CN106344920A - Adjuvant for vaccines and application thereof - Google Patents

Adjuvant for vaccines and application thereof Download PDF

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Publication number
CN106344920A
CN106344920A CN201510420446.4A CN201510420446A CN106344920A CN 106344920 A CN106344920 A CN 106344920A CN 201510420446 A CN201510420446 A CN 201510420446A CN 106344920 A CN106344920 A CN 106344920A
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antigen
adjuvant
vaccine
pig
acrylic acid
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CN106344920B (en
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张许科
孙进忠
王莹
田克恭
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Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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Abstract

The invention provides an adjuvant for vaccines. The adjuvant comprises polyacrylic acid polymer and cellulose ether, and further comprises an immunoenhancer. The adjuvant for vaccines provided by the invention can effectively promote effects of the vaccines, and not only can be used as an adjuvant for inactivated vaccines, but also can be used as a diluent for living vaccines. A vaccine composition containing the adjuvant is used for only once immunization to achieve the effect of twice immunization by using a commercial product, so as to greatly simplify the immune process and also avoid the occurrence of side effects; the adjuvant provided by the invention also makes protein stable without degradation or irreversible changes and can be used as a protein stabilizer.

Description

A kind of vaccine adjuvant and its application
Technical field
The invention belongs to veterinary biologicses technical field is and in particular to be used for enhancement antigen immunity The adjuvant of response.
Background technology
Antibacterial, virus and parasitic infection are distributed widely in humans and animals, are drawn by these infectious agent The disease rising generally has resistance to chemical substances such as antibiotic, therefore not effective treatment side Method.Therefore, this area is more and more many controls infection using vaccinology method, particular by With pathogen, the pathogen through inactivation or its product lived or through genetic engineering means by cause of disease Body protein subunit is prepared into vaccine to carry out vaccination and carrys out inducing specific immunity.However, working as Individually give to be difficult to during some antigen to produce enough stimulations to immune system.Therefore, must The protectiveness to obtain q.s for the immunological adjuvant that can make immune response increase must be added Antibody.
Immunological adjuvant is the requisite ingredient of vaccine, can not only affect body and vaccine is exempted from The intensity of epidemic disease response, and maximally effective type of immune response can be induced for special pathogen (mbow ml,de gregorio e,valiante nm,et al.new adjuvants for human vaccines.current opinion in immunology,2010,22(3):411-416). At present, the immunological adjuvant type of research is a lot, such as oily adjuvant, Freund adjuvant, microorganism and its (dey ak, the srivastava such as metabolite, nucleic acid and the like, cytokine, liposome ik.novel adjuvants and delivery systems for enhancing immune responses induced by immunogens.expert review of vaccines,2011,10: 227-251), but due to there is different degrees of toxic and side effects or potential safety hazard etc., some can not The defect that avoids and be difficult to practical application (mbow ml, de gregorio e, valiante nm, rappuoli r.new adjuvants for human vaccines.current opinion in immunology,2010,22(3):411-416；batista-duharte a,lindblad eb, oviedo-orta e.progress in understanding adjuvant immunotoxicity Mechanisms.toxicology letters, 2011,203 (2): 97-105), lead to these adjuvants The demand of new generation vaccine development far can not be met.Therefore, find safely, effectively, new Immunological adjuvant is a focus (schijins vejc, the lavelle ec. of current vaccines research field teends in vaccine adjuvants.expert review of vaccines,2011,10(4): 539-550).And, immunological adjuvant research has been listed in the pioneer field of vaccine research (harandi am,medaglini d,shattock rj.vaccine adjuvants:a priority for vaccine research.vaccine,2010,28(12):2363-2366；harand am, brewe j,schijn v.conference scene:recent advancements in immunopotentiators for modern vaccines.immunotherapy,2011,3(11): 1297-1301).
The vaccine (as Mycoplasma hyopneumoniae bacterin, Haemophilus parasuis bacterin) of commercialization at present Be by the culture medium containing serum grow gained organism be prepared from, cause for Serum component present in immune material is (as immune complex or non-immunogenic specific protein There is (see Chinese patent cn104334186a) in induction side reaction in vain).
In addition, in the research of veterinary biologicses technical field clinical trial, finding albumen Repeatability and stability are particularly significant, and good stability is the reliable important symbol of data. But, in clinical trial, the less stable of some albumen, easily degrade under general condition Or degeneration, directly affect the accuracy of result of the test.Therefore, the stability how keeping albumen is Key technology in clinical trial.
Content of the invention
The technical problem underlying that the present invention solves is to provide a kind of effective vaccine adjuvant, can The immune efficacy of effective enhancement antigen.Described vaccine adjuvant is used in conjunction with antigen, Neng Goujian Change immune programme for children it is only necessary to primary immune response can reach the effect of commercial vaccine immunity twice.
The present invention relates to a kind of vaccine adjuvant, described adjuvant includes acrylic acid series polymeric compounds and fibre The plain ether of dimension.
The invention still further relates to a kind of vaccine adjuvant, described adjuvant include acrylic acid series polymeric compounds and Cellulose ether and immunostimulant；Described immunostimulant is the compositionss containing saponin, preferably For immunostimulating complex.
The invention still further relates to a kind of vaccine combination, wherein, described vaccine combination includes immunity The acrylic acid series polymeric compounds of the antigen of amount and the present invention and cellulose ether adjuvant.
The invention still further relates to a kind of vaccine combination, wherein, described vaccine combination includes immunity The acrylic acid series polymeric compounds of the antigen of amount and the present invention, cellulose ether and immunostimulant adjuvant.
Another technical problem that the present invention solves is to provide a kind of antigen protein stabilizer, described anti- Former protein stabiliser long-term in preparation process and under felicity condition can keep original point of albumen Son amount, structure and/or biological activity.
The present invention relates to a kind of antigen protein stabilizer, described stabilizer includes acrylic polymeric Thing and cellulose ether.
The invention still further relates to a kind of antigen protein stabilizer, it is poly- that described stabilizer includes acrylic acid seriess Compound, cellulose ether and the compositionss containing saponin.
The adjuvant of the present invention is antigenic content is relatively low, single immunization when animal also can be made to obtain preferably Immunne response level, decrease the stress of pig, reduce the generation of side reaction；Described Adjuvant can be also used for diluting live vaccine, and makes live vaccine with intramuscular injection form immunity pig, Make easy and simple to handle.Additionally, described adjuvant also acts as protein stabilized liquid preserves albumen, make albumen Stablize non-degradable or irreversible change does not occur.
Specific embodiment
Hereinafter, embodiments of the present invention are illustrated.
Term " adjuvant " refers to, when with antigen together with administration, make the immunity to this antigen for the experimenter The compound of increased response.
Term " acrylic acid series polymeric compounds " refers to any polymer containing acrylic acid seriess part or copolymerization Thing, including but not limited to polyacrylic acid, polymethylacrylic acid, polymethacrylates, polypropylene Amide, polyacrylate, polyacrylonitrile and polyacrylic Arrcostab.Acrylic acid series polymeric compounds Example include as poly- (acrylamide -co- butyl methacrylate), acrylic acid-methacrylic acid, Acrylic acid-acrylamide and poly- (methacrylate).The example bag of commercially available acrylic acid series polymeric compounds Include carbopol carbopol (Goodrich Corporation b.f.goodrich co., Ohio, USA gram profit Husband orchid cleveland ohio), Kappa match carboset (Goodrich Corporation b.f.goodrich co., Ohio, USA Cleveland cleveland ohio), Ni Aoke neocryl (peace myriagram company Avecia inc., Wilmington,State of Delaware, US wilmington del.) and Eudragit RS 100 eudragit (rom scientific ＆ technical corporation rohm tech, inc., U.S. Ma Shazhusai state horse two steps on malden mass.).The acrylic acid series polymeric compounds being particularly well-suited in adjuvant of the present invention are Carbomer (carbomer, trade name carbopol), generally also refers to and known in described technical field It is the acrylic acid water-soluble polymer crosslinked with polyallylsucrose, these compounds are shown in Phameuropa (1996,8 (2)), those skilled in the art are referring also to United States Patent (USP) Us2909462, which depict this kind of acrylic acid series polymeric compounds, and it is handed over poly- hydroxylated compound Connection, described compound has at least 3 hydroxyls, preferably more than 8,3 hydroxyls of wherein at least The hydrogen atom of base is had the unsaturated lipid alkyl of at least 2 carbon atoms (aliphatic radical) and is taken Generation, the preferably group containing 2-4 carbon atom, such as vinyl, pi-allyl and other olefinic Unsaturated group (ethylenically unsaturated group).Described unsaturated group itself can Comprise other substituent groups, such as methyl.These products are sold with the name of carbopol, with bf goodrich (ohio, usa) is especially suitable, they with allyl sucrose or with Allyl pentaerythritol (allyl Pentaerythritol) crosslinked, can be mentioned that among these carbopol 941,980,981,940,2020, 974p, 934p and 971p, most preferably with carbopol 971p.
Term " cellulose ether " refer to cellulose ether derivative such as carboxymethyl cellulose cmc, Hydroxyethyl cellulose hec, hydroxypropyl methyl cellulose hmpc, also including its salt is carboxylic Methylcellulose salt.Described carboxymethyl cellulose salt include but is not limited to sodium carboxymethyl cellulose, Potassium carboxymethylcellulose, carboxymethylcellulose calcium, preferably sodium carboxymethyl cellulose.
