CN103893750B - A kind of resisting pstudorabies, swine flue vaccine combination and application thereof - Google Patents

A kind of resisting pstudorabies, swine flue vaccine combination and application thereof Download PDF

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CN103893750B
CN103893750B CN201210590377.8A CN201210590377A CN103893750B CN 103893750 B CN103893750 B CN 103893750B CN 201210590377 A CN201210590377 A CN 201210590377A CN 103893750 B CN103893750 B CN 103893750B
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vaccine combination
vaccine
antigen
piglet
hypotype
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CN103893750A (en
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张许科
孙进忠
白朝勇
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Pulaike Biological Engineering Co Ltd
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Abstract

The present invention relates to a kind of vaccine combination of anti-PRV and SIV disease, said composition, except for except preventing pseudorabies and swine flue, also can prevent the clinical manifestation of the PRDC caused by other pathogen infection.Present invention also offers a kind of described vaccine combination in the application prevented and/or control in immune piglet PRDC disease medicament.Not only there is not interference between antigen in use procedure in said composition, and has unexpected preventive effect for the sick syndrome of porcine respiratory.Therefore, use this vaccine combination, can immune programme for children be simplified, reach the effect that two diseases prevented by a pin.After resisting pstudorabies, swine flue vaccine combination immunity piglet, effectively can prevent that PRDC's is popular.

Description

A kind of resisting pstudorabies, swine flue vaccine combination and application thereof
Technical field
The invention belongs to herding class biological pharmacy technical field, relate to the obtained vaccine combination of pseudorabies live virus antigen and swine influenza virus inactivation antigen and application thereof.
Background technology
Porcine pseudorabies (Pseudorabies, PR) be the infectious disease that many animals is suffered from altogether, cause the infectious disease of domestic animal and wild animal acute fatal, with heating, very itch and encephalomyelitis for cardinal symptom, pig is its natural susceptible host, and mainly cause in-pig miscarriage, stillborn fetus and mummy tire, Infection in Piglets is nervous symptoms and acute death mainly, Adult Pig is then based on respiratory tract infection, and this disease has become the Etiological of kind of pig breeding dysfunction syndrome and piglet mortality.
Swine flue (Swineinfluenza, SI) is a kind of important respiratory system disease of current harm pig industry, and main manifestations is the features such as high heat, cough and dyspnea, is Large-scale pig farm ubiquity and is difficult to one of mass-sending epidemic disease eradicated.According to bibliographical information both domestic and external, influenza A blood serum subtype in pig body has H1N1, H1N2, H1N7, H3N2, H3N6, H4N6, H5N1 and H9N2, domestic swine influenza virus blood serum subtype mainly based on H1N1, H1N2, H3N2, H5N1 and H9N2, and wherein based on H1N1 and H3N2 hypotype.
The sick syndrome (Porcinerespiratorydiseasecomplex, PRDC) of porcine respiratory is that disease the most serious is endangered on current pig farm.The reason of PRDC is caused to be many-sided, normally caused by the many factors such as cause of disease (virus, antibacterial, mycoplasma, parasite), environment and feeding and management interact, and be mostly caused by two or more cause of disease mixed infections.PRV (Pseudorabies virus) and swine influenza virus are as main PRDC primary pathogen, except infecting separately, can also mixed infection and other pathogen infection of secondary, exacerbate the order of severity of epidemic disease, the mortality rate of affected animal is caused to increase, huge to the harm of pig industry.Immunity inoculation is the important measures of the anti-system of porcine pseudorabies and swine flue, but sell without porcine pseudorabies, swine flue bigeminy vaccine or vaccine combination on the market at present, its main cause is: PRV (Pseudorabies virus) can cause immunosuppressant, during with other vaccine conbined usage, the immune effect of vaccine can be caused to reduce; In addition, current swine flue vaccine is inactivated vaccine, and pseudorabies is live-virus vaccine, inactivator (as formaldehyde, beta-propiolactone) in inactivated vaccine and vaccine adjuvant (as mineral oil adjuvant) will have deactivation or interference effect to live virus, by reducing the immune effect of vaccine, therefore, pseudorabies, swine flue are prepared into bigeminy vaccine or vaccine combination, need to solve the interference problem between inactivator, adjuvant, antigen, to reach certain immune effect.
