CN103665152A - Canine parvovirus single-domain antibody, and preparation method and application thereof - Google Patents
Canine parvovirus single-domain antibody, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a canine parvovirus single-domain antibody, and a preparation method and an application thereof. The canine parvovirus single-domain antibody has a nucleotide sequence as shown in SEQIDNO.1. The preparation method comprises the steps: immunizing an alpaca by a canine parvovirus inactivated vaccine, and taking peripheral blood to obtain alpaca anti-canine parvovirus serum; extracting RNA in the peripheral blood, and carrying out reverse transcription to generate cDNA; utilizing specific primers for amplification, and purifying a VHH fragment; connecting the VHH fragment with a phagemid vector, transforming TG1, and constructing a canine parvovirus single-domain antibody library; and enriching the antibody library by utilizing a canine parvovirus structural protein VP2, introducing into an expression bacterial strain E.coil HB2151, and utilizing IPTG to induce the canine parvovirus single-domain antibody VHH having the biological activity to be expressed. The prepared canine parvovirus single-domain antibody has the detection activity of 15 [mu]g/ml and the detection sensitivity of 250 ng/ml, and can be used as drugs applied for treating dogs with canine parvovirus disease.
Description
Technical field
The invention belongs to clinical animal doctor and antibody engineering technical field, relate to a kind of antiviral antibody, the heavy chain antibody that particularly relates to a kind of main support alpaca is realized the canine parvovirus single domain antibody of its biological expression and the preparation method and application of this antibody.
Background technology
Canine parvovirus disease is by canine parvovirus (Canine parvovirus, CPV) cause, the sickness rate of take is high, mortality ratio is high, infectivity is by force feature, all can occur throughout the year, the mortality ratio of hemorrhagic enteritis type and myocarditis type canine parvovirus disease is respectively 10%～50%, 60%～100%, can cause serious financial loss.The vaccine of preventing canine parvovirus mainly contains: deactivation vaccine, weak malicious seedling and multi-joint seedling, the immune programme for children of optimization can be obtained good immune effect.But the immune effect of vaccine on market is detected to investigation, find, only 20% vaccine can produce the antibody of 100% protection.Data shows, China's pet dog quantity reaches 1.5 hundred million, along with the quantity of pet dog increases, and the diagnosis of research canine parvovirus disease and prevent and treat significant.
The diagnostic kit of being combined with antibodies specific based on antigen, can detectable antigens, also detectable antibody, susceptibility is high, high specificity, good stability, easy and simple to handle fast, result is easy to analyze and judges.Specific antibody is most important for the production of diagnostic kit, and conventional antibody has monoclonal antibody, polyclonal antibody at present.Aspect treatment, apply in early days canine parvovirus monoclonal antibody and canine parvovirus antiserum(antisera) etc. and carry out specific treatment, can obtain good effect.But monoclonal antibody large-scale production length consuming time, antiserum(antisera) large-scale production can not guarantee the market requirement and also quality unstable, and monoclonal antibody and sero-fast immunogenicity strong, in therapeutic process, all there is side effect.Therefore the antibody that, research relative specific is strong, avidity is high, immunogenicity is low, be produced on a large scale is significant.
Common IgG antibody (immunoglobulin G) is most important antibody in serum, accounts for 75%～80% of Immunoglobulin in Serum total amount, plays " main force's immunity " effect in humoral immunization.IgG has 2 heavy chains (H chain) and 2 light chains (L chain), and whole antibody molecule is divided into constant region (C district) and variable region (V district), and molecular weight is 150～160kDa.Research shows, in alpaca serum except common IgG antibody, having nearly half IgG is heavy chain antibody, only there is variable region of heavy chain (VH), hinge area and two constant regions (CH2 and CH3), and being built, the variable region clone of heavy chain antibody only have the antibody of a variable region of heavy chain to be called single domain antibody (variable domain of heavy chain of heavy-chain antibody, VHH), molecular weight is only 15kD, there is molecular weight little, structure is single, penetrance is good, high specificity, avidity is high and solubility is high, stability is strong, easily clone, high expression level, the features such as easy purifying.Therefore, VHH can reduce the side effect in therapeutic process, can be used as biosensor diagnosis and uses target reagent, can be used for building Immune Fusion thing, carries out targeted therapy, can be used for commercialization scale operation.At present, antibody is only limiting to cell surface target aspect the treatment of disease, and whole antibody is because molecular structure is complicated, in cell inner expression difficulty, limited it in intracellular application, VHH immunogenicity is low, be difficult to the immune response of activated t cell, can be used as therapeutic antibodies and long-term prescription.In a word, VHH is as a kind of genetic engineering antibody of miniaturization, and unique physics and chemistry and biological characteristics make it in fields such as fundamental research, drug development and medical diagnosis on disease treatments, have broad application prospects.