Term " sodium carboxymethyl cellulose " (sodium carboxymethylcellulose) is to be based on The biopolymer of cellulose, and by right in the sodium hydroxide solution containing sodium monochloroacetate Cellulose carries out being esterified producing (feddersen etc., sodium carboxymethyl cellulose sodium Carboxymethylcellulose, in whistler the rl, " industrial glue of bemiller jn chief editor Many carbohydrates and their derivative " industrial gums polysaccharides and their derivatives, Marcel dekker inc., new york, the third edition (1993): 537-578), give replacement Degree ds refers to esterified hydroxyl in glucose unit as the tolerance of degree of esterification, described substitution value The par of base.There are three reactive hydroxyls to allow to introduce three sodium carboxymethyl groups.Carboxylic The characteristic of sodium carboxymethylcellulose pyce depends on its substitution value and its degree of polymerization (belitz etc., lehrbuch Der lebensmittelchemie.springer-verlag, berlin, fourth edition, 1992), described The degree of polymerization refers to be connected to form the par of the monomer molecule of macromole in the course of the polymerization process.Root According to the present invention, preferred sodium carboxymethyl cellulose has the average molecular mass of 125000g/mol. Described " sodium carboxymethyl cellulose " is also included on the stricti jurise of its definition is not that carboxymethyl is fine The plain sodium of dimension, but because such as cellulose or other multiple sugar are by hydroxylating, carboxylated, esterification And/or be etherified the such as derivatization carrying out and there is the same or similar spy with sodium carboxymethyl cellulose Property, therefore, it is possible to the polysaccharide equally using.
A first aspect of the present invention is to provide a kind of vaccine adjuvant, and described adjuvant includes propylene Sour based polymer and cellulose ether.
As one embodiment of the present invention, in the vaccine adjuvant of the present invention, described propylene Sour based polymer includes polyacrylic acid, polymethylacrylic acid, polymethacrylates, polypropylene Amide, polyacrylate, polyacrylonitrile and polyacrylic Arrcostab.
As a kind of preferred implementation of the present invention, in the vaccine adjuvant of the present invention, described Acrylic acid series polymeric compounds include carbopol, Kappa match, Ni Aoke neocryl, Eudragit RS 100 eudragit.
As one kind more preferably embodiment of the present invention, in the vaccine adjuvant of the present invention, institute State acrylic acid series polymeric compounds include carbopol 941,980,981,940,2020,974p, 934p And 971p.
As one embodiment of the present invention, cellulose ether includes carboxymethyl cellulose, carboxylic first Base sodium cellulosate, potassium carboxymethylcellulose, carboxymethylcellulose calcium, hydroxyethyl cellulose, hydroxyl Propyl methocel.
As one embodiment of the present invention, in the vaccine adjuvant of the present invention, described propylene Sour based polymer is calculated as 2-10mg/ml with Carbomer, and described cellulose ether is 0.1-8mg/ml.
As a kind of preferred implementation of the present invention, in the vaccine adjuvant of the present invention, described Acrylic acid series polymeric compounds are calculated as 4-10mg/ml with Carbomer, and described cellulose ether is 4-8mg/ml.
Term " immunostimulant " (immunopotentiator) refers to that itself does not have immunogen Property, but can be used for stimulating immune system and there is to other immunogenic material answer to improve Answer.When being used together or be applied to same target respectively such as purpose antigen, can be direct or indirect Ground adjusts the immunoreation of described antigen induction or changes the immunoreation type (inclusion of this antigen Such as humoral immunization, cellular immunization).Described immunostimulant including but not limited to contains saponin Compositionss, Lactobacillus, propolis, vitamin e, levamisole, transfer factor, thin in vain Born of the same parents' interleukin, interferon, thymosin, transfer factor, globulin, bacillus calmette-guerin vaccine or a combination thereof.
Term " compositionss containing saponin " can comprise purification or partial purification preparation such as qs21 (business The name of an article is stimulontm), and lipid formulations such as immunostimulating complex iscom.Described Saponin (saponin), also known as saponin, Saponin, alkali soap body or saponarin, is sterol glycoside and three The heterogeneous group of terpene glycosides is it is seen that in the skin of various plants, leaf, stem, root, even spend middle bag Include but be not limited to quilaja saponin, ginseng saponin, panax notoginseng saponin, Platycodon grandiflorum saponin, astragalus saponin, achyranthes saponin, Mentioned saponin (sun hx, xie y, ye in polygala saponin, and the document such as sun yp.advances in saponin-based adjuvants.vaccine,2009,27(12): 1787-1796), preferably quilaja saponin.Described purification or partial purification preparation can use Hplc and rp-hplc purification, has identified specific purified components using these technology, Including quila, qs21, qs7, qs17, qs18, qh-a, qh-b, qh-c, At least one of vaxsap, supersap, gpi-0100, qp uf1000.
Term " immunostimulating complex " (immunostimulating complex, iscom) is The immunostimulating complex referring to not contain antigen is iscom matrix (iscom-matrix) similarly can play the effect of immunological adjuvant after mixing with vaccine, be In glucosides such as Triterpenoid Saponins (specially quil a) and cholesterol, such as European Publication Number Epa109942 and 180564.Typical iscom is the distinct particle containing saponin, cholesterol, Further comprise phospholipid such as cephalin or lecithin, any of saponin can be used in In iscom.Preferably iscom comprises quila (South America quillaja saponin triterpene soap Glycoside), cholesterol, lecithin, mix covalent bond form, under Electronic Speculum be in cage or golf The immune complex of Qiu Shi.Method with regard to saponin and iscom and formation iscom refers to The documents such as barr (barr ig, sjolander a, cox jc.iscoms and other saponin based adjuvants.advanced drug delivery reviews,1998,32(3):247-271). Prepare the iscom for the present invention with standard technique, these technology are well known in the art, It is described in such as U.S. Patent number 4981684,5178860,5679354,6027732, Europe Publication number epa109942,180564,231039, and (coulter a, the wong such as coulter ty,drane d,et al.studies on experimental adjuvanted influenza vaccines:comparison of immune stimulating complexes(isxomstm)and oil-in-water vaccines.vaccine,1998,16(11-12):1243-1253).
As one embodiment of the present invention, in the vaccine adjuvant of the present invention, described adjuvant Also include immunostimulant, described immunostimulant includes compositionss containing saponin, active lactic acid Bacterium, propolis, vitamin e, levamisole, transfer factor, interleukin, interferon, Thymosin, transfer factor, globulin, bacillus calmette-guerin vaccine or a combination thereof.
As a kind of preferred implementation of the present invention, in the vaccine adjuvant of the present invention, described Immunostimulant includes the compositionss containing saponin, and the described compositionss containing saponin are preferably immunostimulation Complex.The described compositionss containing saponin include quila, qs21, qs7, qs17, qs18, Qh-a, qh-b, qh-c, vaxsap, supersap, gpi-0100 and qp uf1000, At least one of immunostimulating complex.
As one embodiment of the present invention, in the vaccine adjuvant of the present invention, described propylene Sour based polymer is calculated as 2-10mg/ml with Carbomer, and described cellulose ether is 0.1-8mg/ml, and The described compositionss containing saponin are calculated as 0.1-10mg/ml with quila.
As a kind of preferred implementation of the present invention, in the vaccine adjuvant of the present invention, described Acrylic acid series polymeric compounds are calculated as 4-10mg/ml with Carbomer, and described cellulose ether is 4-8mg/ml, With the described compositionss containing saponin, 4-10mg/ml is calculated as with quila.
Term " immune effective dose " also known as immunoprotection amount or produce immunne response effective dose, be Can in receiver's body effective inducing immunogenic response amount.Described immunne response may be enough to For diagnostic purposes or other test, or be likely to be suitable for prophylactic sign or symptom, Including the unfavorable healthy result caused by the infection being caused by pathogen or its complication.Body fluid Immunity or all can be induced by both cell-mediated immunity or this.Animal is to immunogenicity The immunne response of compositionss can by for example measurement antibody titer, lymphocyte proliferation assays and Connect assessment, or directly assessed by monitoring sign or symptom after being attacked with wild type strains, And the protective immunity that should be provided by vaccine can be by measuring the clinical symptom of such as experimenter Such as mortality rate, the minimizing of sickness rate, Temperature numerical, experimenter's general physiological state and be totally good for Health to be assessed with performance.Described immunne response may include but be not limited to inducing cell and/or body fluid Immunity.
Term " antigen ", also known as immunogen, refers to any material stimulating immunne response, is a kind of The molecule that can be selectively bound by the antibody is additionally it is possible to induction produces the humoral immunization of b- and/or t- cell Response and/or cellullar immunologic response, also have one or more epi-positions (b- and t- epi-position). Described antigen include killing, inactivation, attenuation the or modified antibacterial of work, virus or Parasite, or its cultured cells preparation, supernatant；Also include subunit antigen, described sub- single Position antigen is other or its any combination of polynucleotide, polypeptide, recombinant protein, synthesis Peptide, protein extract, cell, tissue, polysaccharide, lipid or its fragment, also include resisting The for example anti-experimenter's homologous idiotypic antibody of body or its fragment and can analogue antigen or antigenic determinant (table Position) synthesis peptide mimotope mimotopes.Wherein, described killing, inactivation antibacterial, Virus or parasite refer to containing the infectious organisms being no longer able to duplication or growth or cause of disease Body, pathogen can include freeze thawing, chemical treatment (such as with thimerosal or Fu Er by various methods Malin is processed), sonication, irradiation, hot or any other enough to stop organism from replicating or raw Long common method maintains its immunogenicity to realize inactivation；Described modified work thin Bacterium, virus or parasite refer to promote its variation under artificial condition, lose pathogenic but retain The antigen of immunogenicity, procreation ability and remaining virulence, has certain journey in animal body after inoculation The breeding of degree or duplication, but be not result in fall ill.