Chinese patent CN1624143A discloses the recombinant pseudorabies virus and application of expressing Hemagglutinin Geneof Swine Influenza Virus; this recombinant virus only have expressed H3 hypotype swine influenza virus hemagglutinin (Hemagglutinin; HA) gene; effective protection can not be provided to other hypotype (the H1 hypotype swine flue as popular at present); because swine influenza virus different subtype hemagglutinin differs greatly; thus the cross protection between different subtype is poor, and can to animal dis in potential risks with gene engineering expression.
Therefore, current Problems existing needs research and development a kind of except for except preventing pseudorabies and swine flue, also can prevent the anti-pseudorabies of the clinical manifestation of the PRDC caused by other pathogen infection, the vaccine combination of swine flue.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, the vaccine combination of a kind of prevention or treatment PRV and SIV disease is provided, said composition, except for except preventing pseudorabies and swine flue, also can prevent the clinical manifestation of the PRDC caused by other pathogen infection.Present invention also offers a kind of described vaccine combination in the application prevented and/or control in immune piglet PRDC disease medicament.Not only there is not interference between antigen in use procedure in said composition, and has unexpected preventive effect for the sick syndrome of pig porcine respiratory.Therefore, use this vaccine combination, can immune programme for children be simplified, reach the effect that two diseases prevented by a pin.
For this reason, the invention provides the vaccine combination that a kind of anti-PRV and SIV infects, it comprises at least one PRV antigen and at least one SIV antigen.
According to the present invention, described PRV antigen comprises a kind of or several arbitrarily combination in pseudorabies live virus, improvement/chimeric pseudorabies live virus, the recombinant virus at least containing the immunogen amino acid sequence of PRV (Pseudorabies virus), PRV (Pseudorabies virus) gene delection virus or weak viral disease poison.
Deletion of vaccine totally 3 kinds of live vaccine that PRV live vaccine is divided into natural gene deletion of vaccine, manually Attenuation vaccine and the specific gene relevant with virulence of use technique for gene engineering removal or genetic fragment and obtains.At present, the PRV live vaccine strain that the whole world generally uses has Bartha strain, Bartha-K61 strain, Bucharest strain, BUK strain, SA215 strain and Ea strain.PRV only has a serotype, although different strain there are differences in virulence and biological property etc., its immunogenicity is similar, because PRV live vaccine strain disappearance is virulence gene, has immunogenic albumen and all exists.Therefore, use the live vaccine of different PRV strain, all can obtain good immune effect.
The present invention's " immunity amount " used refers to the amount of vaccine of the immunity provided for porcine pseudorabies, swine flue vaccine combination, depends primarily on following factor: the vaccine whether accepting to resist same virus before the species of immunized animal, kind, age, weight size, health status and animal.
In the present invention, described PRV antigen is totivirus live vaccine.In vaccine combination of the present invention, described PRV antigenic content is>=5.0 × 10 5.0tCID 50/ head part.Preferably described PRV antigenic content is 1.0 × 10 6.0tCID 50/ head part.
In an embodiment of the invention, described PRV antigen comprises PRV (Pseudorabies virus) gene delection SA215 strain (CCTCCNO.V200002), Ea strain (CGMCC, deposit number N0.0907), WKQ-001 strain (CCTCC, preserving number V200511), the sick and weak malicious Bartha strain of pseudorabies, Bartha-K61 strain, Bucharest strain, BUK strain, H strain (GCMCC, preserving number is No.5013) and C strain (CCTCC, deposit number V201114) in a kind of or several arbitrarily combinations.
In the present invention, SIV antigen refers to any compositions comprising at least one antigen, and described antigen can induce, stimulates or strengthen the immunne response of anti-swine flu viral infection after to pig administration.Preferably, described SIV antigen is complete swine flue totivirus, comprise the street strain of clinical separation well known to those skilled in the art, those skilled in the art understand, minor alteration in influenza antigens HA gene order, limited on its immunization impact, the open country poison of the occurring in nature be namely separated by other approach also can realize function of the present invention.
