CN104248759B - Vaccine composition, preparation method and application thereof - Google Patents
Vaccine composition, preparation method and application thereof Download PDFInfo
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- CN104248759B CN104248759B CN201310589600.1A CN201310589600A CN104248759B CN 104248759 B CN104248759 B CN 104248759B CN 201310589600 A CN201310589600 A CN 201310589600A CN 104248759 B CN104248759 B CN 104248759B
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Abstract
The invention provides a vaccine composition, which includes an immune amount of a mycoplasma hyopneumoniae antigen, an immune amount of a swine influenza virus antigen and an immune amount of a porcine reproductive and respiratory syndrome virus antigen. All antigens of the vaccine composition not only do not generate mutual interference or influence of antigen components, but have the effect of mutually enhancing the immune effect instead. One immunization can achieve the immune effect and the antibody continuous level of single antigen twice immunization. Also, the vaccine composition has the advantages of good security, simple preparation method, convenient and fast immunization, and reduction of the immunization cost, etc.
Description
Technical field
The present invention relates to a kind of vaccine combination, and the preparation method and application of the vaccine combination.
Background technology
Porcine reproductive and respiratory syndrome（Porcine Reproductive and Respiratory Syndrome,
PRRS）, it is by porcine reproductive and respiratory syndrome virus also known as pig blue-ear disease（porcine reproductive and
respiratory syndrome virus,PRRSV）What is caused is generated heat with sow, is miscarried, the piglet mortality rate liter before and after wean
Height, all ages and classes pig respiratory disorder etc. for Clinical symptoms disease.Porcine reproductive and respiratory syndrome virus are malicious also known as pig blue-ear disease,
It is a kind of single strand plus RNA virus for having a cyst membrane, virus has 2 serotypes, i.e. american type and Europe class.At present, the disease is tight
Ghost image rings one of important diseases of world's pig industry development.Vaccination is to prevent the sick most efficient method.
Swine flue（Swine influenza, SI）It is a kind of important respiratory system disease for currently endangering pig industry, it is main
The features such as hyperpyrexia, cough and dyspnea are shown as, is Large-scale pig farm generally existing and the mass-sending epidemic disease for being difficult to eradicate
One of disease.Swine flue is mainly by A type swine influenza viruses（Swine influenza virus,SIV）Cause, according to text both domestic and external
Report is offered, the influenza A blood serum subtype in pig body has H1N1, H1N2, H1N7, H3N2, H3N6, H4N6, H5N1 and H9N2,
Domestic swine influenza viruses blood serum subtype mainly based on H1N1, H1N2, H3N2, H5N1 and H9N2, and wherein with H1N1 with
Based on H3N2 hypotypes.Because swine influenza viruses have the close preferendum of high special to airway epithelial, airway epithelial tube wall is easily made
It is impaired, cause Actinobacillus pleuropneumoniae, pasteurellosis bacilluss, haemophilus parasuises, streptococcus, Porcine reproductive and respiratory syndrome disease
Poison, the secondary infection of pig circular ring virus or mixed infection, increase the complexity of epidemic situation, and to pig industry greatly economic damage is caused
Lose.
Mycoplasmal pneumonia（Mycoplasma pneumonia of swine, MPS）It is by mycoplasma hyopneumoniae
A kind of chronic pneumonia that (Mycoplasma hyopneumoniae, Mhp) causes, is also pig also known as epidemic swine pneumonia
One of important pathogen of prdc.For a long time, primary disease is considered as always to cause great economy to pig industry
Lose, occur most frequently, prevalence is most wide, be most difficult to one of important epidemic disease of purification.Mycoplasma hyopneumoniae is conditionality pathogenic bacterium, is occurred
This reduces can Abwehrkraft des Koepers, easily secondary infection pig blue-ear disease poison, swine influenza viruses etc. after being ill, aggravate disease and disease
Dead rate is raised.
Since the mid-90 in 20th century, by porcine reproductive and respiratory syndrome virus, mycoplasma hyopneumoniae, swine flue disease
The PRDC that the main primary pathogen such as poison causes causes serious loss to countries in the world pig industry.In clinical PRDC, on
Three kinds of cause of diseases i.e. porcine reproductive and respiratory syndrome virus, mycoplasma hyopneumoniae, swine influenza viruses are stated usually in mixed infection form
Occur.And the infection currently for these three cause of diseases there is no method to realize that a kind of vaccine immunity resists the mixing that these three cause of diseases cause
Infection.
For the immunity of many cause of disease mixed infection associated diseases, using multiple injection is needed during the immunity of single Seedling, due to antibacterial
The use of medicine can usually cause drug resistance and medicament residue problem, and single Seedling multiple injection not only can cause stress ask with safety
Topic, and in the case of mixed infection, immunoprophylaxises effect Quality Down.Therefore, this area needs can produce protectiveness and exempt from
Epidemic disease response reduces the sense with the combined vaccine of opposing mycoplasma hyopneumoniae, pig blue-ear disease poison and swine influenza virus infection simultaneously
The incidence rate of dye and/or the seriousness of the mitigation infection, or prevent the clinical manifestation relevant with the infection.
Compared with single vaccine, combined vaccine not only facilitates, multiple-effect, low cost, can also reduce vaccination number of times, keeps away
Exempt from that because of leakage kind Full-access immunization can not be obtained；In addition, vaccine is mostly thermo-labile, it produces, transports, storing, selling or even be whole
It is both needed to carry out at a lower temperature using process, i.e., so-called " cold chain ", this cold chain running all linked with one another, expense is high,
Make vaccine cost remain high, and use combined vaccine, then can substantially reduce the expense of cold chain running, therefore with significant
Superiority.
The content of the invention
To solve the deficiencies in the prior art, present invention is primarily targeted at a kind of vaccine combination is provided, the vaccine
Compositionss include the pig blue-ear disease poison of the mycoplasma hyopneumoniae antigen of immunity amount, the swine flue antigen of immunity amount and immunity amount
Antigen.
