CN103784951A - Antigen composition for preventing and treating secondary infected respiratory system diseases of pigs, preparation method and application thereof - Google Patents
Antigen composition for preventing and treating secondary infected respiratory system diseases of pigs, preparation method and application thereof Download PDFInfo
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- CN103784951A CN103784951A CN201210435109.9A CN201210435109A CN103784951A CN 103784951 A CN103784951 A CN 103784951A CN 201210435109 A CN201210435109 A CN 201210435109A CN 103784951 A CN103784951 A CN 103784951A
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Abstract
The invention provides an antigen composition for preventing and treating secondary infected respiratory system diseases of pigs. The antigen composition comprises at least one porcine reproductive and respiratory syndrome antigen with immune amount, and at leas one haemophilus parasuis antigen with immune amount and an adjuvant, wherein the porcine reproductive and respiratory syndrome antigen and the haemophilus parasuis antigen respectively and independently comprise attenuated live holoantigen, inactivated holoantigen, subunit antigen, recombinant live carrier antigen and DNA (deoxyribonucleic acid) carrier antigen. The antigen composition cannot cause mutual immune interference or influence of two antigen components, and enables the immune effects of porcine reproductive and respiratory syndrome antigen and the haemophilus parasuis antigen to be strengthened, and is convenient in vaccination process and good in safety.
Description
Technical field
The present invention relates to vaccine combination of a kind of prevention and treatment Haemophilus parasuis and Porcine reproductive and respiratory syndrome and preparation method thereof, belong to live vaccine field.
Background technology
Porcine reproductive and respiratory syndrome (Porcine Reproductive and RespiratorySyndrome; PRRS); claim again pig blue-ear disease; to be caused by the breeding of sub-thread positive chain RNA pig and respiratory disorder syndrome virus; take the breeding difficulty (miscarriage, stillborn fetus, mummy tire) of in-pig and various age pig particularly the respiratory tract disease of piglet as feature, existing one of the main epidemic disease of large-scale pig farm that become.The highly pathogenic PRRS of China's some areas outburst in 2006 is by pig breeding and the acute high lethal epidemic disease of one that respiratory disorder syndrome (being commonly called as reproductive and respiratory syndrome) virus variant causes, belongs to national two class animal epidemics.There is new epidemic characteristic in China in this disease, virus morphs, and pathogenicity strengthens, and occurs that large-scale outbreak is popular in some areas.Clinical take high heat, skin cyanosis, eye conjunctivitis, Respiratory symptoms, nervous symptoms and death as main, after dissecting, visible lungs are interstitial pneumonia, lymphadenectasis with hemorrhage hyperemia, show as rapid onset, serious symptom, pathological changes obvious, mortality rate is high, in short time, sow is miscarried in a large number, has brought huge economic loss to China's pig industry.Until today, epidemic disease situation is still severe, the same with other viral diseases, there is no at present the treatment of effective curative drug for reproductive and respiratory syndrome, and vaccine virus immunization is this sick most effectual way of anti-system.
Haemophilus parasuis is polyserositis and the arthritis of the pig that causes of haemophilus parasuis (Haemophilus parasuis, Hps), and this disease is called again pig leather and draws Ze Shi disease (Glasser ' sDisease).Haemophilus parasuis can affect the young pig from 2 week age to 4 monthly ages, mainly after wean, falls ill with the child care stage, conventionally sees the pig in 5～8 week age, and sickness rate can reach 40%, and mortality rate can reach 50%.Main clinic symptoms shows as cough, dyspnea, becomes thin, walks lamely and the thick unrest of quilt hair; Cut open inspection visible fibrinous pleurisy, pericarditis, arthritis and meningitis, there is fibrinous exudate in visible abdominal organs surface also, causes between intestinal adhesion etc.In addition, haemophilus parasuis also can cause septicemia, and may leave sequela after actute infection, i.e. sow miscarriage, the chronic limping of boar.On a large amount of bases that separate haemophilus parasuis, find that the serotype complexity of this bacterium is various, by Kieletein-Rapp-Gabriedson (KRG) agar diffusion serotype method, at least haemophilus parasuis can be divided into 15 serotypes; According to the evaluation of China's seroepidemiological survey and isolated strains, the most popular with 4,5 types.In recent years, carry out improperly due to the adjustment of the current breeding technology of China, and the new respiratory syndrome that happens suddenly, makes this disease increasingly popular, endangers day by day serious.
Show according to the result of pig farm epidemiological study in recent years, respiratory system disease is more and more, shows as clinically respiratory symptom, and principal character is heating, growth retardation, appetite depression, dyspnea, and dead pig extremity, ear are blue; Fervescence before sow miscarriage, anorexia, especially the sow of the first tire is more common.Dead pig is cutd open inspection and presents pericarditis, pleuritis, peritonitis and arthritis, pneumonia, and lymph node is hemorrhage, enlargement.From morbidity pig, isolating modal pathogen is porcine reproductive and respiratory syndrome virus and haemophilus parasuis, but, the inventor finds in the time reappearing the respiratory system disease of performance above-mentioned symptom for one, when only infecting haemophilus parasuis in swinery, can't be diseases induced, only have when existing in swinery in the situation of reproductive and respiratory syndrome, just can cause the respiratory system disease of above-mentioned symptom.The sick pig that shows these symptoms is to be caused by these two kinds of pathogen synergism, and once secondary infection haemophilus parasuis is diseases induced, various antibacterials are all difficult to prove effective.
In recent years, it is found that other cause of diseases of the easy secondary infection of pig that porcine reproductive and respiratory syndrome virus infects, particularly encroach on the pathogen of respiratory system, modal is clinically pasteurella multocida, mycoplasma pneumoniae, streptococcus, haemophilus parasuis, Actinobacillus pleuropneumoniae, bordetella bacilli and Salmonella etc., causes swinery high incidence and high mortality.Yang Hanchun (" epidemic characteristic of pig immunosuppressive disease and control countermeasure ", China's animal and veterinary, 2004,31(5): 41-43), Wan Suiru (" prevention and control pig immunosuppressant technical measures ", raise pigs, 2009,1:42-46) etc. research finds that the persistent infection of reproductive and respiratory syndrome virus in pig body also can cause the humoral immunization of swine Fever Vaccine to be subject to obvious inhibition, affect the immune effect of swine fever attenuated vaccine, therefore think that Porcine reproductive and respiratory syndrome is a kind of immunosuppressive disease.
There is not the combination-vaccine of Haemophilus parasuis and reproductive and respiratory syndrome in existing market, and the two time combine use, this is that possible reason has because exist haemophilus parasuis inactivated vaccine to exert an influence to reproductive and respiratory syndrome live vaccine immune effect: the one, and the oil adjuvant viscosity that inactivated vaccine uses can not be diluted live vaccine as diluent too greatly; The 2nd, the toxic component that inactivated vaccine part is contained: inactivator is as formaldehyde; Antiseptic is as thimerosal, sodium azide; As a large amount of endotoxins, cultivation process metabolite and foreign protein etc., on live vaccine poison valency, impact significantly makes live vaccine effect decline to toxin.And commercial goods vaccine need repeatedly immunity, cost is higher, to pig stress be larger.
Summary of the invention
Problem to be solved by this invention is for providing a kind of antigen composition that can prevent and treat the respiratory system disease of the secondary infection of pig, described antigen composition comprises the Porcine reproductive and respiratory syndrome antigen of at least one immunity amount and the haemophilus parasuis antigen of at least one immunity amount, and described Porcine reproductive and respiratory syndrome antigen and haemophilus parasuis antigen comprise the antigen of holoantigen, subunit antigen, live recombinant vectors antigen and the DNA vector of attenuated live holoantigen, deactivation independently of one another.Described respiratory system disease principal character is heating, growth retardation, appetite depression, dyspnea, and dead pig extremity, ear are blue.Dead pig is cutd open inspection and presents pericarditis, pleuritis, peritonitis and arthritis.Should be appreciated that, for given combination, not necessarily use same form.In addition, known as people, antigen product can comprise veterinarily acceptable carrier or excipient, and can select to comprise veterinarily acceptable adjuvant.
Another problem to be solved by this invention is for provide a kind of vaccine combination that can prevent and treat Porcine reproductive and respiratory syndrome and Haemophilus parasuis simultaneously, giving pig Pigs Inoculated reproductive and respiratory syndrome vaccine, and simultaneous inoculation haemophilus parasuis vaccine.Can be understood as to pig Pigs Inoculated reproductive and respiratory syndrome vaccine and haemophilus parasuis vaccine simultaneously.To solve the problems referred to above that faced in the time preventing this two kinds of diseases, and find unexpectedly in this process, the Porcine reproductive and respiratory syndrome live vaccine of haemophilus parasuis inactivated vaccine dilution for inoculation simultaneously, do not affect the generation of anti-haemophilus parasuis antibody, between antigen, do not occur phase mutual interference.After associating use, can alleviate Porcine reproductive and respiratory syndrome strong virus attack time clinical symptoms, and Haemophilus parasuis killed vaccine antigen content reduce by half also can reach independent use time immune effect.
Theme of the present invention also relates to immunogenic composition or the vaccine of the respiratory system disease of the secondary infection of resisting a boar, above-mentioned Porcine reproductive and respiratory syndrome antigen+haemophilus parasuis antigen product that it comprises the effective dose among veterinarily acceptable carrier or excipient, and can select to comprise veterinarily acceptable adjuvant.Immunogenic composition causes immunne response.Therefore, term " immunogenic composition " comprises " vaccine combination ".
