Anti- African swine fever virus and CD dual-target pig source antibody, the preparation method and applicationTechnical field
The present invention relates to the technical field of biological medicine engineering, more specifically to a kind of anti-African swine fever virus andCD dual-target pig source antibody, the preparation method and application.
Background technique
African swine fever (African swine fever, ASF) is by African swine fever virus (African swine feverVirus, ASFV) caused by one kind is acute, deadly infectious disease, the infectiousness with height, disease incidence is high, and the death rate is more up to100%；The pig breeding industry in China and the whole world is not only seriously endangered, it is also related to seriously affect the relevant international trade of live pig industry and phaseThe other industry of pass.World Organization for Animal Health (Office international des é pizooties, OIE) arranges ASFFor that must report animal epidemic, China is classified as a kind of animal epidemic, is the exotic disease of emphasis prevention and control.
It is a kind of disease of complexity in view of African swine fever, understands fast propagation around and invade food chain and cause greatSocioeconomic problem.By taking Spain as an example, government is to put out the project (1985-1995) of ASF to take nearly 100,000,000 dollars, is hadA little countries once all slaughtered the pig in the whole nation to eliminate African swine fever.
Resistivity of the African swine fever virus in tissue and environment is strong, now both without remedy measures, also without effectiveVaccine control the disease, global most efficient method is exactly to kill epidemic-stricken area and all pigs in the neighbourhood；To pig raisingThe consequence of enterprise and bringing on a disaster property of pig farmer.
In recent years, countries in the world scientist constantly tackles key problems, and has been developed that some African swine fever vaccines；But these are AfricanAlthough hog cholera vaccine can induce the antibody for generating certain level, do not have the ability for neutralizing African swine fever virus but, be unable to reachThe purpose of effective prevention and control African swine fever.This is mainly due to African swine fever virus is a very unique DNA arboviruse, solelySpy is mainly manifested in the following aspects: first is that mainly directly or indirectly breaking using the mononuclear phagocytic cells of pig as target cell infectionThe immuning tissue of bad host, and serious immunosupress is caused, make vaccine failure；Second is that viruses adsorption and entrance target cell are not onlyIt is mediated by multiple protein moleculars, and receptor-mediated encytosis is not unique mechanism；Third is that rare heredity variabilityDNA virus, be in particular in the diversity of different separation strain gene group length, the heterogeneity of infection same head swine disease poison clone andThe mutability of viral antigen in infection and succeeding generations；Fourth is that the anti-host protein that group/cording quantity is numerous, mechanism is complicated, referred to asDNA " AIDS virus ".Therefore, making the Control Technology of African swine fever becomes world's hang-up.
Present African swine fever has been passed to China, and epidemic situation constantly spreads and spreads；This fatal swine fever virus mayAsia other countries, FAO's warning, for this time epidemic situation of China's outburst, instant response can be propagated at any timeMeasure is to eliminate it as early as possible.
Therefore, a kind of drug that can be effectively prevented and treated African swine fever is researched and developed, this deadly infectious disease is eliminatedIt damages, is the challenge and a task the most urgent to China's medical science circle most serious.
Summary of the invention
The object of the present invention is to provide a kind of anti-African swine fever virus and CD dual-target pig source antibody, the preparation method and answerWith, infecting and spreading for African swine fever can not be fought to solve the prior art, lead to not prevention it is even more impossible to treat African swine feverThe problem of.
The scheme that the present invention solves above-mentioned technical problem is to provide a kind of anti-African swine fever virus and CD dual-target pig sourceAntibody preparation method, includes the following steps:
S1, mouse-pig chimeric antibody light full-length gene order and mouse-pig chimeric antibody heavy full-length gene sequence are preparedColumn；
S2, by the mouse-pig chimeric antibody light full-length gene order and the mouse-pig chimeric antibody heavy full-length geneSequence is cloned in carrier, and expression obtains anti-African swine fever virus and CD dual-target pig source antibody.
In preparation method of the invention, before step S1 further include:
S0, hybridoma is prepared: by African swine fever virus antigen recombinant protein, or expression African swine fever virus antigenMouse is immunized, then cell fusion in the recombinant virus of albumen together with pig CD recombinant protein, and anti-African swine fever disease can be secreted by being madeThe hybridoma of poison and CD dual-target pig source antibody.
It is major structural protein in view of African swine fever virus VP73 albumen and there is stable immunogenicity, P54 is being escapedIt plays a crucial role in the mechanism of host defense system, P72 participates in the absorption and entrance of virus with cell；A238L is a kind of double functionEnergy albumen, is strong immunosuppressor, has and promotees cells apoptosis；CD2V is virus attachment protein, is spread in virusAnd it plays an important role during impairment of lymphocyte.Therefore, the present invention shoots the arrow at the target preferably this five kinds of albumen as representativeAntigen protein targetedly carries out multinomial innovation as target spot, and it is big basic to solve " immunosupress " and " immunologic escape " twoProperty problem.
Utilize five kinds of African swine fever virus VP73, P54, P72, A238L, CD2V etc. representative antigen proteins or African pigThe other protein expressions of pestivirus are in eukaryocyte or prokaryotic cell and purified, or by African swine fever virus VP73,The gene of five kinds of representativeness antigen proteins such as P54, P72, A238L, CD2V or the gene point of the other albumen of African swine fever virusIt is not inserted into the genome of the viral vectors such as papovavirus, adenovirus, herpesviral, poxvirus, RNA virus, building is sameWhen expression VP73, P54, P72, A238L, CD2V or the other albumen of African swine fever virus recombinant virus and purified, obtainTo the recombinant virus of purifying；
For example, utilizing Bacillus coli expression pig CD3 recombinant protein or other type pig CD recombinant proteins (such as CD2 and CD8)And Purification of Pig CD3 recombinant protein or other type pig CD recombinant proteins (such as CD2 and CD8)；
By in recombinant protein one of which (eukaryocyte or procaryotic cell expression and purify VP73, P54, P72,Any one recombinant protein in five kinds of A238L, CD2V etc. representative antigen proteins or the other albumen of African swine fever virus),The recombinant virus either expressing VP73, P54, P72, A238L, CD2V or the other albumen of African swine fever virus and purifying, with pigOne of CD3 recombinant protein or other type pig CD (such as CD2 and CD8) recombinant protein, which are uniformly mixed, adds adjuvant, by normalMouse is immunized in rule method, then cell fusion, and one of anti-African swine fever virus and CD dual-target pig source can be secreted by being madeThe hybridoma of antibody.