Preferably, the mycoplasma hyopneumoniae antigen is the full pathogen antigen of mycoplasma hyopneumoniae, the shape of work of deactivated form
The mycoplasma hyopneumoniae antigen of formula, mycoplasma hyopneumoniae subunit antigen, mycoplasma hyopneumoniae live recombinant vectorses antigen and Pulmonis Sus domestica
The antigen of scorching mycoplasma DNA vector；The swine flue antigen is the swine influenza viruses totiviruss antigen of deactivated form, work
The swine flue antigen of form, swine influenza viruses subunit antigen, swine influenza viruses live recombinant vectorses antigen and swine flue disease
The antigen of malicious DNA vector；The pig blue-ear disease poison antigen is pig blue-ear disease poison totiviruss antigen, the form of work of deactivated form
Pig blue-ear disease poison antigen, pig blue-ear disease poison subunit antigen, pig blue-ear disease poison live recombinant vectorses antigen and pig blue-ear disease poison DNA are carried
The antigen of body.
Term used herein " vaccine " or " vaccine combination " are used interchangeably, and refer to comprising at least one in animal
The pharmaceutical composition of the immunogenic substance of middle induction immunne response.
Term " mycoplasma hyopneumoniae antigen " is referred in the present invention can induce after being administered to pig, stimulate comprising at least one
Or any combinations thing of the antigen of the immunne response of the anti-mycoplasma hyopneumoniae infection of enhancing.Preferably, the mycoplasma hyopneumoniae
Antigen is the full cause of disease of mycoplasma hyopneumoniae, the full cause of disease of mycoplasma hyopneumoniae of preferred deactivated form, the mycoplasma hyopneumoniae of improvement
The mycoplasma hyopneumoniae cause of disease of cause of disease living or attenuated forms thereof；Immunogen amino acid sequence containing at least mycoplasma hyopneumoniae
Embedded viruses；The polypeptide of any other immunogen amino acid sequence containing at least mycoplasma hyopneumoniae, subunit or other into
Point.
Mycoplasma hyopneumoniae antigen can also include any one or several antigens in following vaccine combination：Such as Bo Linge
Abundant lattice writing brush animal health（The U.S.）The biological big pharmaceutical factory of the mycoplasmal pneumonia of swine inactivated vaccine of company limited's production, Spain Hai Bolai
Happiness J strain mycoplasmal pneumonia of swine inactivated vaccines, the mycoplasmal pneumonia of swine inactivated vaccine of Schering Plough company of the U.S. that can relax（Myco
Silencer）, the MycoGard vaccines of Portec Inc. of the U.S., the MH P-5722-3 of the RespiFend of Pfizer Inc.
The 168 of BQ14 strain vaccines that strain vaccine, the pig gram of Cimmeria animal health company breathe heavily, the mycoplasma hyopneumoniae of Nanjing day nation production
Vaccine prepared by the detached mycoplasma hyopneumoniae HN0613 strains of strain vaccine and Pulaike Biological Engineering Co., Ltd..
In the present invention term " swine flue antigen " refer to comprising at least one can induce after being administered to pig, stimulate or
Strengthen any combinations thing of the antigen of the immunne response of anti-swine flu virus infection.Preferably, the swine flue antigen is
Swine flue totiviruss antigen, including the street strain being clinically separated well known to those skilled in the art, the preferably pig of deactivated form
The swine influenza viruses of influenza all-virus, the swine flue live viruses of improvement or attenuated forms thereof；Immunity containing at least swine influenza viruses
The embedded viruses of immunogenic amino acid sequence；Any at least polypeptide of the immunogen amino acid sequence containing swine influenza viruses, Asia
Unit or other compositions.
Term in the present invention " pig blue-ear disease poison antigen " is referred to can be induced comprising at least one after being administered to pig, stimulate or
Strengthen any combinations thing of the antigen of the immunne response of anti-pig blue-ear disease poison infection.Preferably, the pig blue-ear disease poison antigen is
Pig indigo plant ear totiviruss antigen, including the street strain being clinically separated well known to those skilled in the art, the preferably pig of deactivated form
Blue ear totiviruss, the pig indigo plant ear live viruses of improvement or the pig blue-ear disease poison of attenuated forms thereof；Immunity containing at least pig blue-ear disease poison
The embedded viruses of immunogenic amino acid sequence；The polypeptide of any other immunogen amino acid sequence containing at least pig blue-ear disease poison,
Subunit or other compositions.
It is highly preferred that in vaccine combination of the present invention, the mycoplasma hyopneumoniae antigen is propped up for the hyopneumoniae of deactivated form
Substance J strains or the full pathogen antigen of HN0613 strains；The swine flue antigen is the swine influenza viruses of deactivated form（H1N1 is sub-
Type）ZJS strains or swine influenza viruses（H3N2 hypotypes）One plant or two plants of totiviruss antigen in WX strains；The pig blue-ear disease poison is anti-
Originally be form living pig blue-ear disease poison R98 strains, ATCCVR-2332 strains or CH-1R strains in one plant or several plants totiviruss resist
It is former.
Mycoplasma hyopneumoniae（Mycoplasma hyopneumoniae,Mhp）J strains, are preserved in American Type Culture Collecti
ATCC 25934。
Mycoplasma hyopneumoniae HN0613（Mycoplasma hyopneumoniae strain HN0613）, in Chinese allusion quotation
Type culture collection（Referred to as：CCTCC；Address：Wuhan, China Wuhan University）Carry out preservation, preservation date：2012 6
The moon 13, preserving number is CCTCC NO:M2012230.
Swine influenza viruses（H1N1 hypotypes）ZJS strains（Swine influenza virus（H1N1 subtype）strain
ZJS）And swine influenza viruses（H3N2 hypotypes）WX strains（Swine influenza virus（H3N2subtype）strain WX）,
In China typical culture collection center（Referred to as：CCTCC；Address：Wuhan, China Wuhan University）Carry out preservation, preservation day
Phase：On 08 13rd, 2012, preserving number was respectively CCTCC NO:V201233 and CCTCC NO:V201234.
Pig blue-ear disease poison R98 strains, be disclosed in the isolation identification of porcine reproductive and respiratory syndrome virus R98 low virulent strains with
ORFs3～7 genetic characteristics research（Chinese veterinary's journal, in January, 2008, volume 28, the 1st phase）
Pig blue-ear disease poison ATCCVR-2332 strains, are preserved in American Type Culture Collecti, and preserving number is ATCCVR-2332.