Along with the further investigation to genetic expression, the antibody preparation that appears as of antibody library provides a kind of new means, utilize Antibody library can realize the mass-producing preparation of antibody, relevant display technique of bacteriophage is also a new technology utilizing phage expression foreign gene of setting up in recent years and developing, it is the technology that a kind of gene expression product and affine screening combine, its ultimate principle is that goal gene is connected with the gene of coding bacteriophage coat protein, be inserted on the expression vector of phage, thereby make polypeptide or protein and coat protein amalgamation and expression and be illustrated in the surface of phage, the polypeptide being demonstrated or protein can keep relatively independent space structure and biological activity, utilize phage to be easy to separated amplification in intestinal bacteria system, can be by selecting the phage particle of expressing corresponding acceptor with the combination of part, by the enrichment process of " absorption-wash-out-amplification ", specific antibody gene can be therefrom filtered out, and some biologic activity of shown polypeptide or protein can be directly measured.These technology are existing application in antibody engineering.
Summary of the invention
The object of the invention is the defect for specific treatment preparations such as existing canine parvovirus monoclonal antibody and canine parvovirus antiserum(antisera)s, provide that a kind of molecular weight is little, immunogenicity is low, high specificity, stability are strong, the easily canine parvovirus single domain antibody of clonal expression, and the preparation method and application of this canine parvovirus single domain antibody.
To achieve the above object of the invention, the present invention utilizes display technique of bacteriophage, from the peripheral blood of canine parvovirus immunity alpaca, extract RNA, build CPV-VHH canine parvovirus single domain antibody storehouse, and screening acquisition has bioactive single domain antibody from antibody library.
Canine parvovirus single domain antibody of the present invention has the nucleotide sequence shown in SEQ ID NO.1.
The above-mentioned canine parvovirus single domain antibody of the present invention adopts following steps to be prepared:
1), extract the RNA in alpaca peripheral blood after canine parvovirus immunity, reverse transcription generation cDNA; Special primer amplification purification canine parvovirus single domain antibody VHH fragment;
2), canine parvovirus single domain antibody VHH fragment is connected structure phage recombinant vectors, transformed competence colibacillus e. coli tg1, structure canine parvovirus phage single domain antibody storehouse with phagemid vector;
3), with canine parvovirus structural protein VP2, add in the canine parvovirus phage single domain antibody storehouse of activation the competitive wash-out of glycine solution, enrichment phagemid recombinant vectors importing competent cell
e.coilin HB2151 bacterial strain, IPTG induction produces has bioactive canine parvovirus single domain antibody VHH albumen.
More specifically, step 1) in, be with variable region of heavy chain special primer CALL01/ CALL02 and VHH special primer VHH For2/VHH Rev2, to carry out for the first time respectively and pcr amplification for the second time, reclaim purifying canine parvovirus single domain antibody VHH fragment.
Step 2) in, first with canine parvovirus single domain antibody VHH fragment, to be connected with plasmid vector pMD-18T simple to build plasmid expression vector pMD-18T-VHH, transform intestinal bacteria competence DH5 α, reclaim purifying VHH, build phage recombinant vectors with phagemid vector, transformed competence colibacillus e. coli tg1, builds canine parvovirus phage single domain antibody storehouse.
Wherein, described phagemid vector is pCANTAB 5E, and the phage recombinant vectors of structure is pCANTAB 5E-VHH.
The canine parvovirus single domain antibody that the present invention obtains can be used as medicine, is applied in the treatment of canine parvovirus disease dog.
The present invention is based on the heavy chain antibody of alpaca uniqueness; utilize Antibody library, phage display technique; with canine parvovirus immunity alpaca, set up canine parvovirus single domain antibody storehouse; and therefrom obtained and there is bioactive canine parvovirus single domain antibody, can realize the mass-producing preparation of antibody.