Theme of the present invention also relates to antigen product or immunogenic composition or the antigen product of vaccine and opposing haemophilus parasuis or the immunity of immunogenic composition or vaccine or the vaccination test kit of the upper opposing Porcine reproductive and respiratory syndrome separately of packing.Described test kit can have the various features of above-mentioned definition antigen product, immunogenic composition and vaccine.
Immunity or the method for vaccination of the respiratory system disease of the secondary infection of a boar resisted in addition in theme of the present invention, comprise and use the opposing immunogenic composition of Porcine reproductive and respiratory syndrome or immunogenic composition or the vaccine of vaccine and opposing haemophilus parasuis, or be applied in same preparation, comprise a kind of to all bigeminy immunogenic composition or vaccines of special antigen product of two kinds of cause of diseases.Particularly, described immunity or inoculation method use above-mentioned vaccine.
Theme of the present invention is also particularly antigen product or immunogenic composition or the vaccine of opposing haemophilus parasuis defined above of opposing, combine antigen product or immunogenic composition or the vaccine of resisting Porcine reproductive and respiratory syndrome, the application in the pharmaceutical composition of respiratory system disease of the secondary infection for the preparation of opposing one boar.
In combined immunization or vaccination in the works, the immunity of opposing Porcine reproductive and respiratory syndrome and haemophilus parasuis or vaccination also can be combined immunity or the vaccination of the pathogen that other porcine pathogen of opposing particularly may be relevant with the respiratory system disease of secondary infection.Therefore, can comprise with corresponding other of other porcine pathogen and tire according to immunogenic composition of the present invention or vaccine, these porcine pathogens are selected from mycoplasma hyopneumoniae and/or mycoplasma hyorhinis and/or porcine contagious pleuropneumonia and/or atrophic rhinitis and/or swine influenza virus and/or Pseudorabies virus and/or pig circular ring virus and/or hog cholera and combination thereof.Therefore, can use any type of immunogenic composition or vaccine, particularly any obtainable commercial vaccine, so that it combines use with immunogenic composition or the vaccine of opposing Porcine reproductive and respiratory syndrome as herein described and haemophilus parasuis.
Therefore, theme of the present invention is also multivalent immunogenic compositions and vaccine, multi-vaccine test kit and makes to adopt combined immunization or the method for vaccination of this combined immunization or vaccination plan.
Technical scheme
The invention provides the antigen composition of the respiratory system disease of the secondary infection of prevention and treatment one boar, wherein, haemophilus parasuis antigen and the adjuvant of the Porcine reproductive and respiratory syndrome antigen that described antigen composition comprises at least one immunity amount and at least one immunity amount, described Porcine reproductive and respiratory syndrome antigen and haemophilus parasuis antigen comprise the antigen of holoantigen, subunit antigen, live recombinant vectors antigen and the DNA vector of attenuated live holoantigen, deactivation independently of one another.
Described " Porcine reproductive and respiratory syndrome antigen " refers to any compositions that contains at least one antigen, and the immunne response that opposing porcine reproductive and respiratory syndrome virus infects can be induced, stimulates or be strengthened to described antigen, when to pig administration.Preferably, in vaccine combination of the present invention, Porcine reproductive and respiratory syndrome antigen is totivirus antigen, comprise the street strain of clinical separation well known to those skilled in the art, contain, be preferably the Porcine reproductive and respiratory syndrome totivirus of deactivation form, the Porcine reproductive and respiratory syndrome live virus of improvement or the porcine reproductive and respiratory syndrome virus of attenuation, contain at least embedded virus of the immunogenicity aminoacid sequence of porcine reproductive and respiratory syndrome virus, any other contains at least polypeptide of the immunogenicity aminoacid sequence of porcine reproductive and respiratory syndrome virus, subunit or other compositions, Porcine reproductive and respiratory syndrome antigen can also comprise any antigen of following compositions: as Boehringer Ingelheim animal health (U.S.) company limited produce Porcine reproductive and respiratory syndrome live vaccine (
pRRS MLV), the Porcine reproductive and respiratory syndrome inactivated vaccine inactivated vaccine (CH-1a strain) that the Porcine reproductive and respiratory syndrome inactivated vaccine (NVDC-JXA1 strain) that Chengdu medical instruments factory of Zhongmu Industry Co.,Ltd, Deng Duo company of Qilu Animal Health Products Co., Ltd. produce and Harbin Wei Ke biotechnology development company produce, the Porcine reproductive and respiratory syndrome live vaccine (CH-1R strain) that Harbin Wei Ke biotechnology development company produces, high-pathogenicity porcine reproductive and respiration syndrome live vaccine (JXA1-R strain) that Guangdong Dahuanong Animal Health Products Co., Ltd. etc. produce, pig breeding and breathing syndrome live vaccine (R98) that Jiangsu Nannong High Science Co., Ltd etc. produce, high-pathogenicity porcine reproductive and respiration syndrome live vaccine (HuN4-F112 strain) that Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. etc. produce, high-pathogenicity porcine reproductive and respiration syndrome live vaccine (TJM-F92 strain) that Xinjiang Tiankang Raise Livestock Biotechnology Co., Ltd produces.
Preferably, described Porcine reproductive and respiratory syndrome antigen is NVDC-JXA1-R attenuation whole virus vaccine, described haemophilus parasuis antigen is the deactivation whole-bacterial-vaccine of serotype 4 type JS strains and Serotype 5 ZJ strain, described haemophilus parasuis serum 4 type JS strain preserving numbers are CCTCCNo.M2011172, and described haemophilus parasuis Serotype 5 ZJ strain preserving number is CCTCCNo.M2011173.
More preferably, in vaccine combination of the present invention, described Porcine reproductive and respiratory syndrome totivirus antigen is every part viral level 10
4.5tCID
50～10
7.0tCID
50; More preferably, described totivirus antigen is every part viral level 10
4.8tCID
50～10
6.5tCID
50; Again preferably, described totivirus antigen is every part viral level 10
5.0tCID
50.
Described " haemophilus parasuis antigen " refers to any compositions that comprises at least one antigen, and the immunne response that anti-haemophilus parasuis infects can be induced, stimulates or be strengthened to described haemophilus parasuis antigen after to pig administration.Preferably, described haemophilus parasuis antigen is the complete full bacterium of haemophilus parasuis, comprise the wild bacterial strain of clinical separation well known to those skilled in the art, contain, be preferably the full bacterium of haemophilus parasuis of deactivation form, the haemophilus parasuis viable bacteria of improvement or the haemophilus parasuis of attenuation, contain at least chimeric bacterial strain of the immunogenicity aminoacid sequence of haemophilus parasuis, any other contains at least polypeptide of the immunogenicity aminoacid sequence of haemophilus parasuis, subunit or other compositions, haemophilus parasuis antigen can also comprise any antigen of following compositions: as the Haemophilus parasuis inactivated vaccine (Z-1517 strain) of Boehringer Ingelheim animal health (U.S.) company limited production, the Haemophilus parasuis inactivated vaccine (serum 1 type SV1+ serum 6 type SV6) that the biological large pharmaceutical factory of Spain Hai Bolai produces, Hua Zhong Agriculture University, Wuhan Keqian Animal Biological Products Co., Ltd., the Haemophilus parasuis inactivated vaccine (serum 4 type MD0322+ Serotype 5 SH0165) that Zhongmu Industry Co.,Ltd produces, the Haemophilus parasuis inactivated vaccine (serum 1 type LC strain+Serotype 5 LZ strain) of Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricul.
More preferably, described serum 4 type haemophilus parasuis totivirus antigenic contents are deactivation front 5 × 10
8cFU/ head part～8 × 10
9cFU/ head part; More preferably, described haemophilus parasuis totivirus antigenic content is before deactivation to be 4 × 10
9cFU/ head part, more preferred described serum 4 type haemophilus parasuis totivirus antigenic contents are deactivation front 2 × 10
9cFU/ head part.
Described Serotype 5 haemophilus parasuis totivirus antigenic content is deactivation front 5 × 10
8cFU/ head part～8 × 10
9cFU/ head part; More preferably, described haemophilus parasuis totivirus antigenic content is deactivation front 4 × 10
9cFU/ head part, more preferred described Serotype 5 haemophilus parasuis totivirus antigenic content is deactivation front 2 × 10
9cFU/ head part.
Preferably, described adjuvant comprises nanorize aluminium hydroxide gel, mineral oil, carbomer, MONTANIDE GEL ST, propolis, cytokine, liposome, immunostimulating complex, containing CpC deoxy-oligonucleotide, nanorize ISA206, ISA760VG, nanorize aluminum phosphate, saponin, sorbitan, vegetable oil, ethyl oleate, two (caprylic/capric) propylene glycol ester, three (caprylic/capric) glyceride, two oleic acid propylene glycol esters, isostearate, sorbitan, mannide, glycerol, polyglycereol, propylene glycol, oleic acid, isostearic acid, castor oil acid, hydroxy stearic acid ester, polyoxypropylene, polyoxyethylene block copolymer, acrylic or methacrylic acid polymer, maleic anhydride and thiazolinyl derivant copolymer, the polymer of the crosslinked acrylic or methacrylic acid of the polyalkenyl ether of sugar or polyhydric alcohol one or more combination thing.
Preferably, described adjuvant is aqueous adjuvant, and term aqueous adjuvant is that oil-containing or oil content are not the general designation of the adjuvant of 5% (w/v).MONTANIDE GEL ST aqueous adjuvant, nanorize aluminium hydroxide gel that for example carbomer aqueous solution, SEPPIC company produce.More preferably, described adjuvant is aqueous adjuvant, and described aqueous adjuvant comprises carbomer aqueous solution, 206 adjuvants, nanorize aluminium hydroxide gel adjuvant, Montanide IMS1313 VG, Montanide ISA15A VG, Montanide GEL 01 PR and Montanide GEL ST polymer adjuvant.