Term " pig circular ring virus antigen " refers to containing at least one pig circular ring virus (porcine Circovirus, pcv) antigen forms any combinations thing, described pig circular ring virus antigen can lure Lead, stimulate or strengthen the immunne response of opposing Infection of Porcine circovirus, described antigen forms include But it is not limited to inactivateing, alive or subunit antigen.Described pig circular ring virus antigen comprises sh Strain (preserving number is cgmcc no.23890, referring to Chinese patent cn101240264a), Sd strain (preserving number is cgmcc no.7707, referring to Chinese patent cn103421748a), D strain (preserving number is cgmcc no.7245, referring to Chinese patent cn103275938a), (preserving number is cctcc no.v201333, referring to Chinese patent for wh strain Cn103409374a), (preserving number is cgmcc no.6391, referring to China for zj/h strain Patent cn102787100a), dbn-sx07 strain (preserving number be cgmcc no.3064, Referring to Chinese patent cn101549155a), v201312 strain (preserving number be cctcc No.v201312, referring to Chinese patent cn103436498a), following combination can also be comprised Any one antigen such as Cimmeria company merial of thingPfizer pfizer 'sPcv2, the porcine circovirus 2 type of Harbin Wei Ke biotechnology development company Inactivated vaccine (lg strain), the 2 porcine circovirus of Zhejiang Nuobeiwei Biotechnology Co., Ltd. Type inactivated vaccine (zj/c strain), Boehringer Ingelheim company boehringer-ingelheim ingelvac
Term " mycoplasma hyopneumoniae antigen " refers to containing at least one mycoplasma hyopneumoniae Any combinations thing of (mycoplasma hyopneumoniae, mhp) antigen forms, described Mycoplasma hyopneumoniae antigen can induce, stimulate or strengthen the immunity of opposing mycoplasma hyopneumoniae infection Response, described antigen forms include but is not limited to inactivation, alive or subunit antigen, or Its culture supernatant.Described mycoplasma hyopneumoniae antigen comprises hn0613 strain, and (preserving number is Cctcc no.m2012230, referring to Chinese patent cn103083655a), j strain is (purchased from U.S. It is 25934 that state Type Tissue Collection atcc, atcc number), nj strain (preservation Number be cctcc no.m2012286, referring to Chinese patent cn103585622a), (preserving number is cgmcc no.8096, referring to Chinese patent for hdzk-mhp57 strain Cn103484414a), (preserving number is cctcc no.m2012431, ginseng for an306 strain See Chinese patent cn103740625a), dj-166 strain (preserving number be cgmcc no.4545, Referring to Chinese patent cn103184171a) and nm04/41259 strain (preserving number is Nm04/41259 strain, referring to Chinese patent cn102458462a), can also comprise following Auspicious times of suitable respisure of any one antigen of compositionss such as company of Harbin Pharmaceutical Group and auspicious times be suitable-and prosperous Respisureone, peace hectogram m+pac of Intervet company, Boehringer Ingelheim companyM.hyo, the mycogard of Portec Inc. of the U.S., Pfizer Inc. Respifend mh, the large Rui Fute-prosperous suvaxyn rising (Shanghai) animal health-care product company limited Mh-one, the pig gram of Cimmeria animal health company breathe heavily the happiness of sprintvac, Hai Bolai company Yifeng mypravac suis, the z strain live vaccine of domestic production, 168 plants of live vaccine and rm48 Strain live vaccine.
Term " haemophilus parasuises antigen " refers to containing at least one haemophilus parasuises Any combinations thing of (haemophilus parasuis, hps) antigen forms, described pair pig is thermophilic Blood bacteroides antigen Pigs Inoculated can induce, stimulate or strengthen the immunity of opposing haemophilus parasuises infection Response, described antigen forms include but is not limited to inactivation, alive or subunit antigen, or Its culture supernatant.Described haemophilus parasuises antigen includes well known to those skilled in the art facing The detached street strain of bed, comprises current 1-15 serotype of identification or cannot measure serum The haemophilus parasuises of type, hybrid antigen such as 4 type comprising at least two serotypes is (as js Strain, preserving number is cctcc no.m2011172, referring to Chinese patent cn103083655a) (such as zj strain, preserving number is cctcc no.m2011173, referring to Chinese patent with 5 types Cn103083655a hybrid antigen), can also comprise any one antigen of following compositionss Ingelvac hp-1, Hai Bolai company hipra as Boehringer Ingelheim company Animal before hiprasuis-glasser, Chengdu medical instruments factory of Zhongmu Industry Co., Ltd and Wuhan section Haemophilus parasuises inactivated vaccine (the 4 type md0322 strains+5 of biological product Co., Ltd Type sho165 strain).
A second aspect of the present invention is to provide a kind of vaccine combination, wherein, described vaccine group Compound includes the antigen of immunity amount and the adjuvant of the present invention.
As one embodiment of the present invention, in the vaccine combination of the present invention, described antigen Numerous including pig circular ring virus antigen, mycoplasma hyopneumoniae antigen, haemophilus parasuises antigen, pig Grow and breath syndrome virus antigen, PPV Antigen Using, Pasteurella multocida antigen, pig Streptococcal antigen, Staphylococcus hyicus antigen, bronchitis Boulder spy's bacterium antigen, lobar pneumonia Actinobacillus antigen, bacillus coli antigen, atrophic rhinitiss antigen, PRV (Pseudorabies virus) resist Former, Salmonella choleraesuls antigen, Salmonella enteritidis antigen, pig erysipelothrix rhusiopathiae antigen, Mycoplasma hyorhinis antigens, mycoplasma hyosynoviae antigen, pig Leptospira bacteria antigen, pig exhale Inhale road Bovine Coronavirus Antigen, Porcine epidemic diarrhea virus antigen, porcine rotaviruses antigen, pig biography Metachromia gastroenteritis virus antigen, pig thin circovirus virus antigen, inclusion-body rhinitis virus of pigs antigen, Intestinum Sus domestica disease In malicious antigen, encephalomyocarditis viruses antigen, swine flue antigen, CSFV antigen one Plant or multiple；And described adjuvant includes acrylic acid series polymeric compounds and cellulose ether.
As one embodiment of the present invention, in the vaccine combination of the present invention, described antigen Including above-mentioned antigen one or more；And described adjuvant includes acrylic acid series polymeric compounds, fibre The plain ether of dimension and the compositionss containing saponin.
As a kind of preferred implementation of the present invention, in the vaccine combination of the present invention, described Antigen is made up of pig circular ring virus antigen, mycoplasma hyopneumoniae antigen, haemophilus parasuises antigen, Described adjuvant includes acrylic acid series polymeric compounds and cellulose ether.
As a kind of preferred implementation of the present invention, in the vaccine combination of the present invention, described Antigen is made up of pig circular ring virus antigen, mycoplasma hyopneumoniae antigen, haemophilus parasuises antigen, Described adjuvant includes acrylic acid series polymeric compounds, cellulose ether and the compositionss containing saponin.
As a kind of most preferred embodiment of the present invention, in the vaccine combination of the present invention, institute State antigen to be inactivated by pig circular ring virus sh strain inactivation antigen, mycoplasma hyopneumoniae hn0613 strain Antigen, haemophilus parasuises 4 type js strain and 5 type zj strain inactivation antigens composition, described adjuvant Including acrylic acid series polymeric compounds, cellulose ether and immunostimulating complex.
As one embodiment of the present invention, in the vaccine combination of the present invention, described adjuvant Content be 5%-30%v/v.
As a kind of preferred implementation of the present invention, in the vaccine combination of the present invention, described The content of adjuvant is 10%-25%v/v.
Term " albumen " includes the albumen of chemosynthesis, the natural conjunction of the gene code of cultured cells The albumen becoming and the recombiant protein secreted by cell.Described recombiant protein is by molecular biosciences skill Art introduce cell transgenes encoding those, can by chemical method or translate after during Enzyme is modified.Described albumen includes animal proteinum, is those being produced by cell culture, Also include other source the such as expression such as insecticide, antibacterial, plant albumen, and mutation, Albumen that is artificial, synthesis, merging or being fitted together to.According to the present invention, described albumen includes But it is not limited to the vp60 albumen of rabbit hemorrhagic disease virus rhdv, Canine Parvovirus ppv Vp2 albumen, cap, rep albumen of pig circular ring virus pcv2, mycoplasma hyopneumoniae p46、p65、nrdf、p97r1、p102、pdha、xylf、p78、eutd、mhp145、 P132, mhp389 albumen, the pila of haemophilus parasuises hps, omp, hbpa, oppa, Pala albumen, the nsp2 of porcine reproductive and respiratory syndrome virus prrsv, gp3, gp4, Gp5, m, n albumen, the vp2 albumen of pig parvoviral ppv, Pasteurella multocida The hyae albumen of p.multocide, the 38kda of Streptococcus suis s.suis, saoa, mrp, Hm6, gapdh, omp40, virb4, sly albumen, Staphylococcus hyicus s, hyicus's Exha, exhb, exhc, exhd albumen, the p68 albumen of bronchitis Boulder spy bacterium bb, Omp20, ompw, omp27, omp27, ompa1 of Actinobacillus pleuropneumoniae app With ompa2 albumen, the k88 of escherichia coli e.coli, k99,987p, f41, f17, Ltb, orf353, tolc albumen, the pmt of atrophic rhinitiss ar, PRV (Pseudorabies virus) The hfq albumen of gb, gc, gd, ge of prv, Salmonella enteritidis s.enteria, pig is red Spaa, lipo albumen of speckle Erysipelothrix e.rhusiopathiae, mycoplasma hyorhinises mhr's P37 albumen, s, n albumen of PRCV (Porcine Respiratory Coronavirus) prcv, Porcine Epidemic Diarrhea S, m, n albumen of malicious pedv, vp4, vp6, vp7 egg of porcine rotaviruses prv In vain, s, m, n albumen of transmissible gastro-enteritis viruss tgev, pig thin circovirus virus ttsuv Cap albumen, the gb albumen of inclusion-body rhinitis virus of pigs pcmv, porcine encephalomyocarditis virus emcv Vp1, vp2,3ab albumen, ha, np, na, m, ns egg of swine influenza viruses In vain, one or more of e0, e1, e2 albumen of swine fever virus csfv.
