CN103665152B - Canine parvovirus single domain antibody and its preparation method and application - Google Patents
Canine parvovirus single domain antibody and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of canine parvovirus single domain antibody and its preparation method and application, does described canine parvovirus single domain antibody have SEQ? ID? nucleotide sequence shown in NO.1, be with canine parvovirus immunity alpaca, get peripheral blood and obtain alpaca anti-dog parvovirus serum; Extract RNA in peripheral blood, reverse transcription generates cDNA; Specific primers amplify purifying VHH fragment; VHH fragment be connected with phagemid vector and transform TG1, building canine parvovirus single domain antibody storehouse; Utilize antibody library described in the enrichment of canine parvovirus Viral structural protein VP2, do you import expression strain <i>E.coil</iGrea tT.GreaT.GT? HB2151, IPTG induction has bioactive canine parvovirus single domain antibody VHH and expresses.Canine parvovirus single domain antibody detection of active 15 μ g/ml prepared by the present invention, detection sensitivity 250ng/ml, can be used as medicinal application in the treatment of canine parvovirus disease dog.
Description
Technical field
The invention belongs to clinical veterinarian and antibody engineering technical field, relate to a kind of antiviral antibody, particularly relate to a kind of heavy chain antibody of main support alpaca to realize 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 (Canineparvovirus, CPV) cause, high with sickness rate, 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 consequences.The vaccine of preventing canine parvovirus mainly contains: deactivation vaccine, Attenuate vaccine and multi-joint seedling, and the immune programme for children of optimization can obtain good immune effect.But carry out detection investigation to the immune effect of vaccine on market to find, only the vaccine of 20% can produce the antibody of 100% protection.Data shows, China's pet dog quantity reaches 1.5 hundred million, and along with the increasing number of pet dog, the Diagnosis and treatment of research canine parvovirus disease is significant.
Based on the diagnostic kit that antigen is combined with antibodies specific, 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.In treatment, early stage application canine parvovirus monoclonal antibody and canine parvovirus antiserum(antisera) etc. carry out specific treatment, can obtain good effect.But monoclonal antibody large-scale production length consuming time, antiserum(antisera) large-scale production can not ensure that the market requirement and quality are unstable, and monoclonal antibody and sero-fast immunogenicity strong, all there is side effect over the course for the treatment of.Therefore, the antibody that relative specific is strong, avidity is high, immunogenicity is low, be produced on a large scale is studied 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, except common IgG antibody in alpaca serum, the IgG of nearly half is had to be heavy chain antibody, only there is variable region of heavy chain (VH), a hinge area and two constant regions (CH2 and CH3), and only have the antibody of a variable region of heavy chain to be called single domain antibody (variabledomainofheavychainofheavy-chainantibody to the variable region clone structure 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, easy 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 target reagent, can be used for building Immune Fusion thing, carrying out targeted therapy, can be used for commercialization scale operation.At present, antibody is only limitted to cell surface target in the treatment of disease, and whole antibody is due to molecular structure complexity, in cell inner expression difficulty, limit it in intracellular application, VHH immunogenicity is low, be difficult to the immune response of activated t cell, can as therapeutic antibodies 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 have broad application prospects in fields such as fundamental research, drug development and medical diagnosis on disease treatments.
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 the new technology utilizing phage expression foreign gene set up in recent years and develop, it is the technology that a kind of gene expression product and affinity selection combine, its ultimate principle is connected by the gene of goal gene with encode bacteriophage coat protein, be inserted on the expression vector of phage, thus make polypeptide or protein and coat protein amalgamation and expression and be illustrated in the surface of phage, the polypeptide be demonstrated or protein can keep relatively independent space structure and biological activity, phage is utilized to be easy to be separated amplification at E. coli system, can by selecting the phage particle of expressing corresponding acceptor to the combination of part, by the enrichment process of " absorption-wash-out-amplification ", can therefrom filter out specific antibody gene, and directly can measure some biologic activity of shown polypeptide or protein.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, there is provided that a kind of molecular weight is little, immunogenicity is low, high specificity, stability be strong, canine parvovirus single domain antibody that is easily 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 obtains and has bioactive single domain antibody from antibody library.