The present invention also provides strain serum 4 type haemophilus parasuis JS strains, and preserving number is CCTCCNo.M2011 172; With a strain Serotype 5 haemophilus parasuis ZJ strain, preserving number is CCTCCNo.M2011173.
Optimal way of the present invention relates to the heat-resisting lyophilized protecting agent of vaccine combination, is preferably the one or more combination thing of gelatin, trehalose, polyvinylpyrrolidone, sodium glutamate, bovine serum albumin, sorbitol, sodium carboxymethyl cellulose, potassium dihydrogen phosphate, sodium hydrogen phosphate, lactose, skim milk.
Preferably; described NVDC-JXA1-R attenuation whole virus vaccine also comprises heat-resisting lyophilized protecting agent; described heat-resisting lyophilized protecting agent comprises 1%～2% gelatin, 5%～10% trehalose, 1%～2% polyvinylpyrrolidone (PVP), 0.5%～2% sodium glutamate, 0.1%～2% bovine serum albumin, 0.1%～1% sorbitol, 0.1%～0.2% sodium carboxymethyl cellulose, 0.164% potassium dihydrogen phosphate and 0.052% sodium hydrogen phosphate, and all the other are water.
As the preferred embodiment of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine composition of the present invention, described haemophilus parasuis inactivated vaccine part, bacterium solution preparation culture medium is the preferred haemophilus parasuis culture medium of this laboratory (MHPs): polyprotein peptone (5～10g/L), yeast extract (3～10g/L), sodium glutamate (2～8g/L), lactoalbumin hydrolysate (1～5g/L), sodium chloride (1～6g/L), dipotassium hydrogen phosphate (1～5g/L), glucose (0.1～4g/L), with NaOH adjust pH to 6.8～8.0, 116～121 ℃ of sterilizing 10～40min, add again porcine blood serum (5～100ml/L), NADH(1～50ml/L of 1%), while preparing solid medium, before sterilizing, add agar powder (1～20g/L).Psma ligand is than being the 4 type JS strains of 1:1(serum: haemophilus parasuis Serotype 5 ZJ strain), antigen cumulative volume is 75%～90% of described vaccine cumulative volume, in vaccine, content is that front haemophilus parasuis serum 4 types of deactivation and 5 type viable counts are 5.0 × 10
8cFU/ head part～8.0 × 10
9cFU/ head part.
As the preferred embodiment of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine composition of the present invention, described Porcine reproductive and respiratory syndrome live vaccine part, the weak poison of PRRS be domestic popular strain through chemistry or additive method cause weak and prove not return strong CH-1R strain, JXA1-R strain, R98 strain, HuN4-F112 strain or TJM-F92 strain, adding heat-resisting lyophilized protecting agent is 1%～2% gelatin, 5%～10% trehalose, 1%～2% polyvinylpyrrolidone (PVP), 0.5%～2% sodium glutamate, 0.1%～2% bovine serum albumin, 0.1%～1% sorbitol, 0.1%～0.2% sodium carboxymethyl cellulose, 0.164% potassium dihydrogen phosphate, 0.052% sodium hydrogen phosphate, after lyophilizing, be finished product (every part viral level 10
4.5tCID
50～10
7.0tCID
50).
A kind of method that the invention provides antigen composition of respiratory system disease of the secondary infection of preparing prevention and treatment pig, described method comprises the step of preparing Porcine reproductive and respiratory syndrome live vaccine and the step of preparing haemophilus parasuis inactivated vaccine:
The described step of preparing Porcine reproductive and respiratory syndrome live vaccine comprises the described porcine reproductive and respiratory syndrome virus of propagation, is mixed with heat-resisting lyophilized protecting agent, and lyophilizing obtains Porcine reproductive and respiratory syndrome live vaccine;
The described step of preparing haemophilus parasuis inactivated vaccine comprises the described haemophilus parasuis of propagation, and deactivation, with adjuvant mixed preparing.
The preparation of Haemophilus parasuis inactivated vaccine comprises the steps: (1) respectively the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain culture propagations to be cultivated, and obtains haemophilus parasuis 4 type JS strain bacterium liquid, 5 type ZJ strain bacterium liquid; (2) respectively (1) step is cultivated to the haemophilus parasuis 4 type JS strain bacterium liquid that obtain, 5 type ZJ strain bacterium liquid and add 0.3% formalin by total amount separately, place 37 ℃ of deactivation 24h, stir during this time 3～5 times, according to count results ultrafiltration bacterium liquid; (3) the haemophilus parasuis of above-mentioned deactivation, ultrafiltration 4 type JS strain bacterium liquid 1:1 ratios are mixed, then carry out emulsifying with adjuvant, adjuvant content is 10%～25%; Finished product Haemophilus parasuis killed vaccine antigen content is that front haemophilus parasuis serum 4 types of deactivation and 5 type viable counts are 5.0 × 10
8cFU/ head part～8.0 × 10
9cFU/ head part.(4) with the novel Haemophilus parasuis inactivated vaccine of appropriate volume subpackage.(1) the preparation method of Porcine reproductive and respiratory syndrome live vaccine comprises the steps:, in tidal type microcarrier suspension culture bioreactor, to add 1.0 × 10
9～2.0 × 10
9the Marc-145 cell of cells/g microcarrier or its clonal cell line passage cell and cell growth medium, subculture cell, working procedure: the upper and lower running frequency of carrier is 0～10mm/s, carrier upper and lower end points time of staying in carrier bottle is 0～2min.(2) passage cell is cultured to 1.5 × 10
10cells/L～2.0 × 10
10when cells/L, using cell maintenance medium instead, is M.O.I.=0.0001～1.0 inoculation porcine reproductive and respiratory syndrome virus according to infection multiplicity, cultivates porcine reproductive and respiratory syndrome virus; (3) gather in the crops porcine reproductive and respiratory syndrome virus liquid and add heat-resisting lyophilized protecting agent to carry out lyophilizing, make Porcine reproductive and respiratory syndrome live vaccine.
As the preferred embodiment of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine composition of the present invention, described compositions will be carried out consistency check, respectively get 2 bottles of live vaccine parts, use respectively Haemophilus parasuis inactivated vaccine part (diluent to be checked) and with reference to diluent (sterile water for injection), restore according to operation instruction (1 part/bottle of live vaccine part, 1 part of inactivated vaccine part (2ml)/bottle).Place 2h, check according to Porcine reproductive and respiratory syndrome live vaccine titration method for 20 ± 3 ℃.By with comparison with reference to diluent, assess the impact of diluent to be checked on virus activity component, the difference of the titration results of virus component should be no more than 0.7log10.
The present invention also provides a kind of described antigen composition to apply in the medicament for treating respiratory system thing of the secondary infection of preparation prevention and treatment pig.
Preferably, described application is restored described Porcine reproductive and respiratory syndrome live vaccine with described haemophilus parasuis inactivated vaccine before being included in application.
The present invention also provides a kind of test kit, and described test kit comprises the antigen product of the opposing Porcine reproductive and respiratory syndrome separating on the packaging or antigen product or immunogenic composition or the vaccine of immunogenic composition or vaccine and opposing haemophilus parasuis.
technique effect
The present invention has adopted haemophilus parasuis serum 4 types, 5 types and Porcine reproductive and respiratory syndrome strain, inventor is surprised to find that not only the immune effect of a few strain strains is good, and do not affect each other, can effectively prevent pig blue-ear disease that the Haemophilus parasuis that caused by haemophilus parasuis and porcine reproductive and respiratory syndrome virus cause and the mixed infection of the two thereof; Inactivated vaccine part of the present invention has adopted water-soluble polymer class adjuvant and the suitable Seedling technology of joining to prepare Haemophilus parasuis inactivated vaccine; can dilute Porcine reproductive and respiratory syndrome live vaccine with it as diluent; the two as compositions combine use safe, quality controllable; immune effect is good, and counteracting toxic substances protective rate reaches 80%～100%.
The present invention at least has the following advantages:
1) solved prior art difficulty, understanding prejudice when having changed people porcine reproductive and respiratory syndrome virus and haemophilus parasuis being infected simultaneously, first Porcine reproductive and respiratory syndrome antigen and haemophilus parasuis antigen are combined to use according to suitable ratio, especially, first haemophilus parasuis inactivation antigen is used as this vaccine compound mode of dilution Porcine reproductive and respiratory syndrome active antigen, and before the present invention, never people thinks that two kinds of diseases can be prevented and then these two kinds of vaccines can be combined use simultaneously.
2)porcine reproductive and respiratory syndrome of the present invention and haemophilus parasuis antigen composition and use prevention prepared by described antigen composition and bigeminy vaccine that treatment Porcine reproductive and respiratory syndrome and haemophilus parasuis infect, not only can not produce mutual immune interference or the impact of two kinds of antigen compositions, and after the unexpected antigen mixing of finding Porcine reproductive and respiratory syndrome and haemophilus parasuis, when protective immune response that the antigen composition of Porcine reproductive and respiratory syndrome and haemophilus parasuis can not only produce, also unexpected discovery Porcine reproductive and respiratory syndrome and haemophilus parasuis antigen have the effect of mutual enhancing immune effect, and prove as subsequent embodiment of the present invention, particularly exist wherein in a kind of situation of antigen, another kind of antigen amount reduces by half and still can maintain the immune effect of this antigen, and this exceeds those of ordinary skills' expectation.