The present invention will be thin as the immune hybridoma obtained of complex antigen with pig CD3 recombinant protein using VP73 recombinant proteinBorn of the same parents are named as 1A, and the hybridoma obtained is immunized as complex antigen using using VP73 recombinant protein and pig CD2 recombinant proteinIt is named as 1B, is ordered using VP73 recombinant protein and pig CD8 recombinant protein as the immune hybridoma obtained of complex antigenEntitled 1C；It is named as using P54 recombinant protein with pig CD3 recombinant protein as the immune hybridoma obtained of complex antigen2A is named as 2B as the immune hybridoma obtained of complex antigen for using P54 recombinant protein and pig CD2 recombinant protein,2C is named as the immune hybridoma obtained of complex antigen using using P54 recombinant protein and pig CD8 recombinant protein；It will adoptIt uses P72 recombinant protein and pig CD3 recombinant protein to be named as 3A as the immune hybridoma obtained of complex antigen, will useP72 recombinant protein and pig CD2 recombinant protein are immunized the hybridoma obtained as complex antigen and are named as 3B, will use P72Recombinant protein and pig CD8 recombinant protein are immunized the hybridoma obtained as complex antigen and are named as 3C；It will be using A238L weightHistone and pig CD3 recombinant protein are immunized the hybridoma obtained as complex antigen and are named as 4A, will be recombinated using A238LAlbumen and pig CD2 recombinant protein are immunized the hybridoma obtained as complex antigen and are named as 4B, will recombinate egg using 238LIt is white to be named as 4C as the immune hybridoma obtained of complex antigen with pig CD8 recombinant protein；CD2V recombinant protein will be usedBe named as 5A as the immune hybridoma obtained of complex antigen with pig CD3 recombinant protein, will using CD2V recombinant protein withPig CD2 recombinant protein is named as 5B as the immune hybridoma obtained of complex antigen, will use CD2V recombinant protein and pigCD8 recombinant protein is named as 5C as the immune hybridoma obtained of complex antigen；The rest may be inferred.
In anti-African swine fever virus of the invention and CD dual-target pig source Antibody preparation method, the African swine fever virus is anti-Original weight histone is the African swine fever virus representativeness antigen protein of eukaryocyte or procaryotic cell expression and purifying；By African pigIn the genome of the gene insertion viral vectors of pestivirus representativeness antigen protein, the expression African swine fever virus antigen is obtainedThe recombinant virus of albumen；The African swine fever virus representativeness antigen protein includes in VP73, P54, P72, A238L, CD2VIt is at least one.
In anti-African swine fever virus of the invention and CD dual-target pig source Antibody preparation method, the pig CD recombinant protein isEukaryocyte or procaryotic cell expression and purify representative pig CD albumen, it is described representativeness pig CD albumen include pig CD3 albumen,At least one of pig CD2 albumen, pig CD8 albumen.
It is in step sl, specific to wrap in anti-African swine fever virus of the invention and CD dual-target pig source Antibody preparation methodInclude step:
S101, the total serum IgE for extracting hybridoma, reverse transcription prepare cDNA, expand source of mouse antibody's light chain variable region respectivelyWith the sequence of source of mouse antibody heavy chain variable region；Pig lymphocyte is taken to extract its total serum IgE, reverse transcription prepares cDNA, expands pig respectivelySource antibody light chain constant region and pig source heavy chain constant region sequence；
Source of mouse antibody's light chain variable region described in S102, overlap-extension PCR and pig source antibody light chain constant region obtain mouse-pigChimeric antibody light full-length gene order；Source of mouse antibody heavy chain variable region described in overlap-extension PCR and pig source heavy chain of antibody are constantArea obtains mouse-pig chimeric antibody heavy full-length gene order.
It is also specific in step s 102 in anti-African swine fever virus of the invention and CD dual-target pig source Antibody preparation methodInclude:
According to source of mouse antibody's light chain variable region and source of mouse antibody heavy chain variable region sequence and pig source antibody light chain constant region andPig source heavy chain constant region sequence, design primer；
Source of mouse antibody's light chain variable region and source of mouse heavy chain of antibody are expanded respectively using primer LV-F/LV-R and HV-F/HV-RThe sequence of variable region；Pig source antibody light chain constant region and pig source antibody are expanded respectively using primer HC-F/HC-R and LC-F/LC-RThe sequence of heavy chain constant region；
The source of mouse antibody's light chain variable region and pig source antibody light chain are expanded using primer LV-F/LC-R overlap-extension PCRConstant region；The source of mouse antibody heavy chain variable region and pig source heavy chain of antibody are expanded using primer HV-F/HC-R overlap-extension PCRConstant region.
It is in step s 2, specific to wrap in anti-African swine fever virus of the invention and CD dual-target pig source Antibody preparation methodIt includes:
The mouse-pig chimeric antibody light full-length gene order and the mouse-pig chimeric antibody heavy full-length gene order,It is cloned with virus gene carrier or non-viral gene vector, it is raw by insect cell or other cell large scale fermentationsIt produces, anti-African swine fever virus and the CD dual-target pig source antibody of genotype is obtained after purified；
Alternatively, by the mouse-pig chimeric antibody light full-length gene order and the mouse-pig chimeric antibody heavy overall length baseRecombinant plasmid is obtained because sequence is cloned in respectively under two promoters of expression vector；The Transfected Recombinant Plasmid CHO is thinBorn of the same parents express in insect cell expression system, obtain anti-African swine fever virus and the CD dual-target pig source antibody of protein I type；
Alternatively, the mouse-pig chimeric antibody light full-length gene is cloned in expression vector using restriction enzyme double digestionUnder one promoter；The mouse-pig chimeric antibody heavy full-length gene is used to another promoter of restriction enzyme double digestionUnder, obtain anti-African swine fever virus and the CD dual-target pig source antibody of protein type Il.