According to the present invention, described SIV antigen comprises a kind of or several arbitrarily combination in complete swine flue totivirus, the embedded virus containing at least immunogen amino acid sequence of SIV, other SIV fragment any.
In one embodiment of the invention, described complete swine flue totivirus comprises the SIV virus of the swine flue totivirus of deactivated form, the SIV live virus of improvement or attenuation.Preferred described complete swine flue totivirus comprises at least one of H1N1 hypotype totivirus antigen or H3N2 hypotype totivirus antigen.
As is known to the person skilled in the art: swine flue inactivated vaccine is based on H1N1 and H3N2 hypotype; between different virus hypotype, cross protection is poor; but between identical hypotype there is cross protection in strain; because the main protective albumen of influenza virus is HA albumen; the HA albumen homology of identical subtype virus is high, has similar epitope.Therefore, the immune effect of subunit vaccine prepared by HA albumen, can reach the immune effect of inactivated virus vaccine to a certain extent.
According to the present invention, in described vaccine combination, before described H1N1 hypotype totivirus antigens inactive>=10 7.0tCID 50/ head part.Be 10 before preferred described H1N1 hypotype totivirus antigens inactive 7.5tCID 50/ head part.In described vaccine combination, before described H3N2 hypotype totivirus antigens inactive>=10 7.0tCID 50/ head part.Be 10 before preferred described H3N2 hypotype totivirus antigens inactive 7.5tCID 50/ head part.
Term " head part " in the present invention refers to the amount of vaccine of every pig injection.
" TCID described in the present invention 50" (50%tissuecultureinfectivedose) refer to half cell culture infective amount, is a kind of representation representing virus infectivity." HA-HI test " described in the present invention refers to that SIV totivirus antigen or HA subunit antigen make the erythrocyte of 0.5% that the maximum dilution multiple of 100% coagulation occur, and is a kind of method for expressing with coagulation antigenic content.
Term used herein " vaccine combination of anti-PRV and SIV disease ", refers to the vaccine combination of prevention and therapy PRV and SIV disease.
In one embodiment of the invention, described H1N1 hypotype totivirus antigen is swine influenza virus (H1N1 hypotype) ZJS strain (Swineinfluenzavirus (H1N1subtype) strainZJS), its deposit number is: CCTCCNO.V201233, is preserved in China typical culture collection center and (is called for short: CCTCC; Address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University) carry out preservation, preservation date: on August 13rd, 2012.
In another embodiment of the present invention, described H3N2 hypotype totivirus antigen is swine influenza virus (H3N2 hypotype) WX strain (Swineinfluenzavirus (H3N2subtype) strainWX), its deposit number is: CCTCCNO.V201234, is preserved in China typical culture collection center and (is called for short: CCTCC; Address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University) carry out preservation, preservation date: on August 13rd, 2012.
According to the present invention, other SIV fragment described comprises the polypeptide of the immunogen amino acid sequence containing SIV, subunit or its variant.Preferably other SIV fragment described is the subunit containing SIVHA gene.Preferred further, other SIV fragment described is contain the subunit by the gene constructed SIVHA albumen of SIVHA.
According to the present invention, described SIVHA albumen comprises the HA albumen of H1N1 hypotype or the HA albumen of H3N2 hypotype.HA-HI test >=the 11Log2 of described H1N1 subtype HA protein.HA-HI test >=the 11Log2 of described H3N2 subtype HA protein.The aminoacid sequence of the HA albumen of described H1N1 hypotype is as shown in SEQNO.3 in sequence table.The aminoacid sequence of the HA albumen of described H3N2 hypotype is as shown in SEQNO.4 in sequence table.
Present invention also offers a kind of according to vaccine combination of the present invention in prevention and/or the application that controls in immune piglet PRDC disease medicament.
In a preferred embodiment of the present invention, described vaccine combination also comprises freeze drying protectant.
According to the present invention, described freeze drying protectant includes but not limited to one or more in lactose, skim milk, gelatin, trehalose, bovine serum albumin, polyvinylpyrrolidone, sorbitol, sodium glutamate and vitamin C.