3)porcine reproductive and respiratory syndrome of the present invention and haemophilus parasuis antigen composition and use prevention prepared by described compositions and bigeminy vaccine that treatment Porcine reproductive and respiratory syndrome and haemophilus parasuis infect, compared with the effect of single vaccine of Porcine reproductive and respiratory syndrome or haemophilus parasuis, in to pig injecting immune process, pig body is without side reaction, can there is not the inflammatory reaction such as redness, suppuration in injection site, therefore, bigeminy vaccine safety of the present invention is better, the untoward reaction that can avoid repeatedly immunoprophylaxis to occur.
4) contain two or more antigen, can prevent the combined vaccine of two or more disease to become the feature of vaccine research of new generation with its convenience, multiple-effect, low cost.Compared with single vaccine, combined vaccine can reduce the inoculation times of vaccine, avoids can not obtaining omnidistance immunity because leaking to plant.
5) bigeminy vaccine of the present invention's prevention and treatment mycoplasma hyopneumoniae relevant disease and Streptococcus suis relevant disease, preparation method is simple, the content of tiring of vaccine is high, immunity is convenient and swift, with repeatedly immunity of the prior art, at least needs to make a call to 2 pins or 3 pins and could prevent and treat vaccine and the immunization method thereof of two kinds of diseases above and compare, the present invention only immunity just can prevent mycoplasma hyopneumoniae and two kinds of pathogen infections of Streptococcus suis for 1 time, reduce immune cost, saved immune programme for children, reliable more economically.
6) in addition, it is to propagate rapidly that the applicant reappears principal character at one, fervescence, appetite depression, dyspnea, dead pig extremity, ear is blue, cut open the visible interstitial pneumonia of inspection, pericardial effusion, the a large amount of fibroid exudates of peritoneum organ surface, intestinal tube and abdominal wall adhesion, arthroncus also has hydrops and finds when the respiratory system disease of lymphadenectasis, when only infecting haemophilus parasuis in swinery, can't there is above a large amount of characteristic clinical symptoms and pathological changes, only have when existing in swinery in the situation of reproductive and respiratory syndrome, just can cause the respiratory system disease of above-mentioned symptom.It is unexpected that the sick pig of finding these symptoms be to be caused by these two kinds of pathogen synergism, and once secondary infection haemophilus parasuis causes mixed infection, various antibacterials are all difficult to prove effective, and morbidity is rapid, mortality rate is high.Porcine reproductive and respiratory syndrome of the present invention and haemophilus parasuis antigen composition are to the described respiratory system disease control action that had good prevention.
bacterial strain preservation explanation:
The 4 type JS strains of Haemophilus parasuis serum
Classification And Nomenclature: haemophilus parasuis serum 4 type JS strains (Haemophilus parasuisSerotype 4, strain JS),
Preserving number is: CCTCC NO:M 2011172;
Preservation date: on May 18th, 2011,
Preservation address: Wuhan, China, Wuhan University, Chinese Typical Representative culture collection center
Depositary institution: Chinese Typical Representative culture collection center (being called for short CCTCC).
Haemophilus parasuis Serotype 5 ZJ strain:
Classification And Nomenclature: haemophilus parasuis Serotype 5 ZJ strain (Haemophilus parasuisSerotype 5, strain ZJ),
Preserving number is: CCTCC NO:M 2011173;
Preservation date: on May 18th, 2011,
Preservation address: Wuhan, China, Wuhan University, Chinese Typical Representative culture collection center
Depositary institution: Chinese Typical Representative culture collection center (being called for short CCTCC).
Accompanying drawing explanation
Fig. 1 is tidal type microcarrier suspension bioreactor schematic diagram;
Fig. 2 is haemophilus parasuis vaccine endotoxin assay result.
Reference numeral
The specific embodiment
The tidal type bioreactor construction adopting in the embodiment of the present invention as shown in Figure 1.
Several pipelines are housed on the lid of carrier tank 8, and the effect of these pipelines comprises: mode is to the interior filling liquid of carrier tank 8 (as inoculating cell suspension etc.) or discharge the liquid in carrier tank 8 manually; Controlled to carrier tank 8 injecting gas by computer controller 7, gas is compressed air, oxygen and CO normally
2the mixture of three kinds, three kinds of gas ratio in mixture can auto-adjustment control, to adapt to cell culture needs.Computer controller 7 can be controlled mist automatically to injection and discharge in carrier tank 8, and provides power for morning and evening tides.Culture fluid bag 6 is in order to splendid attire culture fluid, and communicates with carrier tank 8 bottoms by two pipelines, and two pipelines are respectively liquid perfusion passage, by and carrier tank 8 between liquid flow, thereby complete morning and evening tides process.The perfusion rate of culture fluid in morning and evening tides process can be adjusted by computer controller 7.The liquid measure of changing of culture fluid refers in a morning and evening tides process, pumps into or pump the amount of liquid of carrier tank 8, and the size of changing liquid measure has determined the residing position in carrier tank 8 interior liquid level top and bottom.On the pipeline being connected between culture fluid bag 6 and carrier tank 8, be provided with into/probe tube 11, can use asepsis injector by enter/probe tube 11, culture fluid to be sampled, in order to detect the index such as glucose content and viral level wherein.The interpolation of glucose: if glucose is lower than required standard, measure according to glucose sensor the content that remains glucose in culture fluid, and the cumulative volume of culture fluid calculates the glucose amount that need add, the glucose solution that utilizes asepsis injector to extract preparation is expelled in the conduit between carrier tank 8 and culture fluid bag 6 from enter/probe tube 11, and glucose solution is along with the liquid that changes of carrier tank is able to enter culture fluid bag 6 and do further from conduit mix.Constant temperature oscillation case 5, by heating and vibration, carrys out the homogeneity of the constant and composition of maintain liquid bag 6 interior culture-liquid temps.PH/DO watch-dog 4 can be monitored acid-base value (pH value) and the dissolved oxygen (DO) of culture fluid in culture fluid bag, and by injecting alkaline solution (NaOH or NaHCO
3solution) pH value of culture fluid is controlled in OK range.Rewinding bucket 2 and head tank 1 are all connected with culture fluid bag 6 by automatically presenting material instrument 3, when culture fluid in culture fluid bag 6 need to be gathered in the crops, can enter rewinding bucket 2 by automatic feedback material instrument 3, the culture fluid in head tank 1 can supplement culture fluid bag 6 by automatic feedback material instrument 3.
Method of the present invention does not have specific (special) requirements for unit used or instrument, and other various types of bioreactors also can use method of the present invention to complete.
Specific experiment method of the present invention can be referring to " People's Republic of China's veterinary drug allusion quotation (two 〇 mono-〇 versions) " and appendix.Other not marked reagent or raw material all can be obtained according to prior art by those skilled in the art.
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with describing.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
embodiment 1 tidal type microcarrier suspension bioreactor large-scale production porcine reproductive and respiratory syndrome virus and vaccine preparation
In the present embodiment, microcarrier used is polyester fiber, be that (this strain is disclosed in Chinese patent CN101307305A to NVDC-JXA1-R strain for the preparation of the Strain of reproductive and respiratory syndrome virus antigen, be preserved in Chinese common micro-organisms culture presevation administrative center with preserving number No.1964), reproductive and respiratory syndrome virus is there is to the cell line African green monkey kidney cell (Marc-145 cell) of good sensitivity, as seedling cell line, select 20L carrier bottle tidal type bioreactor.
1). the inoculation of cell and cultivation
First use cell growth medium (containing 5% Ox blood serum MEM culture medium, pH value is 7.25) to utilize rolling bottle amplification cultivation Marc-145 cell.In 20L bioreactor, add sterile carrier 1300g, after cell dissociation in rolling bottle is disperseed take cell density as 1.0 × 10
9in cells/L access 20L bioreactor, make it to be combined with carrier, bioreactor and 37 ℃ of constant temperature oscillation case 5(capacity 500L that growth-promoting media is housed) be connected, condition of culture is absorption program: the liquid level rate of climb 5mm/s of culture fluid, decrease speed 3mm/s, liquid level is 60s/10s in the upper and lower end points of reactor dead time.37.0 ℃ of cultivation temperature, pH value regulates 7.3, and dissolved oxygen regulates 65%, and gas concentration lwevel regulates 10%.
The timing of self-operating absorption program, starts cultivation program: the liquid surface lifting speed of culture fluid is 4mm/s after 3h, liquid level is 50s/50 s in the upper and lower end points of reactor dead time.Perfusion cultures 5 days, 37.0 ℃ of cultivation temperature, adopt 7.5%(W/V) NaHCO
3automatically regulate pH value, make pH value be controlled at 7.2 left and right, dissolved oxygen is 50%, and gas concentration lwevel is 5%.After cell inoculation, get carrier sample and bouillon-like by the time point of every 24h, control concentration of glucose 1000～4500mg/L in culture fluid.
2) virus inoculation with cultivate in 20L tidal type suspension culture bioreactor, cell culture to the 5 days, the density of Marc-145 cell in bioreactor reaches 3.5 × 10
10individual/L, changes the culture fluid in culture fluid bag 6 the standard MEM culture medium that maintenance medium contains 1% Ox blood serum into), be 0.001 inoculation reproduction and breath syndrome virus by M.O.I..Operation connects malicious program: the liquid level rate of climb 4mm/s of culture fluid, and decrease speed 2mm/s, liquid level stops 55s in reactor head, and bottom stops 10s, viruses adsorption 3h.Afterwards, operation Virus culture program: the liquid level rise and fall speed of maintenance medium is 4mm/s, and liquid level stops 50s in reactor head, and bottom stops 50s.37.5 ℃ of cultivation temperature; Adopt 7.5%(W/V) NaHCO
3automatically regulate pH, make pH value maintain 7.2; Dissolved oxygen is 30%, and gas concentration lwevel is 3%.
