sequence table is from the DNA molecular shown in 5 ' end 1-387 position Nucleotide; (2) DNA molecular that the DNA sequence dna hybridization limited with (1) under stringent condition and coding have the albumen of identical activity; (3) DNA sequence dna limited with (1) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity;
In described IBDV-VP2 scFv29 single-chain antibody gene, the DNA molecular of the described variable region of light chain of encoding is following (4) or (5) or (6): the sequence 2 of (4) sequence table is from the DNA molecular shown in 5 ' end 433-753 position Nucleotide; (5) DNA molecular that the DNA sequence dna hybridization limited with (4) under stringent condition and coding have the albumen of identical activity; (6) DNA sequence dna limited with (4) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity;
In described IBDV-VP2 scFv30 single-chain antibody gene, the DNA molecular of the described variable region of heavy chain of encoding is following (1 ') or (2 ') or (3 '): the sequence 4 of (1 ') sequence table is from the DNA molecular shown in 5 ' end 1-405 position Nucleotide; The DNA molecular that the DNA sequence dna hybridization that (2 ') limits with (1 ') under stringent condition and coding have the albumen of identical activity; The DNA sequence dna that (3 ') and (1 ') limit at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity;
In described IBDV-VP2 scFv30 single-chain antibody gene, the DNA molecular of the described variable region of light chain of encoding is following (4) or (5) or (6): the sequence 2 of (4) sequence table is from the DNA molecular shown in 5 ' end 451-768 position Nucleotide; (5) DNA molecular that the DNA sequence dna hybridization limited with (4) under stringent condition and coding have the albumen of identical activity; (6) DNA sequence dna limited with (4) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
5. gene as claimed in claim 4 is characterized in that:
Described IBDV-VP2 scFv29 single-chain antibody gene is following (7) or (8) or (9) or (10): the sequence 2 of (7) sequence table is from the DNA molecular shown in 5 ' end 1-753 position Nucleotide; (8) DNA molecular shown in the sequence 2 of sequence table; (9) DNA molecular that the DNA sequence dna hybridization limited with (7) or (8) under stringent condition and coding have the albumen of identical activity; (10) DNA sequence dna limited with (7) or (8) at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity;
Described IBDV-VP2 scFv30 single-chain antibody gene is following (7 ') or (8 ') or (9 ') or (10 '): the sequence 4 of (7 ') sequence table is from the DNA molecular shown in 5 ' end 1-768 position Nucleotide; DNA molecular shown in the sequence 2 of (8 ') sequence table; The DNA molecular that the DNA sequence dna hybridization that (9 ') limits with (7 ') or (8 ') under stringent condition and coding have the albumen of identical activity; The DNA sequence dna that (10 ') and (7) or (8 ') limit at least has the DNA molecular that 90% above homology and coding have the albumen of identical activity.
6. the expression cassette, recombinant vectors, transgenic cell line or the recombinant bacterium that contain arbitrary described gene in claim 3 to 5.
7. the antibody of other form based on claim 1 or 2 described single-chain antibody.
8. the described single-chain antibody of claim 1, the described single-chain antibody of claim 2 or the application of the described antibody of claim 7 in preparing product; The function of described product is following (I), (II) or (III) or (IV): (I) detects infectious bursal disease virus; (II) assistant identification infectious bursal disease virus; (III) prevents and/or treats the disease that infectious bursal disease virus causes; (IV) prevents and/or treats infectious bursal disease.
9. the product that contains the described single-chain antibody of claim 1, the described single-chain antibody of claim 2 or the described antibody of claim 7; The function of described product is following (I), (II) or (III) or (IV): (I) detects infectious bursal disease virus; (II) assistant identification infectious bursal disease virus; (III) prevents and/or treats the disease that infectious bursal disease virus causes; (IV) prevents and/or treats infectious bursal disease.
10. the described single-chain antibody of claim 1, the described single-chain antibody of claim 2 or the application of the described antibody of claim 7 in the assistant identification infectious bursal disease virus.
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