Term " protein stabilized liquid " is that the stability instigating immunogenic protein composition is able to significantly Improve, in preparation process and under felicity condition, long-term (the such as several months is to the several years) is preferably super Spend 12 months (such as 24 months or 36 months) preserve after, keep the original molecular weight of albumen, At least the 80% preferably at least 90% of structure and/or biological activity is constant, keeps protein biology steady Qualitative, physical stability, chemical stability, will not form chemical degradation products, will not occur Irreversible change (is for example assembled, precipitates or degeneration).Described chemical degradation products and natural egg White structure is compared has potential lower biological value and/or potential more low immunogenicity.
A third aspect of the present invention is to provide a kind of antigen protein stabilizer.
As one embodiment of the present invention, the described antigen protein stabilizer of the present invention includes Acrylic acid series polymeric compounds and cellulose ether.
As a kind of preferred implementation of the present invention, the described antigen protein stabilizer of the present invention Including acrylic acid series polymeric compounds, cellulose ether and the compositionss containing saponin.
As a kind of preferred implementation of the present invention, the described antigen protein stabilizer of the present invention Including acrylic acid series polymeric compounds, cellulose ether and immunostimulating complex.
As one embodiment of the present invention, institute in the described antigen protein stabilizer of the present invention State acrylic acid series polymeric compounds and 2-10mg/ml is calculated as with Carbomer, described cellulose ether is 0.1-8mg/ml, and the described compositionss containing saponin are calculated as 0.1-10mg/ml with quila.
To further describe the present invention, advantages of the present invention and feature with reference to specific embodiment Will be with describing apparent.But these embodiments are only exemplary, not to the present invention Scope constitute any restriction.It will be understood by those skilled in the art that without departing from this Under bright spirit and scope can modifying to the details of technical solution of the present invention and form or Replace, but these modifications and replacement each fall within protection scope of the present invention.
The porcine circovirus 2 type sh strain to inactivate for the pig circular ring virus antigen in the embodiment of the present invention, Mycoplasma hyopneumoniae antigen is propped up former with the mycoplasma hyopneumoniae hn0613 strain inactivateing and the hyopneumoniae of living 168 plants of body, haemophilus parasuises antigen is with the 4 type haemophilus parasuises js strains that inactivate and 5 type pairs It is illustrated as a example haemophilus suis zj strain, but no matter under any circumstance all not this embodiment Constitute limitation of the invention.
In the embodiment of the present invention, pneumonopathy varying index is passed judgment on using 28 point-scores.Described 28 point-scores It is that pathological changes are observed according to the typical pulmonary of mycoplasmal pneumonia of swine, quantization is carried out to occurring degree and sentences Substantive sample pathological changes that are fixed, occurring including apex pulmonis leaf, lobus cardiacus, lobus diaphragmaticus, middle leaf, such as " meat sample Change ", " change of pancreas sample ".Pneumonia pathological changes standards of grading are: 2 sharp leaves, 2 lobus cardiacuses, 2 diaphragms Leaf, 1 middle leaf amount to 7 lobes of the lung, and each lobe of the lung full marks is 4 points, 28 points altogether.Root According to the ratio of this leaf shared by lobe of the lung area that substantive pathological changes occur, each lobe of the lung is carried out beating respectively Point.No pneumonia pathological changes are designated as 0 point；Lesion area ratio is 1%-25%, is designated as 1 point；Disease Variable area ratio 26%-50%, is designated as 2 points；Lesion area ratio is 51%-75%, is designated as 3 Point；Lesion area ratio is 76%-100%, is designated as 4 points.As all ill in lobe of the lung tow sides Become, scored with the one side that lesion area is big.Each lobe of the lung marking summation is this morbidity strain Pneumonia disease become score.After respectively immune group pig and matched group pig being scored, according to following The lobe of the lung pathological changes slip of formula Computation immunity group pig:
Embodiment of the present invention statistical analysis technique is: the pneumonopathy varying index of 7 lobes of the lung of statistics, really Determine lesion degree.Carry out anova analysis with spss computer software, it is poor between each group to compare Different, determine the effectiveness of pathological changes difference.
Ph7.2pbs solution formula used in the present invention is: adds in 800ml distilled water nacl 8g、kcl 0.2g、na2hpo41.42g、kh2po40.27g, adjusts ph value extremely 7.4, it is settled to 1000ml, through using after 121 DEG C of autoclavings 30 minutes, but this embodiment party No matter formula does not all constitute limitation of the invention under any circumstance.
In the present invention, used chemical reagent is that analysis is pure, and purchased from Chinese medicines group, but this is real Apply no matter mode does not all constitute limitation of the invention under any circumstance.
For making the present invention easier to understand, with reference to specific embodiment, this is expanded on further Invention.It should be understood that these embodiments are only used for the present invention rather than limit the scope of the present invention. Experimental technique of the present invention, if no specified otherwise, is conventional method；Described biology Material, if no specified otherwise, all commercially obtains.
The preparation of embodiment 1 antigen and identification
The preparation of 1.1 pig circular ring virus antigens and assay
1.1.1 the preparation of porcine circovirus 2 type sh strain
By seed culture of viruses porcine circovirus 2 type sh strain mem fluid medium (with purchased from the U.S. The mem dehydrated medium by specification of invitrogen company is prepared) make 1:9v/v dilution, Then it is inoculated in pk15 (atcc, preserving number is ccl-33) by the 5% of cell culture fluid volume Cell monolayer, 37 DEG C adsorb 30 minutes, add cell maintenance medium (in mem fluid medium Add the d- glucosamine hydrochloric acid of 4% calf serum and 2mmol/l), cultivate 4 for 37 DEG C, Freeze thawing 2-3 time, harvests virus, and virus titer is 106.5tcid50/ml.
Cultivate virus liquid with rolling bottle cell culture method, the pk15 cell of monolayer will be covered with, and go Cell culture fluid (adds the d- of 6% calf serum and 2mmol/l in mem fluid medium Glucosamine hydrochloric acid), seed culture of viruses liquid is pressed 0.1-0.2tcid50The inoculum concentration inoculation an of/cell On pk15 cell, Spin cells bottle 2 weeks, 37 DEG C adsorb 30 minutes, add cell to maintain Liquid, puts 37 DEG C of rotation (10-12 turns/hour) cultures.Observe 1-2 time daily, cell growth Well, 37 DEG C of culture harvestings on the 4th and Cell sap, freeze thawing 3 times, put less than -20 DEG C and preserve Virus liquid.
By the virus liquid doughnut of preparation filter post (10 μm of millipore company aperture with 0.45 μm) filter, remove cell debriss, then use Mi Libo (millipore company) film bag (molecular retention amount is 300kda) is made 5 times of v/v and is concentrated.
1.1.2 porcine circovirus 2 type sh strain virus liquid hold-up measures
Virus liquid is pressed those skilled in the art's universal method with mem fluid medium and makees 10 It is serially diluted again, take 10-5、10-6、10-73 dilution factors, each dilution factor inoculates 96 respectively Well culture plate pk15 cell monolayer 4 hole, every hole 0.1ml, set up negative control simultaneously, 37 DEG C contain 5%co2Incubator in continue culture 24 hours, change cell maintenance medium, continue culture 24 hours；Fix cell with cold acetone, measure each with indirect immunofluorescence assay ifa dilute Degree of releasing contains the hole count of pcv2 positive cell (in green), calculates disease according to karberShi method Malicious tcid50.Result of calculation shows: the content of porcine circovirus 2 type sh strain is 5×106.0tcid50/ml.
1.1.3 the inactivation of porcine circovirus 2 type sh strain virus liquid and inspection
By check qualified virus liquid add formalin (chemical reagent factory analysis in Luoyang City's is pure, Content is 37%-40%v/v), make the final concentration of 0.2%v/v of formalin, 37 DEG C of inactivations 18 hours, stir within every 4 hours 1 time, 10 minutes every time, inactivate inactivation of viruses after terminating Liquid puts 2-8 DEG C of preservation.
Inactivation of viruses liquid is carried out with inactivation inspection, takes a small amount of inactivation of viruses liquid inoculation to grow up to monolayer Pk15 cell, 37 DEG C of absorption abandons virus liquid after 1 hour, adds new cell maintenance medium, Cultivate 2 for 37 DEG C, acellular pathological changes cpe, continuous blind passage 3 times, change after growing up to cell monolayer Become cell maintenance medium, cultivate 2 for 37 DEG C, with the detection of indirect immunofluorescence ifa, redgreen Pcv2 positive cell produces, and shows that porcine circovirus 2 type sh strain virus liquid inactivates successfully.