4) the results of virus gather in the crops virus liquid on the 3rd day after being cultured to and connecing poison, first carrier bottle 8 is moved to virus liquid and discharge program, make it all to flow in culture fluid bag 6, to the virus liquid in culture fluid bag 6, adopt the automatic material receiving bucket 2 of 50L to collect according to the flow velocity of 5L/min ,-20 ℃ of preservations.Results virus liquid 500L, virus titer remains on 10
9.5more than TCID50/ml.
Marc-145 cell is in the time utilizing the tidal type bioreactor of other volumes, and cell culture and virus multiplication situation are basic identical, results virus also no significant difference of titre also.
5) check of seedling venom: carry out according to high-pathogenicity porcine reproductive and respiration syndrome live vaccine (JXA1-R strain) inspection procedure.
6) join Seedling, subpackage and lyophilizing: by the virus liquid being up to the standards, be mixed in same container, by 1:1(v/v) add the heat-resisting lyophilized protecting agent component of 1%～2% gelatin, 5%～10% trehalose, 1%～2% polyvinylpyrrolidone (PVP), 0.5%～2% sodium glutamate, 0.1%～2% bovine serum albumin, 0.1%～1% sorbitol, 0.1%～2% sodium carboxymethyl cellulose, 0.164% potassium dihydrogen phosphate, 0.052% sodium hydrogen phosphate and water composition and formula (in table 1, precentagewise represents), fully mix quantitative separating; After subpackage, carry out rapidly (every part viral level 10 that gets product after lyophilisation
5.0tCID
50).
Table 1 heat-resisting lyophilized protecting agent component and formula
| Component (%) | |
|
|
Formula 4 |
| Gelatin | 1.5 | 1 | 1.75 | 2 |
| Trehalose | 6.75 | 10 | 8.25 | 5 |
| |
1 | 2 | 0.5 | 1.5 |
| Polyvinylpyrrolidone (PVP) | 1.5 | 2.0 | 1.25 | 1.0 |
| |
1 | 0.1 | 0.5 | 2 |
| |
1 | 0.5 | 0.75 | 0.1 |
| Sodium carboxymethyl cellulose | 0.15 | 0.1 | 0.1 | 0.2 |
| Potassium dihydrogen phosphate | 0.164 | 0.164 | 0.164 | 0.164 |
| Sodium hydrogen phosphate | 0.052 | 0.052 | 0.052 | 0.052 |
| Distilled water | 86.884 | 84.184 | 86.684 | 87.984 |
The preparation method of heat-resisting lyophilized protecting agent, carry out according to the following steps: measure gelatin, trehalose, polyvinylpyrrolidone (PVP), potassium dihydrogen phosphate, sodium hydrogen phosphate heated and stirred by each component in table 1 and be dissolved in a part of distilled water, 116 ℃ of autoclavings 30 minutes; Measure sodium glutamate, bovine serum albumin, sorbitol, sodium carboxymethyl cellulose by each component in table 1 and be dissolved in remaining distilled water, with the membrane filtration degerming in 0.22 micron, aperture; Then by 2 kinds of solution mix homogeneously, the heat-resisting lyophilized protecting agent of system.
Product after above-mentioned lyophilizing is done to following detection:
1. the physical behavior of product, steriling test, mycoplasma check, exogenous virus check, residual moisture mensuration and vacuum are measured and are tested by existing " Chinese veterinary pharmacopoeia " appendix, all conform with the regulations.
2. the safety testing of product: this vaccine is pressed 10～100 multiple doses of using dosage, the Porcine reproductive and respiratory syndrome antigen-antibody jack to jack adapter pig of 5 21 ages in days of musculi colli injection, every day thermometric observing 21, without fervescence and clinical abnormal response.
the screening of embodiment 2 adjuvants
With the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain antigens, join respectively Seedling with white-oil adjuvant, 206 adjuvants, nanorize aluminium hydroxide gel adjuvant, Montanide IMS1313 VG, MontanideISA15A VG, Montanide GEL 01 PR and Montanide GEL ST polymer adjuvant respectively, vaccine finished product antigenic content is for being 2.0 × 10 containing serum 4 type JS strains before deactivation, 5 type ZJ strain viable counts
9cFU/ head part.The PRRS vaccine consistency check that the vaccine of configuration is prepared with embodiment 1 after character check, safety examination and efficacy test.
(1) character check (in table 2)
The various Adjuvanted vaccines outward appearances of table 2, steriling test and viscosimetric analysis
(2) safety: get 5 of the healthy susceptible pigs of the each intramuscular injection of the vaccine of preparation, every 4mL, in 14 days, part vaccine group has part or general reaction, but whole strong living.Assay is as following table 3:
Table 3 vaccine safety result of the test
(3) potency test:
Get the vaccine of preparation, every group of vaccine is respectively with 10 of the healthy susceptible pigs of 28 ages in days, and 1 part of each intramuscular injection (2mL), after 28 days, the immunity test pig of having injected every kind of vaccine is divided into 2 groups at random, uses respectively haemophilus parasuis serum 4 types, 5 type bacterial strain counteracting toxic substances.10 of contrast pigs, divide 2 groups, use respectively haemophilus parasuis serum 4 types, 5 type bacterial strain counteracting toxic substances.
Haemophilus parasuis serum 4 type JS strains: choose respectively each 5 of the haemophilus parasuis Seedling immune swine of preparation, together with 5 of the identical contrast pigs of condition, lumbar injection 7.0 × 10
9the serum 4 type JS strain bacterium liquid 2ml of CFU, observe 14 days, and immune swine more than 4/5 is protected, and contrast pig more than 4/5 falls ill.
Table 4 vaccine potency assay
Note: Haemophilus parasuis morbidity standard: morbidity pig is dead or occur heating (body temperature more than 40.5 ℃, continues 1～5), lethargy, cough, dyspnea, becomes thin, walks lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open to inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
(4) vaccine consistency check is because white-oil adjuvant vaccine can not dissolve live vaccine, so get 12 bottles of live vaccine parts, use respectively Haemophilus parasuis inactivated vaccine part (diluent to be checked) and with reference to diluent (sterile water for injection), according to operation instruction, (1 part/bottle of live vaccine part, 1 part/bottle of inactivated vaccine part (2ml) restores.Place 2h, check according to Porcine reproductive and respiratory syndrome live vaccine titration method for 20 ± 3 ℃.By with comparison with reference to diluent, assess the impact of diluent to be checked on virus activity component, the titration results of virus component is as table 5.
Table 5 vaccine consistency check result
According to existing animal vaccines adjuvant and new vaccine adjuvant by the comparison of above safety, potency test and compatibility test, the safety of Haemophilus parasuis inactivated vaccine dilution live vaccine, immune effect and compatibility the best prepared by result aqueous adjuvant Montanide GEL ST, and it is simple to join Seedling technique, vaccine is easily injected, safety, reliable.
Haemophilus parasuis inactivated vaccine (JS strain+ZJ strain), qualified by deactivation, ultrafiltration and safety check, with the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain antigens, obtain with MontanideGEL 01 ST adjuvant mixed preparing, wherein above-mentioned 2 kinds of antigen ratios are 1:1, account for 90% of vaccine cumulative volume, adjuvant content is 10%, and vaccine finished product antigenic content is for being 2.1 × 10 containing serum 4 type JS strains before deactivation, 5 type ZJ strain viable counts
9cFU/ head part.
1. strain
1.1 haemophilus parasuis serum 4 type JS strains, are separated, are identified by Pulaike Biological Engineering Co., Ltd., carry out preservation, preservation date at Chinese Typical Representative culture collection center: on May 18th, 2011, preserving number is: CCTCC M 2011172.
1.2 haemophilus parasuis Serotype 5 ZJ strains, are separated, are identified by Pulaike Biological Engineering Co., Ltd., carry out preservation, preservation date at Chinese Typical Representative culture collection center: on May 18th, 2011, preserving number is: CCTCC M 2011173.
For the strong strain of acquired immunity, the lyophilizing after responsive pig rejuvenation accreditation of each strain is preserved and is contained.
2. the manufacture of vaccine and the inspection of semifinished product
2.1 produce the preparation with seed
Culture medium
Adopt the homemade haemophilus parasuis culture medium of this laboratory (MHPs): polyprotein peptone (5g/L), yeast extract (5g/L), sodium glutamate (5g/L), lactoalbumin hydrolysate (2.5g/L), sodium chloride (2.5g/L), dipotassium hydrogen phosphate (2.0g/L), glucose (1.0g/L), with NaOH adjust pH to 7.2,116 ℃ of sterilizing 30min, add again porcine blood serum (50ml/L), 1% NADH(10ml/L), while preparing solid medium, before sterilizing, add agar powder (12g/L).
2.1.1 the breeding of first order seed
The 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain freeze-drying lactobacillus, streak inoculation is on the preferred haemophilus parasuis culture medium of this laboratory that contains agar powder (MHPs) flat board respectively, putting 37 ℃ cultivates 18～24 hours, choose satisfactory single bacterium colony, be inoculated in MHPs fluid medium, cultivate 12～16 hours for 37 ℃, as first order seed.
2.1.2 the breeding of secondary seed
The culture of first order seed is got in the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, adds in MHPs fluid medium by 1% amount, cultivate 12～16 hours for 37 ℃, through check pure after as secondary seed.
2.2 seedling culture medium
Adopt the homemade haemophilus parasuis culture medium of this laboratory (MHPs).