In alpaca serum, except common IgG antibody, having nearly half IgG is heavy chain antibody.With subcutaneous multiple spot immunization, two healthy adult alpacas are carried out to canine parvovirus immunity, 4 all after dates of immunity, antiserum titre is higher than 1:64.Test shows, with saturated ammonium sulphate salting-out process initial gross separation IgG from alpaca anti-dog parvovirus antiserum(antisera), by protein g affinity chromatography, can separation and purification go out IgG1 and IgG3, and molecular weight is respectively in 90kDa and 180kDa left and right; In conjunction with IgG, by protein A affinity chromatography, can not isolate IgG2, molecular weight is in 75kD left and right.Each hypotype of alpaca IgG SDS-PAGE electrophoretic band after purifying is single.
The present invention utilizes morphocytology and tetrazolium bromide dyeing, alpaca anti-dog parvovirus antiserum(antisera) prepared by the experimental observation the present invention impact on F81 Growth of Cells and proliferation inhibition rate, result shows, alpaca anti-dog parvovirus antiserum(antisera) group, the tiny hyper-immune serum group of dog Growth of Cells are vigorous, closely be connected, cell homogeneous and transparent; PBS organizes cell quantity minimizing, cell free, is the state of drawing in the net.Cell proliferation inhibition rate detects and shows, alpaca anti-dog parvovirus antiserum(antisera) group, the tiny hyper-immune serum group of dog, PBS group act on after F81 cell 96h with virus simultaneously, and cell proliferation inhibition rate is respectively 3.9%, 6.2%, and 80.8%.
The canine parvovirus single domain antibody of expressing based on alpaca heavy chain antibody prepared by the present invention has that molecular weight is little, structure is single, penetrance is good, high specificity, avidity high.The present invention utilizes five to take turns " absorption-wash-out-amplification " concentration method, and phage antibody library is screened to enrichment, makes phage antibody library enrichment increase nearly 4000 times, and positive rate reaches 92%.The detection of active that the ELISA of take detects canine parvovirus single domain antibody is 15 μ g/ml, and detection sensitivity is 250ng/ml.
Canine parvovirus single domain antibody preparation prepared by the present invention is compared with conventional treatment, can make mortality ratio reduce, and curative ratio improves, and can shorten treatment cycle, makes the expense of curing a canine parvovirus dog reduce by 10%～20%.
Embodiment
Below by embodiment, the present invention is described in further detail.It should be understood that following embodiment is only for more clearly explaining the present invention, but can not limit protection scope of the present invention with this.In the protection domain limiting in the claims in the present invention, the change without substantial variation that it is carried out, modification or equivalence change, all should fall within the scope of protection of the present invention.
1, canine parvovirus immunity alpaca
Get CPV virus liquid and insert in Erlenmeyer flask, add analytical pure formaldehyde and be diluted to final concentration 0.25wt%, limit edged shakes up, add stopper and with sterile gauze bag it, 37 ℃ effect 24～48h; Unstopper, with bundle Erlenmeyer flask mouth, interval is shaken, and disperses to formaldehyde taste.
Equivalent virus liquid and Freund's complete adjuvant emulsification, detection is tired, as immune vaccine for the first time; Equivalent virus liquid and Freund's incomplete adjuvant emulsification, detect its blood clotting valency with hemagglutination test (HA test), and recording result is 2
9, using that this carries out immunity as vaccine to alpaca.
The male alpaca of healthy adult two (B014, D007), subcutaneous multiple spot immunity, every immunity in 10 days once, is divided into four times.The total arterial blood extracting 5mL of each immune foreneck, centrifugal rear collection upper serum, 4 ℃ of preservations.After each immunity is complete, antibody titer is surveyed in hemagglutination-inhibition test (HI test), tires higher than the total arterial blood extracting 10mL of 1:64 collare, and centrifugal rear collection upper serum, obtains alpaca anti-dog parvovirus serum.
2, build canine parvovirus single domain antibody storehouse
In 250 μ L fresh whole bloods, add 1000 μ L erythrocyte cracked liquids, the centrifugal 30s of 12000r/min, in the white corpuscle group staying, add 1mL trizol, Trizol method is extracted peripheral blood RNA, with the long ultraviolet/visible light scanning spectrophotometer of NanoDrop ND-1000 all-wave, measure RNA concentration and OD value, with 1% agarose gel electrophoresis, identify the integrity of RNA.