Canine parvovirus single domain antibody of the present invention has the nucleotide sequence shown in SEQIDNO.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 generates cDNA; Specific primers amplify purifying canine parvovirus single domain antibody VHH fragment;
2), canine parvovirus single domain antibody VHH fragment is connected with phagemid vector and builds phage recombinant vectors, transform competent E. coli TG1, builds canine parvovirus phage single domain antibody storehouse;
3), with canine parvovirus Viral 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 imports competent cell
e.coilin HB2151 bacterial strain, IPTG induces generation to have bioactive canine parvovirus single domain antibody VHH albumen.
More specifically, step 1) in be carry out first time and second time pcr amplification with variable region of heavy chain special primer CALL01/CALL02 and VHH special primer VHHFor2/VHHRev2 respectively, reclaim purifying canine parvovirus single domain antibody VHH fragment.
Step 2) in, first to be connected with plasmid vector pMD-18Tsimple with canine parvovirus single domain antibody VHH fragment to build plasmid expression vector pMD-18T-VHH, transformation of E. coli competence DH5 α, reclaim purifying VHH, phage recombinant vectors is built with phagemid vector, transform competent E. coli TG1, builds canine parvovirus phage single domain antibody storehouse.
Wherein, described phagemid vector is pCANTAB5E, and the phage recombinant vectors of structure is pCANTAB5E-VHH.
The canine parvovirus single domain antibody that the present invention obtains as medicine, can be 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 obtain there is bioactive canine parvovirus single domain antibody, the mass-producing preparation of antibody can be realized.
Except common IgG antibody in alpaca serum, the IgG of nearly half is had to be heavy chain antibody.Carry out canine parvovirus immunity with subcutaneous multiple spot immunization to two healthy adult alpacas, 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), separation and purification can go out IgG1 and IgG3 by protein g affinity chromatography, molecular weight is respectively at about 90kDa and 180kDa; Can isolate IgG2 in conjunction with IgG by protein A affinity chromatography, molecular weight is at about 75kD.Alpaca IgG each hypotype 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 experimental observation the present invention is on the impact of F81 Growth of Cells and proliferation inhibition rate, result shows, alpaca anti-dog parvovirus antiserum(antisera) group, dog tiny hyper-immune serum group Growth of Cells are vigorous, closely be connected, cell homogeneous and transparent; The minimizing of PBS group cell quantity, cell free, in the state of drawing in the net.Cell proliferation inhibition rate detects and shows, after alpaca anti-dog parvovirus antiserum(antisera) group, the tiny hyper-immune serum group of dog, PBS group and virus act on F81 cell 96h simultaneously, cell proliferation inhibition rate is respectively 3.9%, and 6.2%, 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, carries out screening enrichment to phage antibody library, and make phage antibody library enrichment increase nearly 4000 times, positive rate reaches 92%.The detection of active detecting canine parvovirus single domain antibody with ELISA is 15 μ g/ml, and detection sensitivity is 250ng/ml.
Canine parvovirus single domain antibody preparation prepared by the present invention, compared with conventional treatment, can make mortality ratio reduce, and curative ratio improves, and can shorten treatment cycle, makes the expense of a healing 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 only for clearly explaining the present invention, but protection scope of the present invention can not be limited with this.In the protection domain that the claims in the present invention limit, its change without substantial variation carried out, amendment or equivalence are changed, all should fall within the scope of protection of the present invention.
1, canine parvovirus immunity alpaca
Getting CPV virus liquid inserts in Erlenmeyer flask, and 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 DEG C 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 first time immune vaccine; Equivalent virus liquid and Freund's incomplete adjuvant emulsification, detect its HA-HI test with hemagglutination test (HA test), recording result is 2
9, in this, as vaccine, immunity is carried out to alpaca.
The male alpaca two (B014, D007) of healthy adult, 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, collected after centrifugation upper serum, 4 DEG C 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, collected after centrifugation upper serum, obtains alpaca anti-dog parvovirus serum.
2, canine parvovirus single domain antibody storehouse is built
1000 μ L erythrocyte cracked liquids are added in 250 μ L fresh whole bloods, the centrifugal 30s of 12000r/min, 1mLtrizol is added in the white corpuscle group stayed, Trizol method extracts peripheral blood RNA, RNA concentration and OD value is measured, by the integrity of 1% agarose gel electrophoresis qualification RNA with the long ultraviolet/visible light scanning spectrophotometer of NanoDropND-1000 all-wave.
Take RNA as template, reverse transcription generates cDNA, and with β-actinFor, β-actinRev for primer, amplification reference gene β-actin detects cDNA validity.