The preparation of 1.2 mycoplasma hyopneumoniae antigens and assay
1.2.1 the preparation of mycoplasma hyopneumoniae hn0613 strain and assay
After freeze-drying lactobacillus unpacking, inoculate fluid medium, 37 DEG C of shaken cultivation by 10% inoculum concentration 3-7 day, harvest when ph value is down to 6.8 by 7.5, through purely checking, produce as one-level Seed.First order seed is taken to inoculate fluid medium, 37 DEG C of shaken cultivation 3-7 by 5% inoculum concentration Day, harvest when ph value is down to 6.8 by 7.5, through purely checking, as two grades of production seeds.
Wherein, the formula of mycoplasma hyopneumoniae fluid medium: Cor Bovis seu Bubali leachate (bd company) 300ml, distilled water 360m1, correction ph value to 7.4,121 DEG C sterilizes 15 minutes.Again plus Enter the composition of following filtration sterilization: hank's balanced salt solution (10 ×) 40m1,0.25%w/v Phenol red 10m1, horse serum 200m1,5%w/v lactoalbumin hydrolysate 100m1,25%w/v ferment Female leachate 20m1,10000iu/ml penicillin 10ml.
The secondary seed of qualified mycoplasma hyopneumoniae hn0613 strain is pressed respectively 5%v/v is inoculated in fluid medium, 37 DEG C of shaken cultivation 3-7 days, treats that ph value is dropped by 7.5 To results bacterium solution when 6.8.Bacterium solution Mi Libo (millipore company) the film bag that results are arrived (molecular retention amount is 300kda) is made 10 times of v/v and is concentrated.
Measure mycoplasma hyopneumoniae bacterium solution content according to bacterium solution viable bacteria counting method, phenol red instruction will be contained The fluid medium subpackage of agent is to small test tube, if two rows, often arranges 13,1.8ml/ props up, and takes 0.2ml culture to be checked is seeded to first small test tube, after mixing successively 10 times be diluted to 12 small test tubes, the 13rd manages as control tube, puts 37 DEG C of shakes cultures, record is till 21 days Produce the maximum tube number of color change, to judge ccu titre, take the meansigma methodss of two row's results. Result shows: the content of mycoplasma hyopneumoniae hn0613 strain is 1 × 109ccu/ml.
1.2.1.1 the inactivation of mycoplasma hyopneumoniae hn0613 strain bacterium solution and identification
Add formalin in the bacterium solution harvesting, make final concentration of the 0.3% of formalin V/v, 37 DEG C inactivate 24 hours, therebetween every stirring in 4 hours 1 time, 10 minutes every time, Inactivated bacterial liquid is put 2-8 DEG C of preservation after terminating by inactivation.
Inactivation inspection is carried out to the bacterium solution after inactivation, takes 1ml inactivated bacterial liquid inoculation 50ml liquid Culture medium, 37 DEG C of cultures, on the 5th, 10th, each transplanting once, continued after last transplanting Culture is observed 11, and culture medium ph value does not reduce, and culture medium coloring does not all change.
1.2.2 168 plants of antigens of mycoplasma hyopneumoniae
168 plants of live vaccine lyophilized powders of mycoplasma hyopneumoniae, limited purchased from Nanjing sky nation biotechnology Company, every part contains 168 plants of viable bacterias of mycoplasma hyopneumoniae is 1 × 107ccu.
The preparation of 1.3 haemophilus parasuises antigens and identification
1.3.1 the preparation of haemophilus parasuises 4 type js strain and 5 type zj strains
By haemophilus parasuises 4 type js strain and 5 type zj strain streak inoculations in containing 5% new born bovine Serum and the pancreas egg of 0.005% nicotinamide adenine dinucleotide nad (bbi company of the U.S.) White Soy Agar plates (abbreviation tsa/nad flat board), 37 DEG C of culture 24-48 hours, respectively Select more than 5 colonies typicals, purely after the assay was approved, as first order seed.First order seed Picking single bacterium colony, accesses the Tryptose soy meat containing 5% new-born calf serum and 0.005%nad Soup (abbreviation tsb/nad fluid medium), 180 revs/min of shaken cultivation 12 of 37 DEG C of shaking tables Hour, sample Gram’s staining simultaneously, examine under a microscope ne ar uniformly, meet pair The morphological characteristic of haemophilus suis, no any varied bacteria growing, as secondary seed.
Wherein, the preparation method of the tsa/nad flat board containing 5% serum: Tryptose soy fine jade 40 grams of fat (tryptic soy agar, tsa, bd difico product) adds 945ml distillation Water, fully shakes up post-heating and dissolves to abundant, 121 DEG C of high pressure steam sterilizations 15 minutes, temperature It is down to 60 DEG C about, add the Ox blood serum of 50ml filtration sterilization, 5ml filtration sterilization 1%nad, is down flat ware after fully shaking up；Tsb/nad liquid training containing 5% new-born calf serum The preparation method of foster base: pancreas soybean protein meat soup (tryptic soy broth, tsb, bd difico Product) 30g addition 940ml distilled water, fully shake up post-heating and dissolve to abundant, 121 DEG C are high Pressure steam sterilization 15 minutes, adds the Ox blood serum of 50ml filtration sterilization, 10ml filtration sterilization 0.01%nad.
Qualified haemophilus parasuises 4 type js strain and the secondary seed of 5 type zj strains are divided It is not inoculated in tsb/nad fluid medium by 1:100v/v, put 37 DEG C of shaking table 200rpm trainings Support.Cultivate to 12-16 hour, harvest bacterium solution.
By the bacterium solution (haemophilus parasuises 4 type js strain and 5 type zj strains) harvesting respectively with even Again the volume to centrifugation is redissolved with pbs solution after continuous (10000 revs/min) centrifugations of centrifuge, Then use Mi Libo film bag (molecular retention amount is 300kda) to make 5 times of v/v to concentrate.
Bacterium solution samples, and makees 10 times by method well known to those skilled in the art and is serially diluted, takes 10-6、10-72 dilution factors are inoculated in aforementioned tsa/nad solid medium, each board joint Plant 0.1ml, after 37 DEG C are cultivated 24 hours, select flat board between 30 and 300 for the clump count Carry out colony counting.Count results show: haemophilus parasuises 4 type js strain and 5 type zj strains Content be 5 × 109cfu/ml.
1.3.2 the inactivation of haemophilus parasuises 4 type js strain and 5 type zj strain bacterium solution and identification
Add formalin in the bacterium solution harvesting, make final concentration of the 0.3% of formalin V/v, 37 DEG C inactivate 24 hours, therebetween every stirring in 4 hours 1 time, 10 minutes every time, Inactivated bacterial liquid is put 2-8 DEG C of preservation after terminating by inactivation.
Bacterium solution after inactivation is carried out with inactivation inspection, the tsa/nad solid of 6 plates of preparation Culture medium, by the bacterium solution of inactivation, 3 tsa/nad solid mediums are put down wherein for sterile working Deca 1 on ware, with inoculating loop line, puts 37 DEG C of common incubator cultures, sets up 3 simultaneously The individual tsa/nad solid medium not connecing bacterium compares.Observe after 24 hours, in plate no Any bacterial growth, compare simultaneously do not connect 2 culture medium of bacterium also should no bacterial growth, continue Observed result equal no bacterial growth to 48 hours 6 plates.
The preparation of embodiment 2 adjuvant
The preparation of Carbomer mother solution: take Carbomer 971p to be dissolved in distilled water, left at room temperature over night makes it The fully swelling carbomer solution obtaining 20mg/ml, adds 3m sodium hydroxide solution to adjust solution ph It is worth to 7.2, plus appropriate distilled water adjusts carbomer solution, obtains the card ripple of final concentration of 15mg/ml Nurse mother solution.
The preparation of sodium carboxymethyl cellulose mother solution: by 1g sodium carboxymethyl cellulose and 100ml distilled water Mixing, heated and boiled, until being completely dissolved, is subsequently cooled to room temperature, that is, being configured to concentration is The sodium carboxymethyl cellulose mother solution of 10mg/ml.
The preparation of iscom substrate mother solution: take the lipid mixtures storing solution of 12mg/ml to add pbs In solution, add 30mg/ml quila storing solution, make final concentration of quila 15mg/ml, phosphorus Phosphatidylcholine 3mg/ml, cholesterol 3mg/ml, ice-bath ultrasonic process after fully mixing, room temperature is placed After 1.5 hours, it is transferred in bag filter, dialysed 3 days for outer liquid room temperature with pbs solution, through 0.22 μm Filter membrane is degerming to be preserved after 4 DEG C, obtains the iscom substrate mother solution of the final concentration of 15mg/ml of quila.
Above mother solution is all standby after 121 DEG C of autoclavings 30 minutes.
The preparation of embodiment 3 inactivated vaccine composition and animal experiment
The preparation of 3.1 inactivated vaccine compositions
Adjuvant prepared by antigen prepared by embodiment 1, embodiment 2 each become decomposition mother solution, use Pbs solution dilutes, and makes each antigenic solution after dilution and adjuvant final concentration by the epidemic disease inactivateing in table 1 Component mixing contained by Seedling compositionss, stirs 10-15 minute with 500-800 rev/min of rotating speed, The thimerosal (purchased from Chemical Reagent Co., Ltd., Sinopharm Group) of 1%v/v is added before terminating stirring Solution so as to final concentration be less than ten thousand/, fully vibration mix, subpackage, 2-8 DEG C after subpackage Save backup.