The cultivation of 2.3 bacterium liquid
2.3.1 in MHPs culture medium, by the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strain secondary seed solution respectively by 2%(v/v) inoculum concentration adds in culture medium and cultivates respectively, mix rearmounted 37 ℃ and cultivate 16～18 hours, as the concentration OD of bacterium liquid
600reach more than 2.5, DO value start rise, pH value while being reduced to 6.5 below, stop cultivation.
2.3.2 check purely
After cultivation finishes, from four kinds of bacterium liquid of seedling, sample respectively, purely check according to " People's Republic of China's veterinary drug allusion quotation (two 〇 mono-〇 versions) " appendix, should be pure.
2.3.3 count plate
From seedling bacterium liquid, sample respectively, haemophilus parasuis carries out count plate to determine the bacterium number of cultivation with dull and stereotyped cultivation of MHPs.
2.3.4 deactivation
In above-mentioned four kinds of cultured bacterium liquid, by the 0.2%(V/V of total amount) add respectively formalin, be positioned over 37 ℃ of deactivation 24h, stir during this time 3～5 times.
2.3.5 deactivation check
2 kinds of haemophilus parasuis bacterium liquid getting deactivation respectively 5mL are inoculated in 100mL MHPs culture medium, cultivate 24 hours for 37 ℃, then from above-mentioned 100ml, get 1ml and transplant in 50mL culture medium 37 ℃ and cultivate 24 hours; Get 2 kinds of haemophilus parasuis bacterium liquid 0.5mL streak inoculation of deactivation in MHPs plating medium, cultivate 24 hours for 37 ℃.After two kinds of methods are cultivated, all asepsis growths.
2.3.6 safety check
Get above-mentioned four kinds of respectively 5 of Balb/C mices of intravenous injection 18～22g of bacterium liquid of deactivation, every 0.2mL, 5/5 strong living in three days, side for deactivation thorough.
3. the preparation of vaccine
3.1 antigen detoxifications
With cross-flow ultrafiltration system (Millipore Congent M1 TFF; CM05230) carry out ultrafiltration detoxification, and the substrate king crab detection kit detection endotoxin content of producing with company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City, the results are shown in Table 6;
Table 6 endotoxin assay result
3.2 vaccine proportionings (in table 7)
The concrete composition proportion of table 7 vaccine
| Main material component | Content |
| Haemophilus parasuis serum 4 type JS strain antigens | Deactivation front 4.8 × 10 9CFU/ head part |
| |
Deactivation front 4.8 × 10 9CFU/ head part |
| Montanide GEL ST(comprises homologous series adjuvant) | 10% |
3.3 vaccine endotoxin measurements
Measure according to endotoxin detection kit (company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City) description, normal equation is shown in Fig. 4, show that according to equation vaccine endotoxin content is respectively: 8825EU/ml.
3.4 subpackage
1 part/bottle, subpackage.
In the present embodiment, can also adopt other pathogenic haemophilus parasuis serological type strains (such as serum 1 type, serum 2 types, serum 13 types etc.), be not limited to the bacterial strain adopting in embodiment 2.The mode of deactivation also can adopt other ablation methods such as beta-propiolactone (BPL), glycidaldehyde (GDA), 60Co irradiation and heating.
embodiment 4 Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine related checks
1, physical behavior detection, steriling test, endotoxin measurement
3 batches of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis deactivation epidemic diseases (lot number 11001,11002,11003).Through physical behavior detect, steriling test is qualified, detailed results is in table 8.
The check of table 8 vaccine physical behavior, steriling test and endotoxin measurement
Note: T.G represents sulphur glycollate culture medium, G.A represents peptone from casein agar culture medium, G.P dextrose peptone medium; "-" represents asepsis growth.
2 safety testings
With 5 of the porcine reproductive and respiratory syndrome virus in 3～4 week age and haemophilus parasuis antigen, negative antibody piglets, each intramuscular inoculation is with the sample to be checked of 10 part live vaccine parts of 2 part inactivated vaccines dilutions., should not there is not the abnormal response of part or general and all strong alive in Continuous Observation 21 days.Assay is as following table 9:
Table 9 vaccine safety result of the test
3 vaccine consistency checks
Respectively get 2 bottles of live vaccine parts, use respectively Haemophilus parasuis inactivated vaccine part (diluent to be checked) and with reference to diluent (sterile water for injection), according to operation instruction, (1 part/bottle of live vaccine part, 1 part/bottle of inactivated vaccine part (2ml) restores.Place 2h, check according to Porcine reproductive and respiratory syndrome live vaccine titration method for 20 ± 3 ℃.By with comparison with reference to diluent, assess the impact of diluent to be checked on virus activity component, the titration results of virus component is as table 10, difference is all no more than 0.7log10(Europe live vaccine standard).
Three batches of vaccine consistency check results of table 10
| Vaccine batch | Inactivated vaccine dilution (TCID 50） | With reference to diluted (TCID 50) | Difference (log10) |
| 11001 | 10 4.5 | 10 4.8 | 0.3 |
| 11002 | 10 4.6 | 10 4.8 | 0.2 |
| 11003 | 10 4.5 | 10 4.9 | 0.4 |
4 potency tests
15 of the each porcine reproductive and respiratory syndrome virus with 3～4 week age of every group of vaccine and haemophilus parasuis antigen, negative antibody health susceptible pigs, every intramuscular injection 2ml, after 28d days, the immunity test pig of having injected every kind of vaccine is divided into three groups at random, uses respectively the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, the strong malicious NVDC-JXA1 strain of porcine reproductive and respiratory syndrome virus counteracting toxic substances separately.15 of contrast pigs, divide 3 groups, use respectively the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, the strong malicious NVDC-JXA1 strain counteracting toxic substances of porcine reproductive and respiratory syndrome virus.
Haemophilus parasuis serum 4 type JS strains: choose each 5 of immune swine, together with 5 of the identical contrast pigs of condition, lumbar injection 7.0 × 10
9the serum 4 type JS strain bacterium liquid 2ml of CFU, observe 14 days, and immune swine is answered more than 4/5 protection, and contrast pig answers more than 4/5 morbidity.
The strong malicious NVDC-JXA1 strain of porcine reproductive and respiratory syndrome virus: choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, the strong malicious culture fluid (10 of intramuscular injection NVDC-JXA1 strain
4.5tCID
50) 3ml, to observe 21 days, immune swine is answered more than 4/5 protection, and contrast pig 5/5 all should fall ill, and at least 2 death.
Assay is as shown in table 11.
Table 11 vaccine potency assay
Note: Haemophilus parasuis morbidity standard: morbidity pig is dead or occur heating (body temperature more than 40.5 ℃, continues 1～5), lethargy, cough, dyspnea, becomes thin, walks lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open to inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum);
Porcine reproductive and respiratory syndrome morbidity standard: body temperature at least 3rd is more than 41 ℃, or spirit, appetite declines, eye conjunctivitis, the respiratory symptom such as cough, breathe heavily, or substantially cut open inspection, there is lamellar consolidation in pulmonary.
5.1 Haemophilus parasuis, Porcine reproductive and respiratory syndrome Experimental infection
With the piglet in 7～8 week age of 40 haemophilus parasuis negative antibodies, the 1st group is infected 5, every collunarium bacterium liquid 2ml(2 × 10 with the 4 type JS strains of haemophilus parasuis serum
6cFU); The 2nd group is infected 5, every collunarium bacterium liquid 2ml(1 × 10 with haemophilus parasuis Serotype 5 ZJ strain
6cFU); The 3rd group with 4 type JS strains and 5 type ZJ strain mixed infections; The 4th group is infected (10 with the strong malicious NVDC-JXA1 strain of porcine reproductive and respiratory syndrome virus
4.5tCID
50); The 5th group with secondary pig 4 type JS strains and the strong malicious NVDC-JXA1 strain mixed infection of blue ear; The 6th group with secondary pig 5 type ZJ strains and the strong malicious NVDC-JXA1 strain mixed infection of blue ear; The 7th group with secondary pig 4 type JS strains, the 5 type ZJ strains of secondary pig and the strong malicious NVDC-JXA1 strain mixed infection of blue ear (in table 12), carry out the research of cases of infection, the 8th group is matched group, do not carry out any counteracting toxic substances, Continuous Observation 14 days after counteracting toxic substances, cut open and kill all pigs and carry out pathological change, antibacterial (virus) and separate PCR and identify, the results are shown in Table 13.
The grouping of table 12 counteracting toxic substances piglet and counteracting toxic substances dosage
| Group | A piglet number | Counteracting toxic substances bacterium (poison) is planted | Counteracting |
| 1 | 5 | HPs JS strain | 2×10
6 |
| 2 | 5 | |
1×10
6 |
| 3 | 5 | HPs JS strain+ |
2×10 6CFU+1×10 6CFU |
| 4 | 5 | NVDC- |
10
4.5 |
| 5 | 5 | HPs JS strain+NVDC- |
2×10
6CFU+10
4.5 |
| 6 | 5 | HPs ZJ strain+NVDC- |
1×10
6CFU+10
4.5 |
| 7 | 5 | HPs+ |
2×10
6CFU+1×10
6CFU+10
4.5 |
| 8 | 5 - - |
Piglet clinical symptoms and cut open inspection pathological changes result after table 13 counteracting toxic substances
Note: "-" represents not damaged or haemophilus parasuis antigen do not detected at damage location.