Pig blue-ear disease poison CH-1R strains, the strain is disclosed in Chinese patent application CN100523177C, with preserving number CGMCC
No.1883 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
It is further preferred that in vaccine combination of the present invention, the mycoplasma hyopneumoniae antigen is the Pulmonis Sus domestica of deactivated form
The full pathogen antigen of scorching mycoplasma HN0613 strain；The swine flue antigen is the swine influenza viruses of deactivated form（H1N1 hypotypes）
ZJS strains and swine influenza viruses（H3N2 hypotypes）WX strain totiviruss antigens；The pig blue-ear disease poison antigen is that the pig of form living is blue
Otopathy poison CH-1R strain totiviruss antigens.
Preferably, the vaccine combination inactivated vaccine part further includes acceptable adjuvant in veterinary pharmacy, described
Adjuvant is water adjuvant；The vaccine combination live vaccine part also includes freeze drying protectant.
It is highly preferred that the adjuvant is the adjuvants of GEL 01.
Term used herein " water adjuvant ", also known as " water-based adjuvant " or " water-soluble adjuvant ", is a kind of polymeric water-soluble
Dispersion, for improving effect and the safety of water-soluble vaccines, can be by high molecular weight polypropylene acids synthetic polymer group
Into.
Preferably, in vaccine combination of the present invention, content is 10 before the mycoplasma hyopneumoniae antigens inactive7.0～2 ×
1010.0MHDCE/ head parts；Content is 10 before the swine flue antigen inactivation4.0～107.0EID50/ head part；The pig indigo plant ear
Virus antigen content is 104.0～106.0TCID50/ head part.
It is highly preferred that content is 10 before the mycoplasma hyopneumoniae antigens inactive8.0～1010.0MHDCE/ head parts；The pig
Content is 10 before influenza antigen inactivation5.0～107.0EID50/ head part；The pig blue-ear disease poison antigenic content is 105.0~
106.0TCID50/ head part.
Most preferably, content is 10 before the mycoplasma hyopneumoniae antigens inactive9.0MHDCE/ head parts；The swine flue disease
Content is 10 before malicious antigens inactive6.0EID50/ head part；The pig blue-ear disease poison antigenic content is 106.0TCID50/ head part.
Preferably, also including the pathogen antigen of other pigs, it is selected from PRV (Pseudorabies virus) to the vaccine combination（PRV）
One or more in antigen, CSFV antigen, Actinobacillus pleuropneumoniae antigen and pig circular ring virus antigen.
Another object of the present invention is to a kind of preparation method of vaccine combination of the present invention is provided, including it is as follows
Step：
（1）Breeding culture mycoplasma hyopneumoniae, inactivation；
（2）Breeding culture swine influenza viruses, inactivation；
（3）Breeding culture pig blue-ear disease poison；
（4）The inactivation mycoplasma hyopneumoniae antigen and swine flue antigen are mixed in proportion, and it is blue with the pig
Ear live viruses antigen separates preservation.
Concrete grammar is as follows：Respectively breeding culture mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison obtain bacterium solution；
Mycoplasma hyopneumoniae bacterium solution and swine influenza viruses liquid are inactivated respectively, then qualified mycoplasma hyopneumoniae antigen is checked in inactivation
Concentration is carried out respectively with swine flue antigen；By the mycoplasma hyopneumoniae antigen, swine flue antigen and vaccine
Adjuvant is mixed to prepare the bivalent vaccine composition of anti-mycoplasma hyopneumoniae, swine influenza viruses（That is inactivated vaccine part）；Pig is blue
Ear virus antigen is mixed with the vaccine combination of anti-pig blue-ear disease poison with freeze drying protectant（That is live vaccine part）.Can when using
The bivalent vaccine composition of anti-mycoplasma hyopneumoniae, swine influenza viruses is diluted into the vaccine combination of anti-pig blue-ear disease poison, is obtained
The triple vaccine compositionss of anti-mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison, also can be by anti-mycoplasma hyopneumoniae, pig
The vaccine combination of influenza virus is individually used with the vaccine combination of anti-pig blue-ear disease poison.
Preferably, the mycoplasma hyopneumoniae antigen and swine flue antigen are mixed to prepare into content with the adjuvants of Gel 01
For 12%（V/V）Inactivated vaccine part；By pig blue-ear disease poison antigen with freeze drying protectant with 1:1（V/V）Ratio be mixed to prepare
Live vaccine part；Then the live vaccine part is diluted in proportion with the inactivated vaccine part, be preferably in a proportion of 1:1（Head part/
Head part）, make mycoplasmal pneumonia of swine, swine flue and pig blue-ear disease triple vaccine.
Term " head part " in the present invention refers to the amount of vaccine of every pig injection.
A further object of the present invention is that the offer vaccine combination is comprehensive in preparation prevention and treatment porcine respiratory disease
Application in the medicine of simulator sickness, wherein, the application includes mycoplasma hyopneumoniae antigen, the swine flue of immunity amount with immunity amount
The pig blue-ear disease poison antigen immune pig of virus antigen and immunity amount, is 3～4 week old during the pig immunity.
Another object of the present invention is to provide a kind of immune reagent kit, the immune reagent kit is included containing the hyopneumoniae
Antigen composition and the pig blue-ear disease poison antigen of mycoplasma antigen and swine flue antigen, wherein, containing the Pulmonis Sus domestica
The antigen composition of scorching mycoplasma antigen and swine flue antigen separates preservation with pig blue-ear disease poison antigen.
Preferably, mycoplasma hyopneumoniae antigen, swine flue antigen described in above-mentioned immune reagent kit are deactivated form
Full pathogen antigen, pig blue-ear disease poison antigen is the totiviruss antigen of form living.
Technique effect
First, mycoplasmal pneumonia of swine of the invention, swine flue and pig blue-ear disease triple vaccine, in certain antigenic content scope
Inside not only will not produce producing protective immunity after mutual immune interference or the impact of antigen composition, and three kinds of antigen mixing
While response, it has surprisingly been found that the antigen of mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison has mutually strengthen immunity
The effect of effect.As subsequent embodiment of the present invention is proved, triple vaccine immunity once can reach each single Seedling secondary immunity
Antibody continue level, the phenomenon exceed those of ordinary skill in the art expectation.