Take RNA as template, and reverse transcription generates cDNA, and take β-actin For, β-actin Rev is primer, and amplification reference gene β-actin detects cDNA validity.
β-actin?For：5’-ACCCTCATAGATGGGCACAG-3’。
β-actin?Rev：5’-AGCCATGTACGTAGCCATCC-3’。
Take cDNA as template, is that primer increases for the first time with variable region of heavy chain special primer CALL01, CALL02, the VHH-CH2 that specific amplified product is 600bp and the VH-CH1-CH2 of 900bp.
CALL01：5’-GTCCTGGCTGCTCTTCTACAAGG-3’。
CALL02：5’-GGTACGTGCTGTTGAACTGTTCC-3’。
Gel reclaims test kit and reclaims purifying VHH-CH2, take it as template, and VHH special primer VHH For2, VHH Rev2 are that primer increases for the second time, the VHH that specific amplified product is 400bp.Reclaim purifying canine parvovirus single domain antibody VHH fragment, with 1% agarose gel electrophoresis, identify.
VHH?For2：5’-CCTTTCTATGCAGGCCCAGCCGGCCGCATGGCCGAKGTSCAGCT-3’。
VHH?Rev2：5’-GTTATTATTATTCAGATTATTATGCGGCCGCTGGAGACGGTGACCWGGGTCC-3’。
Gel reclaims test kit and reclaims purifying canine parvovirus single domain antibody VHH, connects VHH and plasmid vector pMD-18T simple in 10 μ L linked systems, builds connector plasmid expression vector pMD-18T-VHH.With connector, transform intestinal bacteria competence DH5 α, and screen picking white mono-clonal bacterium colony in 5mL LB substratum with blue hickie, 37 ℃, 200r/min, shaking culture 12～16h, plasmid extraction kit extracts bacterium liquid plasmid, and primer VHH For2, VHH Rev2 carry out PCR evaluation, and carries out Not I and Sfi I enzyme is cut evaluation.
Sfi I/Not I double digestion plasmid expression vector pMD-18T-VHH and phagemid vector pCANTAB 5E, the VHH of recovery purifying, is connected with phagemid vector pCANTAB 5E and builds phage recombinant vectors pCANTAB 5E-VHH.Ethanol precipitation purifying connects product, and transforms the competence e. coli tg1 of preparing through Calcium Chloride Method, picking mono-clonal bacterium colony, and PCR identifies and enzyme is cut after evaluation, and positive colony plasmid transforms TG1 the 2nd time, and picking mono-clonal bacterium colony carries out PCR evaluation.The bacterium liquid of getting twice transformation carries out 10 times, 100 times, 1000 times titre dilutions, coats dull and stereotyped upper cultivation, is inverted overnight incubation for 37 ℃, count colony number next day, 100 times of dilution bacteria growings are good, and mono-clonal is obvious, and the coating bacterium liquid of 100 μ L grows approximately 200 mono-clonals.Random picking mono-clonal bacterial strain, PCR identifies that positive rate is 90%, usable storage 1.8 * 10
7.
3, canine parvovirus single domain antibody is expressed
Utilize the affine absorption principle of antigen-antibody, the canine parvovirus structural protein VP2 of take is capture antigen, the canine parvovirus phage single domain antibody supernatant that adds activation, more than the standing 90min of room temperature, centre is constantly shaken 96 orifice plates repeatedly, discard not in conjunction with phage, with PBS/Tween20 (0.05%), wash, the competitive wash-out of glycine-hydrochloric acid elution buffer, with five, take turns " absorption-wash-out-amplification " concentration method and phage antibody library is screened to enrichment, phage antibody library original storage capacity 1.8 * 10
7cfu, activation storage capacity 3.6 * 10
11cfu, is respectively 7.8 * 10 to the 5th enrichment storage capacity for the first time
11cfu, 1.07 * 10
12cfu, 3.24 * 10
12cfu, 9.8 * 10
13cfu, 3.1 * 10
15cfu.
After the 5th enrichment finishes, get phage antibody plasmid transfection TG1 bacterium incubated overnight, on random choose flat board, single bacterium colony carries out PCR detection, positive rate 92%.