The present invention will be for VP73 recombinant protein and pig CD3 recombinant protein three kinds of double targets as made from " 1A hybridoma "Labeling antibody is respectively designated as the anti-African swine fever virus of 1A genotype and CD dual-target pig source antibody, the anti-African pig of 1A protein I typePestivirus and CD dual-target pig source antibody, the anti-African swine fever virus of 1A protein type Il and CD dual-target pig source antibody；By needle1B is respectively designated as to VP73 recombinant protein and pig CD2 recombinant protein three kinds of dual-target antibody as made from " 1B hybridoma "The anti-African swine fever virus of genotype and CD dual-target pig source antibody, the anti-African swine fever virus of 1B protein I type and CD dual-target pigSource antibody, the anti-African swine fever virus of 1B protein type Il and CD dual-target pig source antibody；VP73 recombinant protein and pig will be directed toCD8 recombinant protein three kinds of dual-target antibody as made from " 1C hybridoma " are respectively designated as the anti-African swine fever disease of 1C genotypePoison and CD dual-target pig source antibody, the anti-African swine fever virus of 1C protein I type and CD dual-target pig source antibody, 1C protein type IlAnti- African swine fever virus and CD dual-target pig source antibody；P54 recombinant protein will be directed to pig CD3 recombinant protein by " 2A hybridizesThree kinds of dual-target antibody made from oncocyte " are respectively designated as the anti-African swine fever virus of 2A genotype and CD dual-target pig sourceization is anti-Body, the anti-African swine fever virus of 2A protein I type and CD dual-target pig source antibody, the anti-African swine fever virus of 2A protein type Il and CD are bis-Target pig source antibody；It will be for P54 recombinant protein and pig CD2 recombinant protein three kinds of double targets as made from " 2B hybridoma "Labeling antibody is respectively designated as the anti-African swine fever virus of 2B genotype and CD dual-target pig source antibody, the anti-African pig of 2B protein I typePestivirus and CD dual-target pig source antibody, the anti-African swine fever virus of 2B protein type Il and CD dual-target pig source antibody；By needle2C is respectively designated as to P54 recombinant protein and pig CD8 recombinant protein three kinds of dual-target antibody as made from " 2C hybridoma "The anti-African swine fever virus of genotype and CD dual-target pig source antibody, the anti-African swine fever virus of 2C protein I type and CD dual-target pigSource antibody, the anti-African swine fever virus of 2C protein type Il and CD dual-target pig source antibody；P72 recombinant protein and pig will be directed toCD3 recombinant protein three kinds of dual-target antibody as made from " 3A hybridoma " are respectively designated as the anti-African swine fever disease of 3A genotypePoison and CD dual-target pig source antibody, the anti-African swine fever virus of 3A protein I type and CD dual-target pig source antibody, 3A protein type IlAnti- African swine fever virus and CD dual-target pig source antibody；P72 recombinant protein will be directed to pig CD2 recombinant protein by " 3B hybridizesThree kinds of dual-target antibody made from oncocyte " are respectively designated as the anti-African swine fever virus of 3B genotype and CD dual-target pig sourceization is anti-Body, the anti-African swine fever virus of 3B protein I type and CD dual-target pig source antibody, the anti-African swine fever virus of 3B protein type Il and CD are bis-Target pig source antibody；It will be for P72 recombinant protein and pig CD8 recombinant protein three kinds of double targets as made from " 3C hybridoma "Labeling antibody is respectively designated as the anti-African swine fever virus of 3C genotype and CD dual-target pig source antibody, the anti-African pig of 3C protein I typePestivirus and CD dual-target pig source antibody, the anti-African swine fever virus of 3C protein type Il and CD dual-target pig source antibody；By needleA238L recombinant protein and pig CD3 recombinant protein three kinds of dual-target antibody as made from " 4A hybridoma " are respectively designated asThe anti-African swine fever virus of 4A genotype and CD dual-target pig source antibody, the anti-African swine fever virus of 4A protein I type and CD dual-targetPig source antibody, the anti-African swine fever virus of 4A protein type Il and CD dual-target pig source antibody；Will for A238L recombinant protein withPig CD2 recombinant protein three kinds of dual-target antibody as made from " 4B hybridoma " are respectively designated as the anti-African swine fever of 4B genotypeVirus and CD dual-target pig source antibody, the anti-African swine fever virus of 4B protein I type and CD dual-target pig source antibody, 4B protein I IThe anti-African swine fever virus of type and CD dual-target pig source antibody；It will be for A238L recombinant protein and pig CD8 recombinant protein by " 4CThree kinds of dual-target antibody made from hybridoma " are respectively designated as the anti-African swine fever virus of 4C genotype and CD dual-target pig sourceChange antibody, the anti-African swine fever virus of 4C protein I type and CD dual-target pig source antibody, the anti-African swine fever virus of 4C protein type Il withCD dual-target pig source antibody；It will be for CD2V recombinant protein and pig CD3 recombinant protein three as made from " 5A hybridoma "Kind dual-target antibody is respectively designated as the anti-African swine fever virus of 5A genotype and CD dual-target pig source antibody, and 5A protein I type is anti-African swine fever virus and CD dual-target pig source antibody, the anti-African swine fever virus of 5A protein type Il and CD dual-target pig sourceization are anti-Body；It will be ordered respectively for CD2V recombinant protein with three kinds of dual-target antibody made from pig CD2 recombinant protein " 5B hybridoma "The anti-African swine fever virus of entitled 5B genotype and CD dual-target pig source antibody, the anti-African swine fever virus of 5B protein I type and CD are bis-Target pig source antibody, the anti-African swine fever virus of 5B protein type Il and CD dual-target pig source antibody；Egg will be recombinated for CD2VIt is white to be respectively designated as the anti-Africa of 5C genotype with pig CD8 recombinant protein three kinds of dual-target antibody as made from " 5C hybridoma "Swine fever virus and CD dual-target pig source antibody, the anti-African swine fever virus of 5C protein I type and CD dual-target pig source antibody, 5C eggThe anti-African swine fever virus of white II type and CD dual-target pig source antibody；The rest may be inferred.