Secondly, the mycoplasmal pneumonia of swine of the present invention, swine flue and pig blue-ear disease triple vaccine and mycoplasmal pneumonia of swine, pig stream
Sense is compared with single vaccine of pig blue-ear disease, and during to pig injecting immune, the stress of pig body is unexpectedly little, therefore
The triple vaccine safety of the present invention more preferably, can avoid the untoward reaction of multiple immunoprophylaxis appearance.
Additionally, the present invention solves the difficulty of prior art, first by mycoplasma hyopneumoniae, swine influenza viruses and pig indigo plant ear
Virus antigen is used in combination according to suitable ratio, it is believed that mycoplasmal pneumonia of swine, swine flue and pig indigo plant ear before changing
Sick these three diseases are difficult to while carrying out preventing further obtaining the understanding prejudice of triple vaccine.
Finally, mycoplasmal pneumonia of swine of the invention, swine flue and pig blue-ear disease triple vaccine contain three kinds of antigens, Ke Yitong
When prevent three kinds of diseases, the combined vaccine that there is the spies such as preparation method simple, convenience, multiple-effect, low cost, vaccine valence content height
Point；Meanwhile, compared with single vaccine, combined vaccine can reduce the inoculation times of vaccine, it is to avoid can not obtain complete because of leakage kind
Journey immunity, and immune cost is reduced, save immune programme for children；Further, since vaccine is mostly thermo-labile, its production, transport, storage
Or even be all both needed to using process carry out at a lower temperature, i.e., so-called " cold chain ", this cold chain running all linked with one another takes
With high, make vaccine cost remain high, and use combined vaccine of the present invention, then can substantially reduce cold chain running
Expense, therefore with significant superiority.
Specific embodiment
The present invention is further described with reference to specific embodiment.These embodiments are merely illustrative of, and are not construed as
Any restriction to overall range of the present invention.Those skilled in the art are without departing from the spirit and scope of the invention to the present invention
Any modification and replacement that the details and form of technical scheme is carried out, each fall within protection scope of the present invention.
Mycoplasma hyopneumoniae HN0613 strains, swine influenza viruses are used in the embodiment of the present invention（H1N1 hypotypes）ZJS strains and pig are flowed
Influenza Virus（H3N2 hypotypes）The present invention is illustrated as a example by WX strains, pig blue-ear disease poison CH-1R strains.
Pneumonopathy varying index standards of grading in the embodiment of the present invention：Evaluated according to 7 lobe of the lung lesion degrees, maximum score
For 28 points.When the lobe of the lung area of specific Damage is 0,0 point is designated as, 1%～25% is designated as 1 point, 26%～50% is designated as 2 points, 51%～
75% is designated as 3 points, is 4 points more than 75%.
Embodiment of the present invention statistical analysis technique is：The pneumonopathy varying index of 7 lobes of the lung of statistics, determines lesion degree.With
SPSS computer softwares carry out ANOVA analyses, and each group difference of comparison determines the effectiveness of pathological changes difference.
Vaccine combination, triple vaccine in the present embodiment refers both to prevent or treat mycoplasma hyopneumoniae, swine influenza viruses
The vaccine combination infected with pig blue-ear disease poison, i.e., it is blue including at least mycoplasma hyopneumoniae antigen, swine flue antigen and pig
The vaccine combination of ear virus antigen.
The preparation of the mycoplasma hyopneumoniae antigen of embodiment 1, swine flue antigen and pig blue-ear disease poison antigen
1st, prepared by mycoplasma hyopneumoniae antigen
1.1 bacterium source：
Manufacture and the mycoplasma hyopneumoniae bacterial strain for checking this product are HN0613 strains, in China typical culture collection center
（Referred to as：CCTCC；Address：Wuhan, China Wuhan University）Carry out preservation, preservation date：On June 13rd, 2012, deposit number：
CCTCC NO:M2012230。
The preparation of 1.2 mycoplasma hyopneumoniae production seeds：
The breeding of first order seed：Freeze-drying lactobacillus（HN0613 strains, deposit number is CCTCC No.M2012230）, use liquid
Culture medium dilutes, and streak inoculation puts 37 DEG C of culture 7d on solid medium plate, and the good bacterium colony of growth selection is inoculated in
In culture medium slant, 37 DEG C of culture 7d, as first order seed.
The breeding of secondary seed：The slant culture that a small amount of fluid medium washes first order seed is taken, liquid culture is inoculated in
In the big pipe of base, 37 DEG C of culture 7d are put, Jing after inspection purely as secondary seed.
The formula of fluid medium（Based on 1065m1）：Cor Bovis seu Bubali leachate 300ml, ddH2O360ml, correction pH value is arrived
7.4,121 DEG C sterilizing 15 minutes.Add the composition of following filtration sterilization:Hank ' s balanced salt solutions (10 ×) 40ml, 0.25%
Phenol red 10ml porcine blood serums 200ml, 5% lactoalbumin hydrolysate 100m1,25% yeast leachate 20ml, 10000IU/ml penicillin 10ml,
1% thaliium acetate solution 25m1.
The formula of solid medium：15g Noble Agar are added in liquid medium within.
The preparation of 1.3 seedling bacterium solutions：
By the mycoplasma hyopneumoniae secondary seed solution of fluid medium culture with 1:10（v/v）It is inoculated in fluid medium
In.Cultivate 3～6 at 37 DEG C, culture declines more than 0.5 pH value, purely after the assay was approved, then expands in the same way
Culture（Subculture time was less than for 6 generations）.After culture terminates, sampling is purely checked, should be pure.
1.4 mycoplasma hyopneumoniae thalline assays：
Culture is counted with PCR method.10 times are serially diluted examined culture, are then PCR, PCR detections
Minimum limitation is 3 × 10-3μg（Thalline equivalent to 1000 or so）, by the count multiples thalline number of dilution（Shen Qingchun, Tan Qing
Pine, Wang Qin, wait .PCR methods determine Mhp culture bacterium numbers [J], Chinese Preventive Veterinary Medicine report, 2006,28（1）：55～57）.Training
Foster thing content should be 1～2 × 109MHDCE/ml.（MHDCE=mycoplasma hyopneumoniae DNA cell equivalents, i.e. 1MHDCE equivalent to
1 mycoplasma hyopneumoniae）.