Phage antibody library (pCANTAB 5E-VHH) and phagemid (pCANTAB 5E) be transfection HB2151 competent cell respectively, picking list bacterium colony is cultivated, carry out bacterium liquid PCR experiment, extract plasmid simultaneously, with Sfi I/Not I double digestion pCANTAB 5E-VHH phagemid, check imports correct object carrier.
Phage recombinant vectors pCENTAB 5E-VHH after enrichment imports to competent cell
e.coliin HB2151 bacterial strain, due to the function of this host bacteria unrestraint amber terminator codon, amber terminator codon normally acts on, and great expression canine parvovirus single domain antibody VHH under IPTG induction, produces and have bioactive soluble protein.
Utilize fused protein label E-tag feature of pCENTAB 5E carrier, design primary antibodie is rabbit source Anti-E-Tag, and two resist for goat anti-rabbit igg-HRP, carry out western-blot detection, all contain the target protein of E-Tag label in protein sample.
96 orifice plates are coated with VP2 albumen (final concentration 15 μ g/ml), and recombinant expressed VHH albumen is carried out to gradient dilution, and ELISA detects the active 15 μ g/ml of single domain antibody.Coated 96 orifice plates of VP2 albumen with different concns, add VHH albumen 50 μ l, and the sensitivity that ELISA detects single domain antibody is 250ng/ml.
By great expression and have after testing bioactive canine parvovirus single domain antibody VHH vacuum lyophilization, in packing ampoule, sealing is preserved.
Alpaca anti-dog parvovirus single domain antibody with above-mentioned preparation is treated 20 canine parvovirus dogs, observes its result for the treatment of, and conventional treatment group is average treats after 9 days continuously, mortality ratio 40%, survival rate 60%; VHH single domain antibody treatment group is average treats after 8 days continuously, mortality ratio 20%, and survival rate is 80%.
China's pet dog quantity reaches 1.5 hundred million, and dog is tiny causes serious financial loss.Canine parvovirus single domain antibody prepared by the present invention is for the treatment of canine parvovirus dog, between every sick dog healing period of conventional treatment group, (9 days) total expense is 800 yuan, and between every sick dog healing period of VHH single domain antibody treatment group, (8 days) total expense is 640～720 yuan.Compare with conventional treatment, the treatment of VHH single domain antibody can make mortality ratio reduce, and curative ratio improves, and can shorten treatment cycle, makes the expense of curing a canine parvovirus dog reduce by 10%～20%.
Claims (7)
1. canine parvovirus single domain antibody, has the nucleotide sequence shown in SEQ ID NO.1.
2. the preparation method of claim 1 canine parvovirus single domain antibody, adopts following steps to be prepared:
1), extract the RNA in alpaca peripheral blood after canine parvovirus immunity, reverse transcription generation cDNA; Special primer amplification purification canine parvovirus single domain antibody VHH fragment;
2), canine parvovirus single domain antibody VHH fragment is connected structure phage recombinant vectors, transformed competence colibacillus e. coli tg1, structure canine parvovirus phage single domain antibody storehouse with phagemid vector;
3), with canine parvovirus structural protein VP2, add in the canine parvovirus phage single domain antibody storehouse of activation the competitive wash-out of glycine solution, enrichment phagemid recombinant vectors importing competent cell
e.coilin HB2151 bacterial strain, IPTG induction produces has bioactive canine parvovirus single domain antibody VHH albumen.
3. preparation method according to claim 2, it is characterized in that described step 1) in, with variable region of heavy chain special primer CALL01/ CALL02 and VHH special primer VHH For2/VHH Rev2, carry out for the first time respectively and pcr amplification for the second time, reclaim purifying canine parvovirus single domain antibody VHH fragment.
4. preparation method according to claim 2, it is characterized in that first with canine parvovirus single domain antibody VHH fragment, being connected with plasmid vector pMD-18T simple structure plasmid expression vector pMD-18T-VHH, transform intestinal bacteria competence DH5 α, reclaim purifying VHH, build phage recombinant vectors with phagemid vector, transformed competence colibacillus e. coli tg1, builds canine parvovirus phage single domain antibody storehouse.
5. according to the preparation method described in claim 2 or 4, it is characterized in that described phagemid vector is pCANTAB 5E.
6. according to the preparation method described in claim 2 or 4, the phage recombinant vectors that it is characterized in that described structure is pCANTAB 5E-VHH.
7. the application of claim 1 canine parvovirus single domain antibody in preparation treatment canine parvovirus medicine.
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