Component contained by table 1 inactivated vaccine composition
The potency test of the pig circular ring virus antigen of 3.2 inactivated vaccine compositions
By the vaccine 1-1 prepared by embodiment 3.1,1-2,1-3,1-6,1-7,1-9,2-1, (pcv2 vaccine, by Pu Laike for 2-2,2-3,2-6,2-7,2-9 and reference vaccine control group Biological engineering limited company produces) difference musculi colli injecting immune 14-21 age in days piglet (exclusion Porcine reproductive and respiratory syndrome, porcine circovirus 2 type and swine fever), 5/group, 2ml/ Head, arranges blank control group, totally 14 groups (totally 70 piglets) simultaneously.21 after immunity inoculation My god, every pig was taken a blood sample in 35 days, 60 days, 90 days, collect serum, be saved in -20 DEG C until Test.
According to serological method, the immune effect of pig circular ring virus antigen part is evaluated, tool Body is:
(1) the pcv2orf2 albumen of elisa antibody test escherichia coli expression (is pressed According to Chinese patent cn101101296a preparation) as antigen, determined by square formation burette test Antigen be most preferably coated concentration.By antigen diluent to being most preferably coated coated elisa plate after concentration, 100 μ l/ holes, latter 4 DEG C are coated overnight；Washing 3 times, each 3-5 minute；Every hole adds 200 μ l 0.15%m/v bsa confining liquid closing plank, 37 DEG C act on 2 hours；Washing；Will be to be checked Serum pbs doubling dilution, each sample a line, every hole adds 1,37 DEG C of effect 1h of 100 μ； Washing；It is subsequently adding enzyme target spa (1:10000 times dilutes), 100 μ l/ holes, 37 DEG C of effect 1h； Washing；Plus substrate solution tmb colour developing, finally use the h of 2mol/l2so4Terminating reaction.Result Judge: serum od to be checked450Value/negative serum od450Value >=2.1 is the positive.
(2) serum neutralization test adopts fixed virus diluted blood therapy for clearing away heat.56 DEG C of serum to be checked plus Heat 30 minutes, 10000 revs/min are centrifuged 5 minutes, carefully suction out supernatant, do 1:2 dilution After carry out doubling dilution；Mix with equivalent pcv2 virus liquid respectively, 37 DEG C of 1h, be inoculated in containing list In 96 orifice plates of layer pk15 cell, 100 μ 1/ hole, each dilution factor inoculates 4 holes, sets simultaneously Cell controls and virus control wells.37 DEG C of culture 12h, with the d- aminoglucose of 300mmol/l Sugar is processed, and 37 DEG C are continued culture 48h, and 80% acetone is fixed cell, used indirect immunofluorescence Measure the hole count that each dilution factor contains fluorescence.So that 50% specificity fluorescent cell number can be suppressed Cell hole serum greatest dilution as serum to be checked neutralization titer, and calculate every group put down Average.
Serological evaluation the results are shown in Table 2.
The efficacy test results of the pig circular ring virus antigen of table 2 inactivated vaccine composition
Note: the pcv2 vaccine with reference to used by vaccine control group is limited by Pu Laike biological engineering share Company produces, and antigenic content is 107.0tcid50/ ml, carries out immunity twice by shop instruction.
As shown in Table 2: a1-1 to a1-3, a2-1 to a2-3 organize the pcv2 of same content During antigen (single Seedling) difference adjuvant content, after immunity, produced antibody is with the increasing of adjuvant content Big and slightly elevated and basic with pcv2 antibody with reference to produced by single Seedling of high antigenic content Unanimously, and can last till and at least 60 days can be continued；Pcv2 connection containing same amount adjuvant After Seedling immunity, produced antibody is basically identical with pcv2 antibody with reference to produced by single Seedling, and Can last till and can continue at least 60 days, show vaccine 1-1,1-2,1-3,1-6,1-7, 1-9,2-1,2-2,2-3,2-6,2-7,2-9 can stimulate after immune animal body to produce respectively Pcv2 specific resistance, effect really, and vaccine 1-6,1-7,1-9,2-6,2-7, Other antigens of 2-9 no affect on the immune effect of pcv2 antigen；And in vaccine combination Adjuvant contain Carbomer, iscom and sodium carboxymethyl cellulose immune effect be better than containing Carbomer, Sodium carboxymethyl cellulose.
The potency test of the mycoplasma hyopneumoniae antigen of 3.3 inactivated vaccine compositions
By the vaccine combination 1-4 prepared by embodiment 3.1,1-6,1-8,1-9,2-4,2-6, 2-8,2-9, (pig breeds and exhales difference musculi colli injecting immune 14-21 age in days piglet for exclusion Inhale syndrome, pig annulus 2 type and swine fever), 5/group, 2ml/ head, setting is with reference to epidemic disease simultaneously Seedling matched group, counteracting toxic substances matched group, totally 10 groups (totally 50 piglets).Observe after immunity inoculation Body temperature, side reaction were to 28 days, and on 28th, all pig tracheas injection mycoplasma hyopneumoniae were helped South is that (preserving number is cvcc354, and depositary institution is that National Veterinary is micro- for malicious cvcc354 strain by force Biological inoculum preservation administrative center), 100mid50/ head, dissects after observing 28 days, and observes Pulmonary lesion, scores to pulmonary lesion according to 28 point-scores.Before and after counteracting toxic substances, respectively to examination Test group piglet to be weighed, and calculate average daily gain.
About 10 days about after counteracting toxic substances, wherein 4 pigs of counteracting toxic substances matched group successively occur cough, The symptoms such as asthma, fur is rough；1 pig is had to occur in that the disease of cough with reference to vaccine control group Shape simultaneously there occurs side reaction；The similar symptom of asthma in the pig of other immune group.Attack Before and after poison, the pneumonia disease of each test group pig becomes average and average daily gain the results are shown in Table 3.
The efficacy test results of the mycoplasma hyopneumoniae antigen of table 3 inactivated vaccine composition
Note 1: the vaccine with reference to selected by vaccine control group is the porcine mycoplasmal lung in existing product Scorching inactivated vaccine (j strain), that is, peace hectogram (m+pac), carries out immunity by shop instruction.
Note 2: body temperature "None" shows not occur transient body temperature to raise, " having " shows to occur transient Body temperature raises, and wherein transient body temperature raises and refers to that the 1-2 days body temperature can be of short duration after two immunity Assume 40.5 degree, and recover normal body temperature in the 3rd day.
Note 3: side reaction "None" shows side reaction, " having " shows side reaction, its Middle side reaction refers to occur that transience spirit is depressed, loss of appetite, in anaphylaxiss at least one Kind.
Note 4: in diversity statistical analysiss, compare between group, alphabetical identical person represents that difference does not show Write, alphabetical difference person represents significant difference (p ＜ 0.05).
As shown in Table 3: b1-1 to b1-4 group (i.e. distinguish immune vaccine 1-4,1-6,1-8, 1-9), b2-1 to b2-4 group (distinguishing immune vaccine 2-4,2-6,2-8,2-9) makes Carry out immunity with single Seedling or connection Seedling of antigen containing mycoplasma hyopneumoniae and same content adjuvant, afterwards Carry out counteracting toxic substances, result display immune swine does not all occur transient body temperature to raise, and does not occur secondary anti-yet Should, and pneumonia pathological changes slip equal ＞ 73%, ＞ 84% respectively, average daily gain is respectively 0.0412-0.0415kg, 0.0445-0.0453kg, the not notable (p of difference between each group of b1, b2 ＞ 0.05)；With compared with vaccine control group, difference is all notable (p ＜ 0.05)；With counteracting toxic substances Matched group is compared, and difference is all notable (p ＜ 0.05)；And adjuvant ripple containing card in vaccine combination The immune effect of nurse, iscom and sodium carboxymethyl cellulose is significantly better than containing Carbomer, carboxymethyl (the p ＜ 0.05) of sodium cellulosate.
The potency test of the haemophilus parasuises antigen of 3.4 inactivated vaccine compositions
By the vaccine combination 1-5 prepared by embodiment 3.1,1-7,1-8,1-9,2-5,2-7, 2-8,2-9, (pig breeds and exhales difference musculi colli injecting immune 14-21 age in days piglet for exclusion Inhale syndrome, pig annulus 2 type and swine fever), 5/group, 2ml/ head, setting is with reference to epidemic disease simultaneously Seedling matched group, counteracting toxic substances matched group, totally 10 groups (be named as c1-1, c1-2, c1-3, c1-4, C2-1, c2-2, c2-3, c2-4, reference vaccine control group 1, counteracting toxic substances matched group 1)；Simultaneously Do repeat test, totally 10 groups (be named as c1-5, c1-6, c1-7, c1-8, c2-5, c2-6, C2-7, c2-8, reference vaccine control group 2, counteracting toxic substances matched group 2), refer to table 4.
Carry out within 28 days after immunity inoculation counteracting toxic substances, noted with haemophilus parasuises 4 type abdominal cavity for first 10 groups Penetrate 3ml and carry out counteracting toxic substances, counteracting toxic substances dosage is 9.0 × 109Cfu/ head；Use the secondary bloodthirsty bar of pig for 10 groups afterwards Bacterium 5 type lumbar injection 3ml carries out counteracting toxic substances, and counteracting toxic substances dosage is 6.0 × 109Cfu/ head, after counteracting toxic substances Observe each group pig clinical manifestation, cut open after observing 14 days and enter row pathological observation.Divide before and after counteracting toxic substances Other test group piglet is weighed, to calculate average daily gain.