The inactivation of 1.5 mycoplasma hyopneumoniae bacterium solutions and concentration：
Qualified mycoplasma hyopneumoniae bacterium solution will be checked, by bacterium solution volume total amount final concentration of 0.2% formaldehyde is slowly added to
Solution（V/V）, 37 DEG C of inactivations are put, therebetween every stirring in 4 hours once, take out after 24 hours, carry out inactivation inspection.By inactivation inspection
Test qualified mycoplasma hyopneumoniae bacterium solution to be concentrated using ultrafiltration concentration technology, antigenic content is 10 after concentration11MHDCE/ml。
2nd, prepared by pig blue-ear disease poison antigen
Bacterium source：Pig blue-ear disease poison CH-1R strains, with preserving number CGMCC No.1883 Chinese microorganism strain is preserved in
Preservation administration committee common micro-organisms center.
The dispersion of Marc145 or MA104 cell line Jing EDTA- pancreatin Digestive system is passed on, continues to cultivate with cell growth medium,
It is standby when forming fine and close monolayer, for continuing to pass on or virus inoculation.By pig blue-ear disease poison CH-1R strain virus liquid, by M.O.I.
=0.001～0.01 dosage accesses the cell bottle for having grown into good cell monolayer, and further preferably connecing toxic agent amount is
After M.O.I.=0.001～0.005, absorption 1 hour, cell maintenance medium is added in cell line monolayer, continue to cultivate, when 70%～
Being harvested after pathological changes occurs in 80% cell, and rearmounted less than -20 DEG C of the venom freeze thawing of results 1～2 time is preserved, and is taken and do on a small quantity semi-finished product inspection
Test, antigen liquid viral level 108.0TCID50/ ml, according to national standard inspection regulation is met.
3rd, prepared by swine flue antigen
3.1 bacterium source：
Swine influenza viruses（H1N1 hypotypes）ZJS strains and（H3N2 hypotypes）WX strains, in China typical culture collection center
（Referred to as：CCTCC；Address：Wuhan, China Wuhan University）Preservation is carried out, deposit number is respectively CCTCC NO:V201233 and
CCTCC NO:V201234。
The preparation of 3.2 swine influenza viruses liquid：
By swine influenza viruses（H1N1 hypotypes）ZJS strains（Hereinafter referred to as " SIV H1N1 "）And swine influenza viruses（H3N2 hypotypes）
WX strains（“SIV H3N2”）Seed culture of viruses is respectively by viral infection multiplicity（multiplicity of infection,M.O.I.）For
0.001 inoculum concentration is inoculated in respectively the MDCK for covering with monolayer（ATCC CCL-34TM）On cell, 37 DEG C adsorb 30 minutes, plus
Enter the D-MEM fluid mediums of the calf serum containing 3% volume ratio（With the D- purchased from Life Technologies companies of the U.S.
MEM dehydrated mediums are prepared according to its description）, put 37 DEG C and continue to cultivate, daily observation 2 times, cell growth is good, and 72h is received
Cell culture, freeze thawing 3 times, results virus are obtained, and SIV H1N1 virus liquid is diluted to into virus titer using PBS and be
108.0EID50/ ml, equally, virus titer is diluted to for 10 using PBS by SIV H3N2 virus liquids8.0EID50/ml。
The process of 3.3 swine influenza viruses liquid, inactivation：
By SIV H1N1 and SIV H3N2 virus liquid doughnuts（0.5 μm～2 μm）Filter post（Purchased from GE
Healthcare Life Sciences）Filter, remove cell debriss, add 0.2%～0.3% 37 DEG C of inactivations of formalin
18h。
The preparation of the mycoplasmal pneumonia of swine of embodiment 2, swine flue and pig blue-ear disease triple vaccine
1st, mycoplasmal pneumonia of swine, the preparation of swine flue inactivated vaccine part
The preparation of 1.1 diluent
Aseptic PBS buffer solution：8g Sodium Chloride, 0.25g potassium chloride, 3.63g phosphoric acid hydrogen are dissolved in 900ml purified water
Disodium, 0.24g potassium dihydrogen phosphates, are then settled to 1L, and 121 DEG C of autoclaving 30min are standby.
The process of 1.2 vaccine adjuvants
The adjuvants of Gel 01 sterilize：The adjuvants of Gel 01 are proceeded to can be in sterilization container, and 121 DEG C of autoclaving 30min are standby.
1.3 match somebody with somebody Seedling
By sterile working, by the concentration mycoplasma hyopneumoniae antigen prepared in embodiment 1 and swine flue antigen with
After adjuvant, preservative, diluent etc. are according to different ratio mixing, with mulser stirring at low speed 30min.Inactivated vaccine part Pulmonis Sus domestica
Scorching mycoplasma antigen content is 107.0～2 × 1010.0MHDCE/ head parts, swine flue antigen content is 104.0~
107.0EID50The content of/head part, wherein mycoplasma hyopneumoniae antigen and swine flue antigen in the inactivated vaccine part is
12%（V/V）.
2nd, the preparation of pig blue-ear disease live vaccine part
Qualified pig blue-ear disease will be checked respectively to receive time virus liquid, same container is mixed in, with freeze drying protectant（2wt% gelatin
Aqueous solution is with 15wt% lactose aqueous solutions with 1:1 (v/v) proportions）With 1:Fully mix after the mixing of 1 (v/v) ratio, quantitative point
Dress, carries out rapidly lyophilisation, and finished product content is 104～106TCID50/ head part.
3rd, the preparation of triple vaccine
Each antigen prepared in embodiment 1 is carried out into vaccine formulation according to the proportioning of each group vaccine in table 1.Using front taking
Inactivated vaccine part of dilution live vaccine part.