β-actinFor：5’-ACCCTCATAGATGGGCACAG-3’。
β-actinRev：5’-AGCCATGTACGTAGCCATCC-3’。
Take cDNA as template, be that primer carries out first time amplification with variable region of heavy chain special primer CALL01, CALL02, specific amplified product is the VH-CH1-CH2 of VHH-CH2 and 900bp of 600bp.
CALL01：5’-GTCCTGGCTGCTCTTCTACAAGG-3’。
CALL02：5’-GGTACGTGCTGTTGAACTGTTCC-3’。
Gel reclaims test kit and reclaims purifying VHH-CH2, and with it for template, VHH special primer VHHFor2, VHHRev2 are that primer carries out second time amplification, and specific amplified product is the VHH of 400bp.Reclaim purifying canine parvovirus single domain antibody VHH fragment, identify with 1% agarose gel electrophoresis.
VHHFor2：5’-CCTTTCTATGCAGGCCCAGCCGGCCGCATGGCCGAKGTSCAGCT-3’。
VHHRev2：5’-GTTATTATTATTCAGATTATTATGCGGCCGCTGGAGACGGTGACCWGGGTCC-3’。
Gel reclaims test kit and reclaims purifying canine parvovirus single domain antibody VHH, in 10 μ L linked systems, connect VHH and plasmid vector pMD-18Tsimple, builds connector plasmid expression vector pMD-18T-VHH.With connector transformation of E. coli competence DH5 α, and with blue hickie screening picking white mono-clonal bacterium colony in 5mLLB substratum, 37 DEG C, 200r/min, shaking culture 12 ~ 16h, plasmid extraction kit extracts bacterium liquid plasmid, and primer VHHFor2, VHHRev2 carry out PCR qualification, and carries out NotI and SfiI enzyme and cut qualification.
SfiI/NotI double digestion plasmid expression vector pMD-18T-VHH and phagemid vector pCANTAB5E, reclaims the VHH of purifying, is connected builds phage recombinant vectors pCANTAB5E-VHH with phagemid vector pCANTAB5E.Ethanol precipitation purifying connects product, and transforms the competence e. coli tg1 prepared through Calcium Chloride Method, picking mono-clonal bacterium colony, and after PCR qualification and enzyme cut qualification, positive colony plasmid transforms TG1 the 2nd time, and picking mono-clonal bacterium colony carries out PCR qualification.The bacterium liquid 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 DEG C, next day counts colony number, 100 times of dilution bacteria growings are good, and mono-clonal is obvious, and the coating bacterium liquid of 100 μ L grows about 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, with canine parvovirus Viral structural protein VP2 for capture antigen, add the canine parvovirus phage single domain antibody supernatant of activation, room temperature leaves standstill more than 90min, 96 orifice plates are constantly shaken repeatedly in centre, discard in conjunction with phage, wash with PBS/Tween20 (0.05%), the competitive wash-out of glycine-HCI elution buffer, take turns " absorption-wash-out-amplification " concentration method with five and screening enrichment is carried out to phage antibody library, phage antibody library original storage capacity 1.8 × 10
7cfu, activation storage capacity 3.6 × 10
11cfu, first time is respectively 7.8 × 10 to the 5th enrichment storage capacity
11cfu, 1.07 × 10
12cfu, 3.24 × 10
12cfu, 9.8 × 10
13cfu, 3.1 × 10
15cfu.
After 5th enrichment terminates, 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 (pCANTAB5E-VHH) and phagemid (pCANTAB5E) be transfection HB2151 competent cell respectively, picking list bacterium colony is cultivated, carry out bacterium liquid PCR experiment, extract plasmid simultaneously, with SfiI/NotI double digestion pCANTAB5E-VHH phagemid, inspection imports correct object carrier.
Phage recombinant vectors pCENTAB5E-VHH after enrichment imports to competent cell
e.coliin HB2151 bacterial strain, due to the function of this host bacteria unrestraint Amber stop codon, Amber stop codon normally acts on, and great expression canine parvovirus single domain antibody VHH under IPTG induction, produces and have bioactive soluble protein.
Utilize the fused protein label E-tag feature of pCENTAB5E 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.
Recombinant expressed VHH albumen, by VP2 albumen (final concentration 15 μ g/ml), is carried out gradient dilution by 96 orifice plate bags, and ELISA detects the active 15 μ g/ml of single domain antibody.With the VP2 albumen bag of different concns by 96 orifice plates, add VHH albumen 50 μ l, the sensitivity that ELISA detects single domain antibody is 250ng/ml.