The efficacy test results of the haemophilus parasuises antigen of table 4 inactivated vaccine composition
Note 1: Haemophilus parasuis inactivated vaccine with reference to used by vaccine control group herds industry stock in being Chengdu medical instruments factory of part company limited produces, and antigenic content is 4.0 × 109Cfu/ head part, according to factory Family's description carries out immunity twice.
Note 2: in diversity statistical analysiss, compare between group, alphabetical identical person represents that difference does not show Write, alphabetical difference person represents significant difference (p ＜ 0.05).
As shown in Table 4: c1-1 to c1-4 group, c1-5 to c1-8 group (are exempted from respectively Epidemic disease vaccine 1-5,1-7,1-8,1-9), c2-1 to c2-4 group, c2-5 to c2-8 Group (distinguishing immune vaccine 2-5,2-7,2-8,2-9) uses antigen containing haemophilus parasuises Carry out immunity with single Seedling of same content adjuvant or connection Seedling, carry out counteracting toxic substances afterwards, result shows to be exempted from Epidemic disease pig obtains and protects completely, average daily gain respectively 0.0311-0.0317kg, 0.0388-0.0344kg, in each group of c1, c2, difference is not notable (p ＞ 0.05) each other； With compared with vaccine control group, antigenic content is low can also to make immunoprotection efficiency high, and puts down All daily gain difference is all notable (p ＜ 0.05)；Compared with counteracting toxic substances matched group, average daily gain is poor Different all notable (p ＜ 0.05)；And in vaccine combination adjuvant contain Carbomer, iscom and The immune effect of sodium carboxymethyl cellulose is significantly better than the (p containing Carbomer, sodium carboxymethyl cellulose ＜ 0.05).
The preparation of embodiment 4 live vaccine composition and animal experiment
The preparation of the mycoplasmal pneumonia of swine live vaccine (168 plants) of 4.1 live vaccine
Live vaccine composition taking mycoplasmal pneumonia of swine live vaccine (168 plants) as a example, by embodiment 1.2 Adjuvant conduct prepared by 168 plants of antigens of mycoplasma hyopneumoniae of preparation, the embodiment 2 through pbs dilution Live vaccine diluent is prepared by table 5, dissolves 1 part porcine mycoplasmal with 2ml live vaccine diluent Pneumonia live vaccine (168 plants).
Contained component and final concentration after the dilution of table 5 porcine mycoplasmal live vaccine
The animal experiment of 4.2 mycoplasmal pneumonia of swine live vaccine (168 plants)
By the vaccine combination prepared by embodiment 4.1, press table 5 respectively and adopt musculi colli to note Penetrate immune 14-21 age in days piglet 45 (exclusion Porcine reproductive and respiratory syndrome, pig annulus 2 Type and swine fever), 5/group, 2ml/ head, setting is with reference to vaccine control group, prior art simultaneously Matched group, counteracting toxic substances matched group, totally 9 groups.
Body temperature, side reaction is observed to 28 days before immunity and after inoculation, and on 28th to all pigs Trachea injection mycoplasma hyopneumoniae Jinan system by force malicious cvcc354 strain (preserving number is cvcc354, Depositary institution is National Veterinary Microbiological Culture Collection administrative center), 100mid50/ head, sees Dissect after examining 28 days, and observe pulmonary lesion, according to 28 point-scores, pulmonary lesion is commented Point？.Before and after counteracting toxic substances, respectively test group piglet is weighed, and calculate average daily gain.
Safety testing result: the no significant change of the body temperature of immune group i-vi, cut open observation lung after killing Obvious pathological change is not found during pathological changes yet.
Challenge test result: about 10 days about after counteracting toxic substances, counteracting toxic substances matched group pig (wherein 4 The symptoms such as cough, asthma only) occur successively, fur is rough；Vii, viii group respectively has One pig occurs in that the symptom of cough；Other groups do not have pig the similar disease of asthma Shape.Before and after counteracting toxic substances, the pneumonia disease of each test group pig becomes average and average daily gain result It is shown in Table 6.
Table 6 porcine mycoplasmal live vaccine observed after immunization result and efficacy test results
Note 1: with reference to vaccine control group be Nanjing Tianbang Bio-industry Co., Ltd. produce prop up must Rather, carry out pulmonary injection by shop instruction；Prior art matched group is according to Chinese patent The mycoplasma hyopneumoniae live vaccine (168 plants) of cn103071151b preparation, carries containing this patent And vaccine special diluting agent, by the immunity of this patent musculi colli injection system, immunity 2 times altogether Interval 2 weeks.
Note 2: body temperature "None" shows not occur transient body temperature to raise, " having " shows to occur transient Body temperature raises, and wherein transient body temperature raises and refers to that the 1-2 days body temperature can be of short duration after two immunity Assume 40.5 degree, and recover normal body temperature in the 3rd day.
Note 3: side reaction "None" shows side reaction, " having " shows side reaction, its Middle side reaction refers to occur that transience spirit is depressed, loss of appetite, in anaphylaxiss at least one Kind.
Note 4: in diversity statistical analysiss, compare between group, alphabetical identical person represents that difference does not show Write, alphabetical difference person represents significant difference (p ＜ 0.05).
As shown in Table 6:
(1) i-th-iii group (i.e. immune live vaccine 1-3) is using containing Carbomer, carboxymethyl cellulose Adjuvant dilution mycoplasma hyopneumoniae live vaccine (168 plants) of plain sodium carries out musculi colli injection and exempts from Epidemic disease, carries out counteracting toxic substances afterwards, and result shows: with the rising immune effect of constituent content each in adjuvant (pneumonia pathological changes slip, average daily gain) increases, and immune pig does not occur transient body temperature Raise, also side reaction does not occur, and immune effect significant difference (p ＜ 0.05) between each group； Compare, except i-th with vii group (i.e. with reference to vaccine control group, immune component is Zhi Bining) Organize outer difference all significantly (p ＜ 0.05), and intramuscular injection more easy and simple to handle than pulmonary injection, can Acceptance is high；Compared with viii group (i.e. prior art matched group), except i-th group of outer difference is equal Significantly (p ＜ 0.05), and immune time is few, reduces immune cost, has saved immune programme for children, Economic and reliable；Compared with counteracting toxic substances matched group, difference is all notable (p ＜ 0.05).
(2) i-th v-vi groups (i.e. immune live vaccine 4-6) using containing Carbomer, iscom, Adjuvant dilution mycoplasma hyopneumoniae live vaccine (168 plants) of sodium carboxymethyl cellulose carries out cervical region Intramuscular injection immunity, carries out counteracting toxic substances, result shows: afterwards with the liter of constituent content each in adjuvant High immune effect (pneumonia pathological changes slip, average daily gain) increases, and immune pig does not occur Transient body temperature raises, and also side reaction, and significant difference (p ＜ 0.05) between each group； Compared with the i-th-iii group, after the adjuvant dilution live vaccine that identical content groups are divided in addition to iscom Immune effect is more excellent, and significant difference (p ＜ 0.05)；With vii group (i.e. with reference to vaccine pair According to group, immune component is Zhi Bining) to compare, difference is all notable (p ＜ 0.05), and muscle Injection is more easy and simple to handle than pulmonary injection, it is high to be subjected to degree；(i.e. prior art compares with viii group Group) to compare, difference is all notable (p ＜ 0.05), and immune time is few, reduces immune cost, Immune programme for children, economic and reliable are saved；Compared with counteracting toxic substances matched group, all notable (the p ＜ of difference 0.05).
The application of the protein stabilized liquid of embodiment 5
The protein stabilized Journal of Sex Research of 5.1rhdv vp60
Prepare rabbit hemorrhagic disease virus according to the method described in Chinese patent cn103555766a The immunogenicity vp60 albumen of (rbabbit hemorrhagic disease virus, rhdv), right Vp60 albumen after expression carries out identification and confirms undoubtedly to carry out afterwards purification, and the o type of employment is red thin The agglutination titer of born of the same parents is detected, testing result shows that the HA-HI test ha of vp60 albumen is 1:217.
It is Carbomer mother solution, isocm substrate mother that adjuvant prepared by embodiment 2 respectively becomes decomposition mother solution Liquid, sodium carboxymethyl cellulose mother solution, in order to prepare the protein stabilized liquid of vp60, dilute preparation with pbs Rhdv vp60 protein solution (solution 1-2), makes each component final concentration of: vp60ha is 1:214, Carbomer is 1mg/ml, and sodium carboxymethyl cellulose is 0.1mg/ml.Meanwhile, prepare solution (solution 3-4) makes each component final concentration of: vp60ha is 1:214, Carbomer is 1mg/ml, Saponin is 0.1mg/ml, and sodium carboxymethyl cellulose is 0.1mg/ml.
Take same dose of rhdv vp60 protein solution each 2 parts deposit 6 respectively at 2-8 DEG C, 12, 18th, 24,30,36 months, and compareed with 2 parts of the vp60 protein solution of pbs dilution, right Protein solution under different preservation conditions carries out Blood coagulation test, the results are shown in Table 7.
Table 7 rhdv vp60 protein stability result of the test
As shown in Table 7: containing Carbomer, sodium carboxymethyl cellulose solution as rhdv vp60 Protein stabilized liquid makes vp60 protein stabilized at least 24 months, containing Carbomer, iscom, carboxymethyl The solution of sodium cellulosate makes vp60 protein stabilized at least 30 as the protein stabilized liquid of rhdv vp60 Individual month, and be gradually lowered using pbs as the matched group ha prolongation in time preserving solution, Show that albumen occurs in that a certain degree of irreversible change of degrading or there occurs.