In practical application, the above antibody can be arbitrarily made with to the compound anti-African swine fever virus of full price genotype and CD is bis-Target pig source antibody and the compound anti-African swine fever virus of full price protein type (dividing I, II) and CD dual-target pig source antibody (byAbove 15 kinds of antibody combinations form)；Excellent pentavalent (containing 1A, 2A, 3A, 4A, 5A) genotype or the anti-African pig of protein type (dividing I, II)Pestivirus and CD dual-target pig source antibody；Excellent trivalent (containing 1A, 2A, 3A) the anti-African swine fever virus of genotype and CD dual-target pigSource antibody or the anti-African swine fever virus of protein type (dividing I, II) and CD dual-target pig source antibody；Multivalence (containing 1A, 1B, 1C and2A, 2B, 2C and 3A, 3B, 3C etc.) genotype or the compound anti-African swine fever virus of protein type (dividing I, II) and CD dual-target pig sourceChange antibody.In addition, unit price (the containing only 1A) genotype or the anti-African swine fever disease of protein type (dividing I, II) of a kind of popular style can be producedPoison and CD dual-target pig source antibody product.
In view of CD3 there are in pig whole T cell, therefore, excellent pentavalent (containing 1A, 2A, 3A, 4A, 5A) genotype or protein typeAnti- African swine fever virus and CD dual-target pig source antibody are most economical effective selections.
The present invention also provides a kind of anti-African swine fever viruses and CD dual-target pig source antibody, use aforementioned preparation process system?.
The present invention also provides the applications of a kind of anti-African swine fever virus and CD dual-target pig source antibody, use aforementioned preparationAntibody made from method, as preparation prevent and remove African swine fever virus to pig infect, and/or for preventing and treating it is non-Application in the drug of continent swine fever, sterile products.
The present invention also provides a kind of composition, comprising above-mentioned anti-African swine fever virus and CD dual-target pig source antibody andPharmaceutically acceptable auxiliary material.
The present invention also provides a kind of carrier, including be cloned in carrier mouse-pig chimeric antibody light full-length gene order andMouse-pig chimeric antibody heavy full-length gene order, the mouse-pig chimeric antibody light full-length gene order are exempted from by related antigenThe antibody light chain sequencing obtained after epidemic disease obtains, and the mouse-pig chimeric antibody heavy full-length gene order is immune by related antigenThe heavy chain of antibody sequencing obtained afterwards obtains.The carrier is genophore or expression vector.
Implement the present invention, with it is following five aspect the utility model has the advantages that
1. changing the traditional approach of vaccine prevention, solve the problems, such as to influence vaccine effect: countries in the world are all mainly at presentHow vaccine prevention African swine fever is used in research, still, first, African swine fever virus is rare hereditary variability DNA diseasePoison, gene type is more, substantial amounts, and vaccine can only be directed to several models, invalid to most of model；Second, African swine fever diseaseMalicious easily variation, the speed of vaccine development does not catch up with virus variation speed, in addition African swine fever virus is in infection and succeeding generationsThe mutability of viral antigen makes the effect of vaccine have a greatly reduced quality；Third, African swine fever virus are a kind of special virus, mainly withThe mononuclear phagocytic cells of pig are target cell infection, directly or indirectly destroy the immuning tissue of host, and cause serious immune suppressionSystem, causes vaccine not work, contained apoptotic proteins can make target cell (macrophage and other immunocytes) apoptosis no matterIt is natural infection or artificial challenge, African swine fever virus does not generate typical neutralizing antibody, and nearest countries in the world scientist grindsStudy carefully improved vaccine although and can induce the antibody for generating certain level, but does not have the ability for neutralizing African swine fever virus, nothingMethod achievees the purpose that effective prevention and control African swine fever, this is the biggest obstacle of vaccine development；4th, African swine fever virus immunologic escapeThe complicated multiplicity of mechanism, can interfere the expression of immunomodulatory gene, escape the removing of host immune cell, this is also to influence vaccine realityAn important factor for border effect；5th, vaccine only has prevention effect, and without therapeutic effect, pig vaccinates and can only avenge once falling illUpper plus frost.The present invention changes the traditional approach for vaccinating and generating antibody in stimulation pig body, and uses directly defeated into pig bodyEnter the passive immunity mode of high activity antibody.Anti- African swine fever virus and CD dual-target pig source antibody of the invention can kill intoEnter intracorporal African swine fever virus, the large dosage of dual-target for preventing the organ of virus damage pig and closing its diffusion, while inputtingAntibody cleavable dissolves infected cell and phagocyte is activated to be removed, and reaches therapeutic effect；To solve the big face of sick pigThe dead problem of product.Since anti-African swine fever virus of the invention and CD dual-target pig source antibody belong to monoclonal antibody oneClass, the potency ratio of monoclonal antibody vaccinate high 200 times or more the universal (note: injection epidemic disease of potency of the antibody generated in vivoThe potency ﹥ 1:40 that seedling generates antibody in vivo is i.e. qualified, and double ten thousand -1:100 of targeting antibodies potency 1:1 are ten thousand)；Thus can be from oneAngle solves the problems, such as that the antibody of vaccine-induced generation does not have the ability for neutralizing African swine fever virus.Therefore, the present invention providesAnti- African swine fever virus and CD dual-target pig source antibody be the more preferably selection for surmounting vaccine, be the wound to traditional precautionary approachNewly.