By testing unexpected discovery, can find out from the result of table 13, only infect with the haemophilus parasuis collunarium of low dosage, pig body can not be fallen ill, and also inorganization degree of impairment occurs, carries out antibacterial isolation identification and fails to detect antigen; And infect at the haemophilus parasuis collunarium with low dosage, and infect porcine reproductive and respiratory syndrome virus simultaneously, there will be fervescence, appetite depression, dyspnea, dead pig extremity, ear are blue, cut open inspection visible interstitial pneumonia, pericardial effusion, a large amount of fibroid exudates of peritoneum organ surface, intestinal tube and abdominal wall adhesion, arthroncus have hydrops and the respiratory system disease of lymphadenectasis, and when only the porcine reproductive and respiratory syndrome virus with same dose infects serious tissue injury; In these tissue injurys, can separation detection arrive a large amount of reproductive and respiratory syndrome virus antigens, but in these identical tissue injurys, can't detect the existence of haemophilus parasuis antigen.Matched group is not observed macroscopic or histological damage yet.Therefore, seem to only have haemophilus parasuis and this combination of reproductive and respiratory syndrome virus can reappear this serious respiratory system disease.The respiratory system disease that the prepared vaccine combination of the present invention causes for this mixed infection of pig, has diagnostic significance clinically.
5.2 vaccine injection pig safety tests
Haemophilus parasuis inactivated vaccine (4 type+5 type) lot number by the production of Wuhan Ke Qian biotech firm of selling on the some bottles of vaccine that to randomly draw Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine lot number of being up to the standards be 11001 and market is 100409, high-pathogenicity porcine reproductive and respiration syndrome live vaccine (JXA1-R strain) lot number that sea, Shanghai profit is produced are 10080, use respectively porcine reproductive and respiratory syndrome virus and the haemophilus parasuis antigen in 6～7 week age, 25 of the healthy susceptible piglets of negative antibody are injected, test as follows: (antigenic content is front 4 types of deactivation and 5 types are each is all no less than 4 × 10 to 2 parts of Wuhan Ke Qian biotech firm Haemophilus parasuis inactivated vaccine (HPS) intramuscular injection vaccine
9cFU/ head part), the high-pathogenicity porcine reproductive of Shanghai Hai Li company and 10 parts of respiration syndrome live vaccine (PRRS) intramuscular injection (every part viral level>=10
5.0tCID
50), (every part is containing JXA1-R strain virus amount>=10 for 2 parts of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine (HPs+PRRS) intramuscular injection prepared by this laboratory
4.8tCID
50, containing the front 4 type JS strains of deactivation with 5 type ZJ strain is each is all no less than 2 × 10
9cFU/ head part), Continuous Observation 14 days after injection, result is as table 14.
The comparative test of table 14 vaccine safety in utilization
Note: side reaction comprises that appetite declines, vomits, trembles, astasia, dyspnea, have loose bowels etc.
Result of the test shows, Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine safety prepared by the present invention is good, can not produce side reaction, and commercially available Haemophilus parasuis inactivated vaccine and Porcine reproductive and respiratory syndrome live vaccine have side reaction to produce, and provide immune programme for children will carry out 2 immunity according to it.
5.3 vaccine immunity pig protest tests
Haemophilus parasuis inactivated vaccine (4 type+5 type) lot number by the production of Wuhan Ke Qian biotech firm of selling on the some bottles of vaccine that to randomly draw Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine lot number of being up to the standards be 11001 and market is 100409, high-pathogenicity porcine reproductive and respiration syndrome live vaccine (JXA1-R strain) lot number that sea, Shanghai profit is produced are 10080, use respectively porcine reproductive and respiratory syndrome virus and the haemophilus parasuis antigen in 3～4 week age, the healthy susceptible piglet of negative antibody carries out immune swine counteracting toxic substances protection contrast test, test following (table 15): according to the operation instruction of vaccine, the 1st, 5 groups, (antigenic content is front 4 types of deactivation and 5 types are each is all no less than 4 × 10 to 1 part of Wuhan Ke Qian biotech firm Haemophilus parasuis inactivated vaccine (HPS) intramuscular injection vaccine
9cFU/ head part), exempted from same dose and approach two after 3 weeks at interval, 2nd, 6 groups, the high-pathogenicity porcine reproductive of Shanghai Hai Li company and 1 part of respiration syndrome live vaccine (PRRS) intramuscular injection (every part viral level>=10
5.0tCID
50), 3rd, 7 groups, (every part is containing JXA1-R strain virus amount>=10 for 1 part of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine (HPs+PRRS) intramuscular injection prepared by this laboratory
4.8tCID
50, containing the front 4 type JS strains of deactivation with 5 type ZJ strain is each is all no less than 2 × 10
9cFU/ head part), the 4th, 8 groups is blank group.
Table 15 vaccine immunity piglet
After vaccine immunity, the 1st group, the 5th group respectively has respectively 1 pig injection site to occur the symptoms such as redness, fervescence, and the pig of other group is all normal.
Counteracting toxic substances when immunity piglet is large at 8 weeks, 4 type JS strains and the protection of 5 type ZJ strain counteracting toxic substances the results are shown in Table 16: haemophilus parasuis serum 4 type JS strain lumbar injection bacterium liquid 2ml(are containing viable count 7.0 × 10
9cFU), the 1st group of HPs immune swine, the 3rd group of HPs+PRRS immune swine counteracting toxic substances 100% are protected as a result, and matched group 80% is fallen ill.Haemophilus parasuis Serotype 5 ZJ strain lumbar injection bacterium liquid 2ml(is containing viable count 5.0 × 10
9cFU), the 3rd group of HPs+PRRS immune swine counteracting toxic substances 100% protected, the 1st group of HPs immune swine protection 80%, and contrast pig 100% falls ill.
The immune piglet 4 type JS strains of table 16 and 5 type ZJ strain protest test results
Note: Haemophilus parasuis morbidity standard: morbidity pig is dead or occur heating (body temperature more than 40.5 ℃, continues 1～5), lethargy, cough, dyspnea, becomes thin, walks lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open to inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum).
Counteracting toxic substances when immunity piglet is large at 8 weeks, 4 type JS strains and the protection of 5 type ZJ strain counteracting toxic substances the results are shown in Table 17: the strong malicious NVDC-JXA1 strain culture fluid 3ml(10 of intramuscular injection porcine reproductive and respiratory syndrome virus
4.5tCID
50), the 7th group of HPs immune swine counteracting toxic substances 100% protected, and the 6th group of PRRS immune swine counteracting toxic substances 80% protected, and matched group 100% is fallen ill, and has 4 death.
The immune piglet NVDC-JXA1 of table 17 strain protest test result
Note: Porcine reproductive and respiratory syndrome morbidity standard: body temperature at least 3rd is more than 41 ℃, or spirit, appetite declines, eye conjunctivitis, the respiratory symptom such as cough, breathe heavily, or substantially cut open inspection, there is lamellar consolidation in pulmonary.
The immune piglet NVDC-JXA1 of table 18 strain counteracting toxic substances clinical symptoms result
According to table 16, table 17, table 18 result of the test, discovery Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine is combined use as compositions, between antigen, there will not be phase mutual interference, surprisingly, after associating use, the clinical symptoms can alleviate the attack of Porcine reproductive and respiratory syndrome strong virus time.In addition, find out from above-mentioned result of the test: (every part is containing JXA1-R strain virus amount>=10 for 1 part of Porcine reproductive and respiratory syndrome live vaccine-Haemophilus parasuis inactivated vaccine (HPs+PRRS) compositions intramuscular injection prepared by this laboratory
4.8tCID
50, containing the front 4 type JS strains of deactivation with 5 type ZJ strain is each is all no less than 2 × 10
9cFU/ head part) (antigenic content is front 4 types of deactivation and 5 types are each is all no less than 4 × 10 with 1 part of commercial Haemophilus parasuis inactivated vaccine (HPS) intramuscular injection vaccine
9cFU/ head part) compare, the haemophilus parasuis killed vaccine antigen content immune effect also can obtain commercialization haemophilus parasuis inactivated vaccine and use separately time that reduces by half.
1, vaccine is according to embodiment 1 use tidal type microcarrier suspension bioreactor large-scale production porcine reproductive and respiratory syndrome virus, and prepares Porcine reproductive and respiratory syndrome live vaccine with four kinds of heat-resisting lyophilized protecting agent formulas of embodiment 1; According to preparation method and the step of embodiment 2 Haemophilus parasuis inactivated vaccines (JS strain+ZJ strain), by the qualified haemophilus parasuis serum 4 type JS strains of deactivation, ultrafiltration and safety check, 5 type ZJ strain antigens, obtain according to different antigenic contents and MontanideGEL ST adjuvant mixed preparing, wherein above-mentioned 2 kinds of antigen ratios are 1:1, and vaccine finished product antigenic content is for containing the front serum 4 type JS strains of deactivation, 5 type ZJ strain viable counts in table 19.
The different antigenic content vaccines of table 19
2, efficacy test
15 of the each porcine reproductive and respiratory syndrome virus with 3～4 week age of every group of vaccine and haemophilus parasuis antigen, negative antibody health susceptible pigs, every intramuscular injection 2ml, after 28 days, the immunity test pig of having injected every kind of vaccine is divided into three groups at random, uses respectively the 4 type JS strains of haemophilus parasuis serum, 5 type ZJ strains, the strong malicious NVDC-JXA1 strain of porcine reproductive and respiratory syndrome virus counteracting toxic substances separately.15 of contrast pigs, divide 3 groups, use respectively above-mentioned haemophilus parasuis serum 4 type JS strains, 5 type ZJ strains, the strong malicious NVDC-JXA1 strain counteracting toxic substances of porcine reproductive and respiratory syndrome virus.