The protein stabilized Journal of Sex Research of 5.2pcv2cap
Prepare porcine circovirus 2 type according to the method described in Chinese patent cn104017813a The immunogenicity cap albumen of (porcine circovirus type 2, pcv2), after expression Cap albumen carries out identification and confirms undoubtedly to carry out purification afterwards, according to (the purchase of bca protein quantification test kit From merck company) concentration of the method for description quantitation cap albumen, result shows: cap egg White concentration is 2mg/ml.
It is Carbomer mother solution, isocm substrate mother that adjuvant prepared by embodiment 2 respectively becomes decomposition mother solution Liquid, sodium carboxymethyl cellulose mother solution, in order to prepare the protein stabilized liquid of cap, dilute preparation with pbs Pcv2cap protein solution (solution 1-2), makes each component final concentration of: cap protein concentration is 0.40mg/ml, Carbomer is 1mg/ml, and sodium carboxymethyl cellulose is 0.1mg/ml.Meanwhile, make Standby solution (solution 3-4) makes each component final concentration of: cap protein concentration is 0.40mg/ml, card Ripple nurse is 1mg/ml, and saponin is 0.1mg/ml, and sodium carboxymethyl cellulose is 0.1mg/ml.
Take same dose of pcv2cap protein solution each 2 parts deposit 6 respectively at 2-8 DEG C, 12, 18th, 24,30,36 months, and compareed with 2 parts of the cap protein solution of pbs dilution, right Protein solution under different preservation conditions carries out bca mensure respectively, the results are shown in Table 8.
Table 8 pcv2cap protein stability result of the test
As shown in Table 8: containing Carbomer, sodium carboxymethyl cellulose solution as pcv2cap egg White stabilizing solution, makes cap protein stabilized at least 24 months, containing Carbomer, iscom substrate, carboxylic The solution of sodium carboxymethylcellulose pyce, as the protein stabilized liquid of pcv2cap, makes cap protein stabilized at least 30 months, and using pbs as preserve solution matched group in cap albumen concentration with preserve when Between prolongation and be gradually lowered, albumen occurs in that a certain degree of irreversible change of degrading or there occurs.
In sum, containing Carbomer, immunostimulating complex and sodium carboxymethyl cellulose adjuvant The immune effect of vaccine combination is significantly better than containing Carbomer, sodium carboxymethyl cellulose, these epidemic diseases Immunne response level after Seedling compositionss (1) immune animal raises with the increase of adjuvant content, And basically identical with the immunne response level of existing commercial vaccine, the immune persistent period is also relatively Long, effect is certain；(2) animal also can be made preferably immunity is obtained when antigenic content is relatively low Response level and protecting completely, immune time makes the stress of pig reduce even no less, and no Side reaction occurs；(3) this adjuvant can be also used for diluting live vaccine, makes live vaccine with muscle Injection form immunity pig, none crosses gonosome temperature rise, side reaction generation to make pig, makes pig Pneumonia disease variability is less, average daily gain rises high good immune effect, better than existing epidemic disease alive Seedling, and make easy and simple to handle, cost reduction, program saving, economic and reliable；(4) this adjuvant Can use as protein stabilized liquid, make protein stabilized non-degradable or not there occurs irreversible change.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, All any modification, equivalent substitution and improvement within the spirit and principles in the present invention, made etc., Should be included within the scope of the present invention.

Claims (10)

1. a kind of vaccine adjuvant, described adjuvant includes acrylic acid series polymeric compounds and cellulose ether.
2. vaccine adjuvant according to claim 1, wherein, described acrylic acid seriess gather Compound include polyacrylic acid, polymethylacrylic acid, polymethacrylates, polyacrylamide, Polyacrylate, polyacrylonitrile and polyacrylic Arrcostab；
Preferably, described acrylic acid series polymeric compounds include carbopol, Kappa match, Ni Aoke Neocryl, Eudragit RS 100 eudragit；
It is highly preferred that described acrylic acid series polymeric compounds include carbopol 941,980,981,940, 2020th, 974p, 934p and 971p.
3. vaccine adjuvant according to claim 1, wherein, described cellulose ether bag Include carboxymethyl cellulose, sodium carboxymethyl cellulose, potassium carboxymethylcellulose, carboxymethyl cellulose Calcium, hydroxyethyl cellulose, hydroxypropyl methyl cellulose.
4. vaccine adjuvant according to claim 1, wherein, described in described adjuvant Acrylic acid series polymeric compounds are calculated as 2-10mg/ml with Carbomer, and described cellulose ether is 0.1-8mg/ml； Preferably, described in described adjuvant, acrylic acid series polymeric compounds are calculated as 4-10mg/ml with Carbomer, institute Stating cellulose ether is 4-8mg/ml.
5. vaccine adjuvant according to claim 1, wherein, described adjuvant also includes Immunostimulant, described immunostimulant includes compositionss containing saponin, Lactobacillus, honeybee Glue, vitamin e, levamisole, transfer factor, interleukin, interferon, thymosin, Transfer factor, globulin, bacillus calmette-guerin vaccine or a combination thereof；
Preferably, described immunostimulant includes the compositionss containing saponin, the described group containing saponin Compound quila, qs21, qs7, qs17, qs18, qh-a, qh-b, qh-c, vaxsap, Supersap, gpi-0100 and qp uf1000, at least one of immunostimulating complex.
6. vaccine adjuvant according to claim 5, wherein, described in described adjuvant Acrylic acid series polymeric compounds are calculated as 2-10mg/ml with Carbomer, and described cellulose ether is 0.1-8mg/ml, and the described compositionss containing saponin are calculated as 0.1-10mg/ml with quila.
7. a kind of vaccine combination, wherein, described vaccine combination includes the antigen of immunity amount Adjuvant with any one of claim 1～6；Described antigen includes pig circular ring virus antigen, Pulmonis Sus domestica Scorching mycoplasma antigen, haemophilus parasuises antigen, porcine reproductive and respiratory syndrome virus antigen, PPV Antigen Using, Pasteurella multocida antigen, Streptococcus suis antigen, Staphylococcus hyicus resist The special bacterium antigen of former, bronchitis Boulder, Actinobacillus pleuropneumoniae antigen, escherichia coli resist Former, atrophic rhinitiss antigen, PRV (Pseudorabies virus) antigen, Salmonella choleraesuls antigen, Salmonella enteritidis antigen, pig erysipelothrix rhusiopathiae antigen, mycoplasma hyorhinis antigens, pig synovial fluid Mycoplasma antigen, pig Leptospira bacteria antigen, PRCV (Porcine Respiratory Coronavirus) antigen, pig stream Row diarrhea viruses antigen, porcine rotaviruses antigen, transmissible gastro-enteritis viruss antigen, pig Thin circovirus virus antigen, inclusion-body rhinitis virus of pigs antigen, Intestinum Sus domestica virus antigen, encephalomyocarditis viruses resist Former, one or more of swine flue antigen, CSFV antigen；
Preferably, described vaccine antigen by pig circular ring virus antigen, mycoplasma hyopneumoniae antigen, Haemophilus parasuises antigen forms；
Most preferably, described antigen propped up by pig circular ring virus sh strain inactivation antigen, hyopneumoniae former Body hn0613 strain inactivation antigen, haemophilus parasuises 4 type js strain and 5 type zj strains inactivation are anti- Former composition, described adjuvant includes acrylic acid series polymeric compounds, cellulose ether and immunostimulating complex.
8. vaccine combination according to claim 7, wherein, described vaccine combination Described in adjuvant content be 5%-30%v/v；Preferably, described in described vaccine combination The content of adjuvant is 10%-25%v/v.
9. a kind of antigen protein stabilizer, described stabilizer includes acrylic acid series polymeric compounds and fibre The plain ether of dimension；Preferably, described stabilizer also includes the compositionss containing saponin.
10. antigen protein stabilizer according to claim 9, wherein, described stabilizer Described in acrylic acid series polymeric compounds 2-10mg/ml is calculated as with Carbomer, described cellulose ether is 0.1-8mg/ml, and the described compositionss containing saponin are calculated as 0.1-10mg/ml with quila.
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CN109806391A (en) * 2017-11-22 2019-05-28 西北民族大学 The preparation method and applications of Water Soluble Compound adjuvant are immunized in a kind of live vaccine
CN108245675A (en) * 2018-02-02 2018-07-06 四川华神兽用生物制品有限公司 A kind of live vaccine dilution and preparation method thereof, application and vaccine product
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CN110404065A (en) * 2018-04-27 2019-11-05 洛阳赛威生物科技有限公司 One boar adjunvant composition and preparation method thereof
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CN110898218B (en) * 2019-12-30 2023-10-10 瑞普（保定）生物药业有限公司 Duck tembusu virus inactivated vaccine and preparation method thereof
CN110898218A (en) * 2019-12-30 2020-03-24 瑞普（保定）生物药业有限公司 Duck tembusu virus disease inactivated vaccine and preparation method thereof
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CN113244390A (en) * 2021-05-28 2021-08-13 西南大学 Triple egg yolk antibody freeze-dried powder and preparation method and application thereof
CN115444933B (en) * 2022-09-14 2023-08-29 贵州福斯特生物科技有限公司 Preparation method and application of water-soluble composite adjuvant for rabbits
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