2. dual-target antibody activates compromised immune system: dual-target antibody increases a specificity compared with common antibodyAntigen binding site, following advantage is shown in terms for the treatment of: two antigen binding sites are respectively in connection with African swine fever virusWith immunocyte and generate bridge joint effect, play two big functions, first function be can directly with African swine fever virus act on,(1) it attacks and presses down to go out and participate in viruses adsorption and invade the P72 albumen of cell, prevent African swine fever virus absorption and intrusion pig bodyInner cell, (two), which attack and press down, goes out as immunosuppressor and has the A238L for promoting cells apoptosis, eliminates immunosupress,Restore immune function, (three) attack and press down the P54 albumen to play a crucial role in immunologic escape that goes out, eliminate immunologic escape, (four)The CD2V albumen for going out and playing an important role during virus diffusion and impairment of lymphocyte is attacked and pressed down, African swine fever virus is blockedOther healthy cells are infected in diffusion, protect lymphocyte not attacked by African swine fever virus, perfect immune system；Second functionIt is the function of activating the immunocytes such as T cell and NK cell, improves part NK cell or T cell concentration, enhancement effect molecule killHurt ability, active cell is immune, and it is more effective to kill African swine fever virus and cracking, remove infected cell, it on the one hand plays and exempts fromThe coordination stimulation of epidemic disease cell and antigen presenting cell discharges cell factor, is pressed down with cellular immunity and goes out and remove African swine feverVirus；On the other hand by dual-target antibody in the intracorporal ADCC effect (Antibody -dependent cell cytotoxicity effect) of pigThe cell infected by African swine fever virus is cracked and dissolved, achievees the effect that treatment, makes sick pig rehabilitation without slaughter and destruction.Since African swine fever virus is using the mononuclear phagocytic cells of pig as target cell infection, can destroy host immuning tissue cause it is seriousImmunosupress, therefore, activating immune system are crucial；Dual-target antibody of the invention makes to cause " immunosupress " and " immune to escapeA238L and P54 and CD2V the antigen protein inactivation of ease ", thus, effective immune cell activated.Meanwhile two antigen binding domainsIt can significantly increase the specificity and effect of antibody, therapeutic effect is the 100-1 of common antibody, 000 times.Therefore, from another angleDegree solves the problems, such as that the antibody of vaccine-induced generation does not have and neutralizes African swine fever virus ability.
Double targeting antibodies are compared with conventional antibodies in tissue penetration, killing African swine fever virus efficiency, toxicity of missing the target hairIt all has a clear superiority in terms of the raw indexs such as rate and clinical indication.Original 1/2 is reduced in view of its dosage, 000, may be used alsoSignificantly reduce treatment cost.
3. antibody pig sourceization extends antibody action time: dual-target antibody pig source it is living to be retained source of mouse antibody by the present inventionProperty area gene order make to combine antigenic capacity constant, replace source of mouse constant region gene sequences with pig source constant region gene sequences, keep awayThe repulsion in pig body to foreign protei antibody is exempted from.
Although the monoclonal antibody of mouse has very strong antibody mediated effect, if by the monoclonal antibody of mouseAnti-mouse antibody reaction can be induced by, which being injected directly into pig body, (refers to that mouse monoclonal antibody there is foreign protei to be immunized other animal bodiesOriginality), can be disposed of quickly in the intracorporal circulatory system of pig, cause in vivo half-life period it is shorter.The present invention will be preparedAnti- African swine fever virus and CD dual-target pig source antibody be transformed, retain the base of high-affinity of the antigen in conjunction with antibodyOn plinth, most of source of mouse gene order is removed, with the replacement of people pig source gene order, which reduces the foreign body of mouse monoclonal antibodyAnd immunogenicity, it reduces murine antibody and reacts in the intracorporal antiantibody of people and strengthen antibody and make in the intracorporal ADCC of pigWith (Antibody -dependent cell cytotoxicity effect), the duration of its half-life period is increased.So that anti-African pig of the inventionThe action time of pestivirus and CD dual-target pig source antibody is enhanced with function and effect.
4. antibody nano liposomesization improve therapeutic index: the present invention is by made anti-African swine fever virus and the bis- targets of CDMicrocapsules and nano-microcapsule or micro emulsion or nano liposomes are made in mark pig source antibody, and the bioavilability of antibody can be improved, andGenerate prolongation of effect effect；It eats when this antibody to be made oral agents or be added in feed for pig, is used in a manner of oral administrationWhen preventing and treating African swine fever, nano liposomes dosage form can reduce the destruction of stomach and digestive enzyme to antibody bioactive.Meanwhile anti-African swine fever virus and the CD dual-target pig source of microcapsules and nano-microcapsule or micro emulsion or nano liposomes is madeThe ability into microcirculatory vascular and permeation cell gap can be enhanced by changing antibody, and antibody is made more effectively to penetrate into lesions positionPress down African swine fever virus pathogen of going out, and the cells play cellular immunity infected by African swine fever virus is entered by encytosisEffect；To further promote therapeutic index.
5. the comprehensive prevention and treatment of Multiple Antibodies optimum organization: the present invention provides 15 kinds of genotype and 15 kinds of anti-Africa of protein typeSwine fever virus and CD dual-target pig source antibody, existing full price, also there is multivalence, more there is popular unit price.Actually answeringIn choosing can be optimized according to the model feature and epidemic situation of local popular African swine fever virus and the state of an illness of morbidity swineryIt selects, using most economical means, achievees the purpose that effectively prevent.
Specific embodiment
Below with reference to embodiment, to anti-African swine fever virus of the invention and CD dual-target pig source antibody and combinations thereofObject, preparation method and application are described further:
Then above-mentioned double targeting antibodies pigs source is carried out scale fermenting and producing antibody egg by Chinese hamster ovary celI by the present inventionIt is white, or simultaneously mass production high-purity contains anti-African swine fever virus and CD dual-target pig source for expression in insect cell expression systemChange the carrier of antibody, then using the anti-African swine fever virus of high-purity of albumen or gene forms and the suppression of CD dual-target pig source antibodyIt goes out African swine fever virus, eliminates African swine fever virus to reach, prevent and treat the purpose of African swine fever.
The preparation method of above-mentioned anti-African swine fever virus and CD dual-target pig source antibody is as described below.
The method for preparing double targeting antibodies has hybridoma technology, chemical crosslink technique and technique for gene engineering etc..The present invention withIllustrate for hybridoma technology and technique for gene engineering, but is not limited to following explanation.