Haemophilus parasuis serum 4 type JS strains: choose each 5 of immune swine, together with 5 of the identical contrast pigs of condition, lumbar injection 7.0 × 10
9the serum 4 type JS strain bacterium liquid 2ml of CFU, observe 14 days, and immune swine is answered more than 4/5 protection, and contrast pig answers more than 4/5 morbidity.
The strong malicious NVDC-JXA1 strain of porcine reproductive and respiratory syndrome virus: choose 5 of immune swines, together with 5 of the identical contrast pigs of condition, the strong malicious culture fluid 10 of intramuscular injection NVDC-JXA1 strain
4.5tCID
50/ 3ml, observes 21 days, and immune swine is answered more than 4/5 protection, and contrast pig 5/5 all should fall ill, and at least 2 death.
Assay is shown in table 20, respectively organizes vaccine and all can reach more than 4/5 protection, and immune effect is good.
Table 20 vaccine potency assay
Note: Haemophilus parasuis morbidity standard: morbidity pig is dead or occur heating (body temperature more than 40.5 ℃, continues 1～5), lethargy, cough, dyspnea, becomes thin, walks lamely and by the thick clinical symptoms such as disorderly of hair.Dying pig is cutd open to inspection, the pathological changes such as visible polyserositis (pleuritis, pericarditis, peritonitis), arthritis and meningitis, there is serosity or fibrinous exudate in each serosal surface (joint capsule, pericardium, pleura and peritoneum);
Porcine reproductive and respiratory syndrome morbidity standard: body temperature at least 3rd is more than 41 ℃, or spirit, appetite declines, eye conjunctivitis, the respiratory symptom such as cough, breathe heavily, or substantially cut open inspection, there is lamellar consolidation in pulmonary.
Can find out according to result of the test, comprising homologous series adjuvant with preferred aqueous adjuvant Montanide GEL ST() Haemophilus parasuis inactivated vaccine and the Porcine reproductive and respiratory syndrome live vaccine made for adjuvant combine use as compositions, not only injection is convenient, safety, and Haemophilus parasuis and Porcine reproductive and respiratory syndrome and the mixed infection thereof that can prevent haemophilus parasuis and Porcine reproductive and respiratory syndrome to cause, there is good preventive effect.
According to those skilled in the art's understanding, select other antigen of technical solution of the present invention also can realize the object of the invention, therefore embodiment does not form the restriction to inventing itself.The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (11)
1. the antigen composition of the respiratory system disease of the secondary infection of prevention and treatment pig, wherein, haemophilus parasuis antigen and the adjuvant of the Porcine reproductive and respiratory syndrome antigen that described antigen composition comprises at least one immunity amount and at least one immunity amount, described Porcine reproductive and respiratory syndrome antigen and haemophilus parasuis antigen comprise the antigen of holoantigen, subunit antigen, live recombinant vectors antigen and the DNA vector of attenuated live holoantigen, deactivation independently of one another.
2. antigen composition according to claim 1, wherein, described Porcine reproductive and respiratory syndrome antigen is NVDC-JXA1-R attenuation whole virus vaccine, described haemophilus parasuis antigen is the deactivation whole-bacterial-vaccine of serotype 4 type JS strains and Serotype 5 ZJ strain, described haemophilus parasuis serum 4 type JS strain preserving numbers are CCTCC No.M2011172, and described haemophilus parasuis Serotype 5 ZJ strain preserving number is CCTCC No.M2011173.
3. antigen composition according to claim 1 and 2, wherein, described Porcine reproductive and respiratory syndrome totivirus antigen is every part viral level 10
4.5tCID
50～10
7.0tCID
50; More preferably, described totivirus antigen is every part viral level 10
4.8tCID
50～10
6.5tCID
50; Again preferably, described totivirus antigen is every part viral level 10
5.0tCID
50.
4. antigen composition according to claim 1 and 2, wherein, described serum 4 type haemophilus parasuis totivirus antigenic contents are deactivation front 5 × 10
8cFU/ head part～8 × 10
9cFU/ head part; More preferably, described haemophilus parasuis totivirus antigenic content is before deactivation to be 4 × 10
9cFU/ head part, more preferred described serum 4 type haemophilus parasuis totivirus antigenic contents are deactivation front 2 × 10
9cFU/ head part;
Described Serotype 5 haemophilus parasuis totivirus antigenic content is deactivation front 5 × 10
8cFU/ head part～8 × 10
9cFU/ head part; More preferably, described haemophilus parasuis totivirus antigenic content is deactivation front 4 × 10
9cFU/ head part, more preferred described Serotype 5 haemophilus parasuis totivirus antigenic content is deactivation front 2 × 10
9cFU/ head part.
5. according to the antigen composition described in claim 1-2 any one, wherein, described adjuvant comprises nanorize aluminium hydroxide gel, mineral oil, carbomer, MONTANIDE GEL ST, propolis, cytokine, liposome, immunostimulating complex, containing CpC deoxy-oligonucleotide, nanorize ISA206, ISA760VG, nanorize aluminum phosphate, saponin, sorbitan, vegetable oil, ethyl oleate, two (caprylic/capric) propylene glycol ester, three (caprylic/capric) glyceride, two oleic acid propylene glycol esters, isostearate, sorbitan, mannide, glycerol, polyglycereol, propylene glycol, oleic acid, isostearic acid, castor oil acid, hydroxy stearic acid ester, polyoxypropylene, polyoxyethylene block copolymer, acrylic or methacrylic acid polymer, maleic anhydride and thiazolinyl derivant copolymer, the one or more combination thing of the polymer of the crosslinked acrylic or methacrylic acid of the polyalkenyl ether of sugar or polyhydric alcohol, preferably, described adjuvant is aqueous adjuvant, and described aqueous adjuvant comprises carbomer aqueous solution, 206 adjuvants, nanorize aluminium hydroxide gel adjuvant, MontanideIMS1313 VG, Montanide ISA15A VG, Montanide GEL 01 PR and Montanide GEL ST polymer adjuvant.
6. antigen composition according to claim 2; wherein; described NVDC-JXA1-R attenuation whole virus vaccine also comprises heat-resisting lyophilized protecting agent; described heat-resisting lyophilized protecting agent comprises 1%～2% gelatin, 5%～10% trehalose, 1%～2% polyvinylpyrrolidone (PVP), 0.5%～2% sodium glutamate, 0.1%～2% bovine serum albumin, 0.1%～1% sorbitol, 0.1%～0.2% sodium carboxymethyl cellulose, 0.164% potassium dihydrogen phosphate and 0.052% sodium hydrogen phosphate, and all the other are water.
7. the method for the antigen composition of the respiratory system disease of the secondary infection of preparation prevention and treatment one boar, described method comprises the step of preparing Porcine reproductive and respiratory syndrome live vaccine and the step of preparing haemophilus parasuis inactivated vaccine:
The described step of preparing Porcine reproductive and respiratory syndrome live vaccine comprises the described porcine reproductive and respiratory syndrome virus of propagation, is mixed with heat-resisting lyophilized protecting agent, and lyophilizing obtains Porcine reproductive and respiratory syndrome live vaccine;
The described step of preparing haemophilus parasuis inactivated vaccine comprises the described haemophilus parasuis of propagation, and deactivation, with adjuvant mixed preparing.
8. preparation method according to claim 7, wherein, the described haemophilus parasuis of described propagation uses MHPs haemophilus parasuis culture medium, described MHPs haemophilus parasuis culture medium comprises 5～10g/L polyprotein peptone, 3～10g/L yeast extract, 2～8g/L sodium glutamate, 1～5g/L lactoalbumin hydrolysate, 1～6g/L sodium chloride, 1～5g/L dipotassium hydrogen phosphate, 0.1～4g/L glucose, pH value is 6.8～8.0, 116～121 ℃ of sterilizing 10～40min, add again 5～100ml/L porcine blood serum, 1～50ml/L NADH, while preparing solid medium, before sterilizing, add 1～20g/L agar powder.
9. apply in the medicament for treating respiratory system thing of the secondary infection of preparation prevention and treatment pig according to the antigen composition described in claim 1-2 any one.
10. application according to claim 9, restores described Porcine reproductive and respiratory syndrome live vaccine with described haemophilus parasuis inactivated vaccine before described application is included in and applies.
11. 1 kinds of test kits, described test kit comprises the antigen product of the opposing Porcine reproductive and respiratory syndrome separating on the packaging or antigen product or immunogenic composition or the vaccine of immunogenic composition or vaccine and opposing haemophilus parasuis.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104055736A (en) * | 2014-07-08 | 2014-09-24 | 安徽医科大学 | Nano aluminum-encapsulating carrier and application thereof |
| CN106215186A (en) * | 2016-08-02 | 2016-12-14 | 甘肃省畜牧兽医研究所 | Sore mouth virus live-vaccine heat-proof protective agent and lyophilized powder thereof and the preparation method of this lyophilized powder |
| CN106215186B (en) * | 2016-08-02 | 2019-10-22 | 甘肃省畜牧兽医研究所 | The preparation method of sore mouth virus live-vaccine heat-proof protective agent and its freeze-dried powder and the freeze-dried powder |
| CN106497791A (en) * | 2016-10-31 | 2017-03-15 | 中国食品发酵工业研究院 | A kind of staphylococcus aureuses lyophilizing is prepared and uses NEW TYPE OF COMPOSITE protective agent |
| CN106497791B (en) * | 2016-10-31 | 2020-01-14 | 中国食品发酵工业研究院有限公司 | Novel composite protective agent for freeze-drying preparation of staphylococcus aureus |
| WO2023113094A1 (en) * | 2021-12-16 | 2023-06-22 | 주식회사 씨티씨백 | Covid-19 vaccine composition with increased immunogenicity |
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