The each group vaccine proportioning of table 1
The different antigenic content mycoplasmal pneumonia of swine of embodiment 3, swine flue and pig blue-ear disease triple vaccine potency test
1 test material
The vaccine 1 prepared in embodiment 2（Mycoplasma hyopneumoniae 107MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）With
Swine influenza viruses（H3N2 hypotypes）Each 104EID50/ head part, pig blue-ear disease poison 104TCID50/ head part）, vaccine 2（Hyopneumoniae original
Body 108MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）And swine influenza viruses（H3N2 hypotypes）Each 105EID50/ head part, pig are blue
Otopathy poison 105TCID50/ head part）, vaccine 3（Mycoplasma hyopneumoniae 109MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）And pig
Influenza virus（H3N2 hypotypes）Each 106EID50/ head part, pig blue-ear disease poison 106TCID50/ head part）, vaccine 4（Mycoplasma hyopneumoniae
1010MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）And swine influenza viruses（H3N2 hypotypes）Each 107EID50/ head part, pig indigo plant ear
Virus 106TCID50/ head part）With vaccine 5（Mycoplasma hyopneumoniae 2 × 1010MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）With
Swine influenza viruses（H3N2 hypotypes）Each 107EID50/ head part, pig blue-ear disease poison 106TCID50/ head part）.
3～4 week old, the ablactational baby pig without mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison antibody.
2 test methods
2.1 safety test
3～4 week old, the ablactational baby pig 30 without mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison antibody are selected, with
Machine is divided into 6 groups, wherein 5 groups of difference musculi collis inject two part（4ml）Vaccine 1, vaccine 2, vaccine 3, vaccine 4 and vaccine 5, remain
Remaining to observe two weeks as control, vaccinated pig should be normal without obvious Temperature changing, spiritual appetite, anti-without other visible clinicals
Should occur.2.2 potency test
3～4 week old, the ablactational baby pig 120 without mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison antibody are selected,
It is randomly divided into 24 groups, the 1st, 2,3,4 groups of immune 1 part vaccines 1, the 5th, 6,7,8 groups of immune 1 part vaccines 2, the 9th, 10,11,12
The immune 1 part vaccine 4 of group the 3, the 13rd, 14,15,16 groups of part vaccine of immunity 1, the 17th, 18,19,20 groups of immune 1 part vaccines 5.
21st, 22,23,24 groups are control counteracting toxic substances group.35 days after immunity, the 1st, 5,9,13,17,21 groups are used mycoplasma hyopneumoniae virulent strain
（CVCC354 strains, purchased from Chinese veterinary microorganism culture presevation administrative center）Carry out trachea injection, 5ml/ heads（2×
109MHDCE/ml）, cut open inspection statistics pneumonopathy becomes after 30 days, judges protection situation；2nd, 6,10,14,18,22 groups of injection swine flue
Virus（H1N1 hypotypes）（ZJS strains, 106.0EID50/0.1mL）, the 3rd, 7,11,15,19,23 groups of injection swine influenza viruses（H3N2 is sub-
Type）（WX strains, 106.0EID50/0.1mL）, per strain by collunarium and each 1mL of intratracheal injection counteracting toxic substances, Continuous Observation 25 after counteracting toxic substances
Day, slaughter after weighing within 25th after counteracting toxic substances, cut open inspection.Vaccine is evaluated to Pigs Inoculated according to the scoring of the pulmonary lesion of test pig
Protected effect；4th, 8,12,16,20,24 groups malicious by force with pig blue-ear disease poison HuN4-F5（3×104.0TCID50, it is disclosed in Chinese special
Sharp CN101280292A）Attack.Continuous Observation 21 days, carries out cut open inspection, record morbidity number（Including death toll）, judge protection feelings
Condition.
3 result of the tests
3.1 safety test results
1, vaccine 2, vaccine 3, vaccine 4 and vaccine 5 are vaccinated to piglet doubling dosage, after injection observe body temperature, appetite,
The mental status, whether there is visible clinical reaction and death condition, and vaccine injection local whether there is the inflammatory reactions such as swelling, Continuous Observation 2
In week, cut open after three weeks and kill, carry out Pathologic Observation, observation the results are shown in Table 2.
Result is observed in the triple vaccine safety test of table 2
Safety test result shows：Vaccinate 4 and the immune group of vaccine 5 have indivedual temperature of pig body to continue to reach 40.5 in 2 days
DEG C, there is 1 pig injection site swelling occur wherein vaccinating 5 immune group, it may be possible to due to antigen in vaccine 4 and vaccine 5
Content is high compared with other vaccines, and antigenic content height causes stress to become apparent from.
3.2 efficacy test results
The efficacy test results of the triple vaccine of different antigenic contents are shown in Table 3.
The efficacy test results of the triple vaccine of the different antigenic contents of table 3
Efficacy test results show：It is 60% to the protective rate vaccine 1 of mycoplasma hyopneumoniae, vaccine 2 is 80%, vaccine 3, epidemic disease
Seedling 4 and vaccine 5 are 100%；It is 40～60% to the protective rate vaccine 1 of swine influenza viruses, vaccine 2 is 80%, vaccine 3 is 100%,
And vaccine 4 is 80～100%, vaccine 5 is 80%；It is 60% to the protective rate vaccine 1 of pig blue-ear disease poison, vaccine 2 is 80%, vaccine 3
For 100%, vaccine 4 is 80%, and vaccine 5 only 60%.As a result show, mycoplasmal pneumonia of swine, swine flue and the epidemic disease of pig blue-ear disease three
Seedling can produce preferable protective effect in the range of certain antigen to corresponding virus, and antigenic content is higher, the protection of generation
Effect is stronger；But when the content of mycoplasma hyopneumoniae antigen reaches 1010During MHDCE/ head parts, the guarantor to pig blue-ear disease poison is found
Shield rate drops to 80%, when its antigenic content reaches 2 × 1010During MHDCE/ head parts, there was only 60% to the protective rate of pig blue-ear disease poison,
And 80% is also dropped to the protective rate of swine influenza viruses.Illustrate, when the too high levels of mycoplasma hyopneumoniae antigen, to flow pig
The immunoprotection of Influenza Virus and pig blue-ear disease poison has certain impact.
The mycoplasmal pneumonia of swine of embodiment 4, swine flue and pig blue-ear disease triple vaccine primary immune response and each single Seedling secondary immunity
Immune effect comparative test
1st, test material
The vaccine 3 prepared in embodiment 2（Mycoplasma hyopneumoniae 109MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）With
Swine influenza viruses（H3N2 hypotypes）Each 106EID50/ head part, pig blue-ear disease poison 106TCID50/ head part）, vaccine 6（Hyopneumoniae original
Body 109MHDCE/ head parts）, vaccine 7（Swine influenza viruses（H1N1 hypotypes）And swine influenza viruses（H3N2 hypotypes）Each 106EID50/ head
Part）, vaccine 8（Pig blue-ear disease poison 106TCID50/ head part）.