S0, preparation double cross oncocyte:
Utilize five kinds of African swine fever virus VP73, P54, P72, A238L, CD2V etc. representative antigen proteins or African pigThe other protein expressions of pestivirus are in eukaryocyte or prokaryotic cell and purified, or by African swine fever virus VP73,The gene of five kinds of representativeness antigen proteins such as P54, P72, A238L, CD2V or the gene point of the other albumen of African swine fever virusIt is not inserted into the genome of the viral vectors such as papovavirus, adenovirus, herpesviral, poxvirus, RNA virus, building is sameWhen expression VP73, P54, P72, A238L, CD2V or the other albumen of African swine fever virus recombinant virus and purified, obtainTo the recombinant virus of purifying；
Using Bacillus coli expression pig CD3 recombinant protein or other type pig CD recombinant proteins (such as CD2 and CD8) and purifyPig CD3 recombinant protein or other type pig CD recombinant proteins (such as CD2 and CD8)；
By in recombinant protein one of which (eukaryocyte or procaryotic cell expression and purify VP73, P54, P72,Any one recombinant protein in five kinds of A238L, CD2V etc. representative antigen proteins or the other albumen of African swine fever virus),The recombinant virus either expressing VP73, P54, P72, A238L, CD2V or the other albumen of African swine fever virus and purifying, with pigOne of CD3 recombinant protein or other type pig CD (such as CD2 and CD8) recombinant protein, which are uniformly mixed, adds adjuvant, by normalMouse is immunized in rule method, then cell fusion, and one of anti-African swine fever virus and CD dual-target pig source can be secreted by being madeThe hybridoma of antibody.
The clone of S1, variable region sequences and constant-region sequences: extracting the total serum IgE of the double cross oncocyte, and reversion is recordedStandby cDNA expands the light chain of mouse antibody variable region, the sequence of heavy chain respectively；Lymphocyte is taken to extract its total serum IgE, reversion is recordedStandby cDNA, expands light chain, the sequence of heavy chain of pig source antibody constant region respectively；
The clone of light chain and heavy chain full-length gene: source of mouse antibody's light chain variable region described in overlap-extension PCR and pig source antibodyConstant region of light chain obtains mouse-pig chimeric antibody light full-length gene order；Source of mouse antibody heavy chain variable region described in overlap-extension PCR andPig source heavy chain constant region obtains mouse-pig chimeric antibody heavy full-length gene order；
Wherein, according to the light chain of mouse antibody variable region and sequence of heavy chain and the light chain and heavy chain sequence of pig source antibody constant regionColumn design specific primer；
Expand the sequence of the light chain of mouse antibody variable region, heavy chain respectively using primer LV-F/LV-R and HV-F/HV-R；The light chain of pig source antibody constant region, sequence of heavy chain are expanded respectively using primer HC-F/HC-R and LC-F/LC-R；
The source of mouse antibody's light chain variable region and pig source antibody light chain are expanded using primer LV-F/LC-R overlap-extension PCRConstant region；The source of mouse antibody heavy chain variable region and pig source heavy chain of antibody are expanded using primer HV-F/HC-R overlap-extension PCRConstant region.
S2, anti-African swine fever virus and CD dual-target pig source antibody are prepared: by the mouse-pig chimeric antibody light overall lengthGene order and the mouse-pig chimeric antibody heavy full-length gene order are cloned in genophore, and purifying obtains gene after amplificationThe anti-African swine fever virus of type and CD dual-target pig source antibody；Alternatively, by the mouse-pig chimeric antibody light full-length gene sequenceColumn and the mouse-pig chimeric antibody heavy full-length gene order are cloned in expression vector and express in expression system, obtain eggThe anti-African swine fever virus of white type and CD dual-target pig source antibody.
Design and constructed using the gene clone method of conventional molecular biology, express that can to target African swine fever simultaneously sickThe pig source dual-target antibody of poison and CD (other type pig CD recombinant proteins such as pig CD3 recombinant protein or CD2 and CD8).
Wherein, the preparation process of the anti-African swine fever virus of genotype and CD dual-target pig source antibody includes: the mouse-Pig chimeric antibody light full-length gene order and the mouse-pig chimeric antibody heavy full-length gene order, are carried with virus geneBody or non-viral gene vector are cloned, and are fermented and are mass produced by insect cell, and the anti-of genotype is obtained after purifiedAfrican swine fever virus and CD dual-target pig source antibody；
The virus gene carrier includes baculoviral；The non-viral gene vector include Plasmid DNA, minicircle dna,Naked DNA, liposome and liposome complex, cation superpolymer, ligand-mediated targeting vector, episomal vector.
Wherein, the preparation process of the anti-African swine fever virus of protein type and CD dual-target pig source antibody include: will be describedMouse-pig chimeric antibody light full-length gene order and the mouse-pig chimeric antibody heavy full-length gene order are cloned in table respectivelyRecombinant plasmid is obtained under up to two promoters of carrier pFast-Bac-Dual；By the Transfected Recombinant Plasmid Chinese hamster ovary celI orIt is expressed in insect cell expression system, anti-African swine fever virus and the CD dual-target pig sourceization that protein type is obtained after purified are anti-Body.
The mouse-pig chimeric antibody light full-length gene can also be cloned in expression using I/EcoR of BamH, I double digestionUnder a promoter of carrier pFast-Bac-Dual；By the mouse-pig chimeric antibody heavy full-length gene using Xho I/Under another promoter of I double digestion of Nhe.
In anti-African swine fever virus provided by the invention and CD dual-target pig source preparation method for antibody, further includes:
Expand culture: selecting highly expressed stable transfected cells strain, is fermented by the cell reactor amplified step by step, filteringIt collects supernatant and removes cell and fragment；
Antibody purification: the supernatant is captured to the anti-African pig of genotype or protein type by cation exchange chromatographyPestivirus and CD dual-target pig source antibody, are further purified and are refined by anion exchange chromatography, then pass through ultrafiltration sideMethod carries out buffer exchange, and anti-African swine fever virus and the CD dual-target pig sourceization that high-purity genotype or protein type is made are anti-Body.
In the preparation method of anti-African swine fever virus provided by the invention and CD dual-target pig source antibody, using as followsThe anti-African swine fever virus and CD dual-target pig source antibody of genotype made from operation processing or protein type: protective agent system is addedStandby Cheng Kangti or antibody protein liquid preparation or liposomal processing are prepared into nano liposomes antibody or antibody protein, orPerson is dried to antibody or antibody protein dry powder.