Great expression is had bioactive canine parvovirus single domain antibody VHH vacuum lyophilization after testing, and in packing ampoule, sealing is preserved.
Treat 20 canine parvovirus dogs with the alpaca anti-dog parvovirus single domain antibody of above-mentioned preparation, observe its result for the treatment of, conventional treatment group is on average treated after 9 days continuously, mortality ratio 40%, survival rate 60%; VHH single domain antibody treatment group is on average treated after 8 days continuously, and mortality ratio 20%, survival rate is 80%.
China's pet dog quantity reaches 1.5 hundred million, and dog is tiny causes serious financial consequences.Canine parvovirus single domain antibody prepared by the present invention is for the treatment of canine parvovirus dog, every only (9 days) total expense 800 yuan between sick dog healing period of conventional treatment group, every only (8 days) total expense 640 ~ 720 yuan between sick dog healing period of VHH single domain antibody treatment group.Compared 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 a healing canine parvovirus dog reduce by 10% ~ 20%.
Claims (7)
1. canine parvovirus single domain antibody is the nucleotide sequence shown in SEQIDNO.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 generates cDNA; Specific primers amplify purifying canine parvovirus single domain antibody VHH fragment;
2), canine parvovirus single domain antibody VHH fragment is connected with phagemid vector and builds phage recombinant vectors, transform competent E. coli TG1, builds canine parvovirus phage single domain antibody storehouse;
3), with canine parvovirus Viral 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 imports competent cell
e.coilin HB2151 bacterial strain, IPTG induces generation to have bioactive canine parvovirus single domain antibody VHH albumen.
3. preparation method according to claim 2, it is characterized in that described step 1) in, first time and second time pcr amplification is carried out respectively with variable region of heavy chain special primer CALL01/CALL02 and VHH special primer VHHFor2/VHHRev2, reclaim purifying canine parvovirus single domain antibody VHH fragment, wherein, the nucleotide sequence of primer CALL01 and CALL02 is respectively as shown in SEQIDNO.4 and SEQIDNO.5, and the nucleotide sequence of primer VHHFor2 and VHHRev2 is respectively as shown in SEQIDNO.6 and SEQIDNO.7.
4. preparation method according to claim 2, it is characterized in that first being connected with plasmid vector pMD-18Tsimple with canine parvovirus single domain antibody VHH fragment building plasmid expression vector pMD-18T-VHH, transformation of E. coli competence DH5 α, reclaim purifying VHH, phage recombinant vectors is built with phagemid vector, transform competent E. coli TG1, builds canine parvovirus phage single domain antibody storehouse.
5. the preparation method according to claim 2 or 4, is characterized in that described phagemid vector is pCANTAB5E.
6. the preparation method according to claim 2 or 4, is characterized in that the phage recombinant vectors of described structure is pCANTAB5E-VHH.
7. the application of claim 1 canine parvovirus single domain antibody in preparation treatment canine parvovirus medicine.
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| US11130800B2 (en) | 2016-05-20 | 2021-09-28 | Novobind Livestock Therapeutics Inc. | Antibodies against microorganisms and uses thereof |
| CN106220729A (en) * | 2016-08-01 | 2016-12-14 | 西北农林科技大学 | A kind of method of the fowl source genetic engineering antibody preparing anti-dog parvovirus |
| CN106478820A (en) * | 2016-10-10 | 2017-03-08 | 米度（南京）生物技术有限公司 | A kind of liver cancer PET diagnosis tracer and preparation method thereof and purposes |
| CN107304230A (en) * | 2017-07-20 | 2017-10-31 | 华南农业大学 | A kind of anti-dog parvovirus refines antibody and preparation method thereof |
| US20220064224A1 (en) * | 2018-11-13 | 2022-03-03 | Novobind Livestock Therapeutics, Inc. | Antibodies against disease causing agents of canines and felines and uses thereof |
| CN113861287B (en) * | 2021-11-12 | 2022-10-25 | 中国农业科学院北京畜牧兽医研究所 | Canine parvovirus nano antibody CPV-VHH-H1 and application thereof |
| CN113861286B (en) * | 2021-11-12 | 2022-10-25 | 中国农业科学院北京畜牧兽医研究所 | Canine parvovirus nano antibody CPV-VHH-D4 and application thereof |
| CN114478805B (en) * | 2022-03-23 | 2024-03-22 | 武汉生物工程学院 | Single-chain antibody for resisting canine parvovirus, preparation method and application thereof |
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