3～4 week old, the ablactational baby pig without mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison antibody.
2nd, test method
3～4 week old ablactational baby pig 60 is chosen, is divided into 12 groups, 5 per group, the 1st, 2,3,4 groups of immune 1 part vaccines 3,
5th group of the 6, the 6th, 7 groups of part vaccine 1 part vaccine 7 of immunity of immunity 1, the 8th group of the 8, the 9th～12 group of part vaccine of immunity 1 is counteracting toxic substances
Matched group；After the 14d of interval, the 5th, 6,7,8 groups of booster immunizations 1 time.Head exempts from 35 days afterwards, and the 1st, 5,9 groups strong with mycoplasma hyopneumoniae
Strain（CVCC354 strains）Carry out trachea injection, 5ml/ heads（2×109MHDCE/ml）, cut open inspection statistics pneumonopathy becomes after 30 days, judges
Protection situation；2nd, 6,10 groups are used swine influenza viruses（H1N1 hypotypes）（ZJS strains, 106.0EID50/0.1mL）By collunarium and trachea
Interior to inject each 1mL of counteracting toxic substances, Continuous Observation 25 days after counteracting toxic substances are slaughtered, cut open inspection after weighing within 25th after counteracting toxic substances；3rd, 7,11 groups of use
Swine influenza viruses（H3N2 hypotypes）（WX strains, 106.0EID50/0.1mL）By collunarium and each 1mL of intratracheal injection counteracting toxic substances, after counteracting toxic substances
Continuous Observation 25 days, slaughters, cut open inspection after weighing within 25th after counteracting toxic substances；4th, 8,12 groups malicious by force with pig blue-ear disease poison HuN4-F5
（3×104.0TCID50）Attack.Continuous Observation 21 days, carries out cut open inspection, record morbidity number（Including death toll）, judge protection situation.
Vaccine immunity and counteracting toxic substances packet situation are shown in Table 4 in immune effect comparative test.
The vaccine immunity of table 4 and counteracting toxic substances packet situation
3rd, result of the test
3.1 immune effect comparative test results
Immune effect comparative test the results are shown in Table 5, table 6 and table 7.
The mycoplasma hyopneumoniae counteracting toxic substances of table 5 protect result
Note：Compare between group, alphabetical identical person represents that difference is not notable, alphabetical difference person represents significant difference（P ＜ 0.05）
The swine influenza viruses counteracting toxic substances of table 6 protect result
Note：Compare between counteracting toxic substances bacterial strain identical group, alphabetical identical person represents that difference is not notable, letter is different
Person represents significant difference（P ＜ 0.05）
The pig blue-ear disease of table 7 poison counteracting toxic substances protection result
Note：Morbidity criterion piglet body temperature is raised（≥40.5℃）, at least continuing 3～4, loss of appetite occurs bright
Aobvious respiratory symptom；Pathological change, cut open inspection piglet lung tissue is presented bronzing graniphyric, does not subside；Lymph node moderate is arrived
Severe enlargement, the obvious enlargement of spleen head of indivedual pigs, tangent plane red pulp hypertrophy；Microscopic pathology changes alveolar septum inhomogeneous broadening,
It can be seen that enlargement, capillary endothelium hypertrophy, and have macrophage and lymphocyte infiltration；With IFA methods detect lungs, spleen and
Lymph node, should detect substantial amounts of PRRSV antigens.
Immune effect comparative test result shows：Mycoplasmal pneumonia of swine, swine flue and pig blue-ear disease triple vaccine are once exempted from
Epidemic disease is to the protection of mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison and the level of protection phase of the secondary immunity of each single Seedling
When.Illustrating the triple vaccine of mycoplasmal pneumonia of swine, swine flue and pig blue-ear disease fine can must resist mycoplasma hyopneumoniae, swine flue
The infection of virus and pig blue-ear disease poison.
The mycoplasmal pneumonia of swine of embodiment 5, swine flue and pig blue-ear disease triple vaccine and single Seedling immune duration comparative test
1st, test material
The vaccine 3 prepared in embodiment 2（Mycoplasma hyopneumoniae 109MHDCE/ head parts, swine influenza viruses（H1N1 hypotypes）With
Swine influenza viruses（H3N2 hypotypes）Each 106EID50/ head part, pig blue-ear disease poison 106TCID50/ head part）, vaccine 6（Hyopneumoniae original
Body 109MHDCE/ head parts）, vaccine 7（Swine influenza viruses（H1N1 hypotypes）And swine influenza viruses（H3N2 hypotypes）Each 106EID50/ head
Part）, vaccine 8（Pig blue-ear disease poison 106TCID50/ head part）.
3～4 week old, the ablactational baby pig without mycoplasma hyopneumoniae, swine influenza viruses and pig blue-ear disease poison antibody.
2nd, test method
3～4 week old ablactational baby pig 25 is chosen, 5 groups are randomly divided into, the 1st group of immune 1 part vaccine 3, the 2nd group of immunity 1
Part vaccine 6, the 3rd group of immune 1 part vaccine 7, the 4th group of immune 1 part vaccine 8, the 5th group is blank control group, vaccine immunity and
TPPA packet situation is shown in Table 8.Each group separates serum respectively at 3 week old, 5 week old, 3 monthly ages, the blood sampling of 6 monthly ages.It is respectively adopted
Competitive ELISA detects the serum antibody of anti-mycoplasma hyopneumoniae, and using indirect ELISA swine influenza viruses antibody horizontal is detected,
Using immuno-fluorescence assay pig blue-ear disease poison antibody horizontal.
The vaccine immunity of table 8 and TPPA packet situation
3rd, result of the test
Respectively TPPA, mycoplasma hyopneumoniae are carried out to the serum of the week old of each group 3,5 week old, 3 monthly ages, the pig at 6 monthly ages
Antibody is shown in Table 9 in different immunization period antibody horizontal measurement results, and swine influenza viruses antibody is in different immunization period antibody horizontals
Measurement result is shown in Table 10, and pig blue-ear disease poison antibody is shown in Table 11 in different immunization period antibody horizontal measurement results.