Wherein following methods can be used in the production of nano liposomes, and the present invention is not limited to this method as example:
First the anti-African swine fever virus and the source antibody low temperature drying of CD dual-target pig of genotype or protein type are made dryPowder adds in micronizer crushing of milling, be processed into size between 1-100nm, granularity be more than >=15000 mesh ultra microParticle.Then, lecithin and cholesterol are dissolved in ether, then by nanosizing genotype or the anti-African swine fever virus of protein type4mmol/L phosphate buffer (PBS) is added with CD dual-target pig source antibody and is made into antibody-solutions, ultrasonic treatment 2min is (everyHandle 0.5min, interval .0.5min), for decompression rotary evaporation to being in gel, vortex oscillation turns gel in a water bath immediatelyPhase is further continued for evaporation and eliminates ether, and then ultracentrifugation (35000r/min, 30min) is separated off the antibody not being packed in, precipitatingIt is washed with water secondary, centrifugation must precipitate, be diluted with 10mmol/L PBS, and the anti-African swine fever for obtaining genotype or protein type is sickPoison and CD dual-target pig source antibody nano liposomes.
In addition, the composition includes anti-African swine fever virus above-mentioned and the bis- targets of CD the present invention also provides a kind of compositionMark pig source antibody and pharmaceutically acceptable auxiliary material.It should be noted that the composition can be by anti-Africa above-mentionedSwine fever virus and CD dual-target pig source antibody are also possible to the dynamic guarantor production of preparation plus the various pharmaceutical preparations etc. of auxiliary material preparationProduct and sterile products etc..
The anti-African swine fever virus and CD dual-target pig source antibody of genotype above-mentioned or protein type or nano liposomesVarious stabilization formulations can be made in the anti-African swine fever virus and CD dual-target pig source antibody changed, including but not limited to following systemsAgent:
Preferably, auxiliary material include it is a kind of in excipient, filler, solvent, cosolvent, surfactant and capsule auxiliary material orIt is a variety of.
Preferably, said preparation is injection, spray (disappears for environment and means of transport disinfection and pig farm and slaughterhousePoison and purification), pulvis (can be made into pig feed additive), tablet, oral solutions, pig mouth spraying agent, nasal spray or capsule etc..
The anti-African swine fever virus and CD dual-target pig source antibody of genotype above-mentioned or protein type or nano liposomesAt least one of water for injection injection and powder-injection is made in the anti-African swine fever virus and CD dual-target pig source antibody changed.ItsIt can be used for following purposes: preventing and treating African swine fever, the purification of pig farm swinery is infected swinery by African swine fever virusTreatment.
Alternatively, anti-African swine fever virus and CD dual-target pig source antibody or the nanometer rouge of genotype above-mentioned or protein typeThe anti-African swine fever virus of plastid and CD dual-target pig source antibody addition auxiliary material or base-material are made liquid drugs injection and powder-injection, coagulateAt least one of glue, washing lotion, tablet, effervescent tablet, capsule, soft capsule can be prepared for eliminating African swine fever virus infectionAnd prevent and treat the biological medicament of African swine fever disease.Since African swine fever virus can be survived in feedstuff more than 30 days,Therefore, anti-African swine fever virus and CD dual-target pig source antibody dry powder can be added in pannage, produce it is a kind of prevent it is non-The bio-safety feed and feed addictive of continent swine fever.
It is described in detail below by specific embodiment.
Embodiment 1: anti-African swine fever virus and CD dual-target pig source antibody are to VP73, P54, P72, A238L, CD2V etc.The antibody combination bioactivity of five kinds of representative antigen proteins.
VP73, P54, P72, A238L, CD2V is respectively adopted as detection antigen, with " ELISA " method (enzyme-linked immunization)The antibody titer of detection obtained anti-African swine fever virus and CD dual-target pig source antibody, as a result as shown in the table:
Note: anti-African swine fever virus and CD dual-target pig source antibody-solutions concentration in test sample are 1mg/mL.
It can be seen that from the above testing result, prepared anti-African swine fever virus and CD dual-target pig source antibody are to there is generationTable VP73, P54, P72, A238L, CD2V proteantigen has very high antibody combination potency.
Embodiment 2: the antibody combination bioactivity of anti-African swine fever virus and CD dual-target pig source antibody to CD.WithFor CD3 antigen.
Using CD3 as detection antigen, with the obtained anti-African swine fever disease of " ELISA " method (enzyme-linked immunization) detectionThe antibody titer of poison and CD dual-target pig source antibody, as a result as shown in the table:
Note: anti-African swine fever virus and CD dual-target pig source antibody in test sample
Solution concentration is 1mg/mL.
It can be seen that from the above testing result, prepared anti-African swine fever virus and CD dual-target pig source antibody are to CD3Antigen has very high antibody combination potency.
The anti-African swine fever virus of embodiment 3:[and CD dual-target pig source antibody injection] prevent and treat African swine fever diseaseThe animal experiment of poison infection.
1. experimental animal: African swine fever virus antigen and double-negative 30 ages in days (12kg) piglet 30 of antibody, random pointIt is three groups, blank control group, saline control group, antibody prevention group, Antybody therapy group.
2. experimental enviroment requires:Bio-safetyPrevention room (It is indoorIn negative pressure state) it carries out.
3. test drug: 1. antibody prevention groups and Antybody therapy group use [anti-African swine fever virus and CD dual-target pigSource antibody injection]；2. saline control group physiological saline injection.
4. test method:
(1) viral dilution method dilutes African swine fever virus positive serum with PBS so that every milliliter of serum dilution instituteIt is 1uL containing former serum amount, is analyzed through quantitative fluorescent PCR, contained African swine fever virus copy number is 20,000, and every pig attacks poisonDosage is 1mL serum dilution.
(2) every pig of malicious method is attacked to carry out attacking poison by injection original serum amount 1uL.
Blank control group (not attacking poison), saline control group (respectively inject a physiology in second day and the 7th day after attacking poisonSalt water, every pig per injection 2mL), antibody prevention group (attacks and injects [anti-African swine fever virus and CD dual-target pig on the day before poisonSource antibody injection], every pig per injection 2mL), Antybody therapy group (it is primary [anti-to attack each injection in second day and the 7th day after poisonAfrican swine fever virus and CD dual-target pig source antibody injection], every pig per injection 2mL).
(3) blood sampling detection on the day before attacking poison and attack after poison blood was collected within the 1st, 2,7,10,21 day and detection.
5. test result
(1) Symptom Observation
(2) African swine fever virus antigen detects
6. conclusion:
(1) [anti-African swine fever virus and CD dual-target pig source antibody injection] can prevent piglet by African swine fever virus senseDye, protective rate is up to 100%；
(2) [anti-African swine fever virus and CD dual-target pig source antibody injection] can treat by African swine fever virus senseThe piglet of dye, cure rate is up to 100%.
Embodiment 4: anti-African swine fever virus and CD dual-target pig source antibody injection are produced
By obtained excellent pentavalent (containing 1A, 2A, 3A, 4A, 5A) anti-African swine fever virus of protein type and CD dual-target pig sourceChange antibody and a kind of novel injection agent is made, for preventing and treating African swine fever.Specific formula and production technology are as follows:
Formula
Technique
(1) formula ratio water for injection is added into 0.1% needle-use activated carbon, 60 DEG C of stirring 15min；Decarbonization filtering；Filtrate is closed100 DEG C of 30min are heated, are cooled to room temperature spare；
(2) distinguish formula ratio biphosphate sodium-hydrate and sodium phosphate dibasic heptahydrate while stirring andIn the sterilized water for injection that the charcoal treated that sodium chloride addition accounts for formula ratio 90% is crossed, dissolve by heating；Separately take polysorbateEster -80 is added PEG400 and mixes；It is added in above-mentioned mixed liquor under stiring；Again plus 0.1% needle-use activated carbon, 60 DEG C of stirrings15min, decarbonization filtering；100 DEG C of 30min of filtrate airtight heating, are cooled to room temperature spare, obtain mixed liquor A；
(3) anti-African swine fever virus and the addition of CD dual-target pig source antibody purification liquid are accounted for into formula ratio while stirringThe sterilized water for injection that 10% charcoal treated is crossed sufficiently dissolves, obtains anti-African swine fever virus and CD dual-target pig sourceAntibody-solutions B.
(4) antibody-solutions B is added in mixed liquor A while stirring with thread, sufficiently dissolves, obtains solution C.
(5) with the pH value of the NaOH solution adjusting solution C of 0.1mol to 6.0~7.5.
(6) use sterilized 0.45 μm 0.22 μm of miillpore filter of series connection by solution C filtration sterilization.In sterile filling and sealing machineEncapsulating is in sterile chamber；Lamp inspection, leak detection, lettering, packaging factory.
Embodiment 5: anti-African swine fever virus and CD dual-target pig source antibody disinfection spray solution are produced (for pigField, slaughterhouse, haulage vehicle disinfection and pig farm purification are used, and can also be divided in aerosol can, be made pig mouth spraying agent and nasal sprayAfrican swine fever virus infection is prevented and treated for swinery).
Formula
Technique
(1) formula ratio seaweed essence, pharmaceutical grade glycerol, essence and 403,503 foaming agents are sterilized with ultraviolet lightDisinfection 24 hours, sterile sealing is spare；
(2) formula ratio distilled water is heated to 90 DEG C, stopped 15 minutes；60 DEG C are cooled to, is successiveed while stirringSeaweed essence is added and pharmaceutical grade glycerol, low speed whisk 30 minutes, until being completely dissolved, is cooled to room temperature, obtains solution A；
(3) essence (rose essence) is added in solution A while stirring, stirs at low speed 60min, until completely moltenSolution, obtains solution B；
(4) 80 DEG C are heated to by 403, are then added dropwise to in 403, stir at low speed slowly by 503 while stirring30min obtains solution C；
(5) it keeps solution C temperature at 80 DEG C, is added dropwise to peppermint oil in solution C slowly while stirring, low speed stirs60min is mixed, solution D is obtained；
(6) solution D is heated to 90 DEG C of high-temperature sterilization 5min, then cooled to room temperature；
(7) solution B is slowly added in solution D while stirring, stirs at low speed 60min；As not formed homogeneous is steadyFixed milky solution then needs to extend mixing time, obtained solution E；
(8) double targeting antibodies of anti-human papilloma virus (anti-HPV) (HPV) and AntiCD3 McAb humanization are slowly added while stirringInto solution E, 60min is stirred at low speed；Such as the milky solution of not formed isotropic stable, then need to extend mixing time, obtained solutionF；
(9) it with the pH value of pH meter measurement solution F, is adjusted using citric acid or pH4.5 disodium hydrogen phosphate-citrate buffer solutionPH value is to 4.5 ± 0.1；
(10) stand a night, until top foam all disappear it is molten after, then solution F liquid is divided in the plastics that cleaning and sterilizing is crossedIn container, labelled factory.
Embodiment 6: anti-African swine fever virus and CD dual-target pig source antibody tablet are produced (with by African swine fever virus senseIt catches an illness the emergency aid and treatment of pig)
Formula
Technique
(1) it is spare twice to cross 60 meshes for D-sorbite；
(2) carboxymethyl cellulose, which is scattered in 30% ethyl alcohol, is made 1% ethanol solution；
(3) by appropriate (2) the item softwood of (1) item, the granulation of 14 mesh screens, 60 DEG C of aeration-dryings, 18 mesh sieves；With 40Mesh sifts out the anti-African swine fever virus of appropriate fine powder and protein type and CD dual-target pig source antibody mixes well；
(4) magnesium stearate is admixed again, is uniformly mixed together with particle by the gross, closed 4 hours or more；
(5) tabletting machine, every 600mg；
(6) after the assay was approved, it packs, tests factory entirely.
The information that the present invention is included, under the spirit and scope without departing from appended claims of the present invention, this hairThe bright accurate description of various deviations, is obvious for those skilled in the art related to the present invention.The present inventionIt is not regarded as to be limited in the range of defined program, property or composition, because preferred embodiment and other descriptions are only usedThe particular aspects of invention are provided at present in explanation.For in chemistry, biochemistry or those skilled in the relevant art, realizeThe present invention should all belong to the protection domain of appended claims of the present invention interior in the description mode of various modifications.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,Within all these improvement or transformation should all belong to the protection domain of appended claims of the present invention.