The immune different times mycoplasma hyopneumoniae antibody horizontal measurement result of table 9
The immune different times swine influenza viruses antibody horizontal measurement result of table 10
The immune different times pig blue-ear disease poison antibody horizontal measurement result of table 11
Antibody horizontal measurement result shows：Before matched group pig and the immunity of immune group pig（3 week old）Antibody test is feminine gender,
Head exempts from latter 14 days（5 week old）The detection of each immune group pig corresponding antibodies is the positive.The mycoplasma hyopneumoniae of the immune swine of vaccine 3,
Swine influenza viruses are in identical variation tendency with the antibody horizontal of each single Seedling with pig blue-ear disease poison antibody horizontal, and antibody horizontal is not
Gradually rise with immunization period.Surprisingly antibody horizontal of the antibody horizontal of the immune swine of vaccine 3 than each single Seedling immune swine
Higher level is risen in advance, and is kept longer compared with the time of High antibody level.Illustrate mycoplasma hyopneumoniae, swine flue and pig
Reproductive and respiratory syndrome triple vaccine only needs primary immune response, can just meet or exceed the immune effect of each single Seedling secondary immunity.Present invention system
Standby triple vaccine, will not only produce mutual immune interference or the impact of antigen composition in the range of certain antigenic content, and
And after three kinds of antigen mixing, while protective immune response is produced, it has surprisingly been found that mycoplasma hyopneumoniae, swine influenza viruses
Play the role of mutually to strengthen immune effect with the antigen of pig blue-ear disease poison.
The above is only the preferred embodiments of the present invention, and any pro forma restriction is not done to the present invention, is appointed
What those skilled in the art, in the range of without departing from technical solution of the present invention, when the technology using the disclosure above
Content is made a little change or is modified to the Equivalent embodiments of equivalent variations, as long as without departing from technical solution of the present invention
Hold, according to any simple modification, equivalent variations and modification that the technical spirit of the present invention is made to above example, all should include
Within protection scope of the present invention.
Claims (6)
1. a kind of vaccine combination, the vaccine combination includes the pig stream of the mycoplasma hyopneumoniae antigen of immunity amount, immunity amount
The pig blue-ear disease poison antigen of Influenza Virus antigen and immunity amount, wherein, the mycoplasma hyopneumoniae antigen is the pig of deactivated form
The full pathogen antigen of mycoplasma pneumoniae HN0613 strains；The swine flue antigen is the serum H1N1 hypotype ZJS strains of deactivated form
With serum H3N2 hypotype WX strain totiviruss antigens；The pig blue-ear disease poison antigen is the pig blue-ear disease poison CH-1R strains of form living
Totiviruss antigen,
Wherein, content is 10 before the mycoplasma hyopneumoniae antigens inactive9.0MHDCE/ head parts；The swine flue antigen is gone out
Content is 10 before living6.0EID50/ head part；The pig blue-ear disease poison antigenic content is 106.0TCID50/ head part,
Wherein, the vaccine combination inactivated vaccine part further includes adjuvant and diluent, and the adjuvant is water adjuvant, institute
It is aseptic PBS buffer solution to state diluent；The vaccine combination live vaccine part also includes freeze drying protectant.
2. a kind of method for preparing vaccine combination according to claim 1, methods described comprises the steps：
(1) breeding culture mycoplasma hyopneumoniae, inactivation；
(2) breeding culture swine influenza viruses, inactivation；
(3) breeding culture pig blue-ear disease poison；
(4) by the inactivation mycoplasma hyopneumoniae antigen and swine flue antigen mixing, and resist with pig indigo plant ear live viruses
Original separates preservation.
3. vaccine combination according to claim 1 is in the medicine for preparing prevention and treatment porcine respiratory disease complex
Application.
4. application according to claim 3, wherein, it is 3～4 week old during the pig immunity.
5. a kind of immune reagent kit, the immune reagent kit includes vaccine combination according to claim 1, the vaccine group
Compound includes antigen composition and the pig blue-ear disease poison containing the mycoplasma hyopneumoniae antigen and swine flue antigen
Antigen, wherein, the antigen composition containing the mycoplasma hyopneumoniae antigen and swine flue antigen and the pig blue-ear disease are malicious
Antigen separates preservation.
6. immune reagent kit according to claim 5, wherein, the mycoplasma hyopneumoniae antigen, swine flue antigen
For the full pathogen antigen of deactivated form, the pig blue-ear disease poison antigen is the totiviruss antigen of form living.
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| CN1652817A (en) * | 2002-05-17 | 2005-08-10 | 惠氏公司 | Mycoplasma hyopneumoniae bacterin vaccine |
| CN101420975A (en) * | 2006-04-10 | 2009-04-29 | 英特威国际有限公司 | Vaccine against mycoplasma PRRSV |
| CN102373181A (en) * | 2010-08-09 | 2012-03-14 | 中山大学 | Swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle, and its preparation method and use |
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| KR20100094587A (en) * | 2007-12-21 | 2010-08-26 | 와이어쓰 엘엘씨 | Methods and compositions for immunizing pigs against porcine circovirus |
| CN102988978B (en) * | 2011-08-01 | 2016-02-03 | 普莱柯生物工程股份有限公司 | Vaccine combination containing porcine circovirus 2 type antigen and haemophilus parasuis antigen and preparation method thereof and application |
| UA114503C2 (en) * | 2012-04-04 | 2017-06-26 | Зоетіс Сервісіз Ллс | PCV AND MYCOPLASMA HYOPNEUMONIA COMBINED VACCINE |
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| CN1652817A (en) * | 2002-05-17 | 2005-08-10 | 惠氏公司 | Mycoplasma hyopneumoniae bacterin vaccine |
| CN101420975A (en) * | 2006-04-10 | 2009-04-29 | 英特威国际有限公司 | Vaccine against mycoplasma PRRSV |
| CN102373181A (en) * | 2010-08-09 | 2012-03-14 | 中山大学 | Swine influenza virus-porcine reproductive and respiratory syndrome virus mixed virus-like particle, and its preparation method and use |
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