Preferably, freeze drying protectant of the present invention is heat-resisting lyophilized protecting agent, mainly include but not limited to containing in gelatin, bovine serum albumin, trehalose, polyvinylpyrrolidone, sorbitol, sodium glutamate and vitamin C one or more.
In another preferred embodiment of the present invention, described vaccine combination can also comprise veterinarily acceptable carrier or excipient, and can select to comprise veterinarily acceptable adjuvant.
Vaccine adjuvant can be divided into oil adjuvant and aqueous adjuvants usually, and oil adjuvant generally refers to that in vaccine, oil content, between 10% ~ 60%, is generally mineral oil.Aqueous adjuvants generally refers to that in vaccine, oil content is lower than 10%, is generally vegetable oil or degradable oil.Oil adjuvant stress be large, produce antibody slow, and aqueous adjuvants stress be little, produces antibody fast.
Preferably, vaccine adjuvant of the present invention can comprise aluminium hydroxide, aluminum phosphate, saponin as QuilA and QS-21, light mineral oil as Marcol52, produced by olefin oligomerisation isoprenoid oil as aqueous vaccine adjuvants such as squalane or Squalene, carbomer, MontanideTMGel.More preferably, vaccine adjuvant described in the present invention is aqueous vaccine adjuvant, i.e. carbomer, the aqueous solution prepared by a certain percentage.
In addition, the present invention still further provides a kind of preparation method of the vaccine combination for prevention and therapy PRV and SIV infection, comprises the steps:
(1) PRV live virus antigen is prepared.
(2) SIV inactivation antigen is prepared.
(3) PRV live virus antigen is added vaccine freeze-drying protective agent vacuum freeze-drying; separately by SIV inactivation antigen and aqueous vaccine adjuvant (aqueous suspensions that carbomer, saponin, levamisole, cholesterol are prepared by a certain percentage) by a certain amount of mixing; during vaccine immunity; by SIV inactivation antigen dilution PR freeze-dried antigen, porcine pseudorabies, swine flue vaccine combination.
Wherein, in step (1), preparation PRV live virus Antigen Method is not limited to use chick embryo fibroblast primitive cell culture, passage cell also can be adopted to cultivate, as ST cell and PK-15 cell.Concrete grammar comprises: be inoculated in by PRV seed culture of viruses on ST cell, and preferred seed culture of viruses inoculum concentration is viral infection plural number (M.O.I.) is 0.001 ~ 0.1, and preferred cultivation temperature is 33 ~ 37 DEG C, preferred incubation time is 2-5 days, harvesting culture, multigelation 3 times, results virus.
Wherein, in step (2), preparation SIV antigen comprises preparation preparation SIV totivirus antigen and subunit antigen, and its method is not limited to use Embryo Gallus domesticus, mdck cell system, Vero cell line, bhk cell system, CEF cell line and use escherichia expression system, baculovirus expression system, yeast expression system etc.
In step (2), SIV antigen preparation procedure comprises: SIVH1N1 hypotype seed culture of viruses and H3N2 hypotype seed culture of viruses are inoculated in respectively and cover with in the mdck cell of monolayer, the inoculum concentration M.O.I. of preferred seed culture of viruses is 0.001 ~ 0.1, preferred cultivation temperature is 33 ~ 37 DEG C, preferred culture medium contains the DMEM culture medium of 2% ~ 10% calf serum, preferred incubation time is 24 ~ 96h, harvesting culture, freeze thawing 3 times, results virus.
Freeze drying protectant described in step (3), preferred heat-resisting lyophilized protecting agent, mainly includes but not limited to containing gelatin, bovine serum albumin, trehalose, polyvinylpyrrolidone, sorbitol, sodium glutamate and vitamin C.Immunological adjuvant is preferably Squalene, carbomer, MontanideTMGel etc., is more preferably aqueous vaccine adjuvant, i.e. the aqueous suspensions prepared by a certain percentage of carbomer, saponin, levamisole, cholesterol.
Visible above, the present invention has following advantage at least:
1) vaccine combination of prevention and therapy PRV and SIV infection of the present invention, comprises at least one PRV antigen and at least one SIV antigen.This vaccine combination not only can not produce mutual immune interference or the impact of two kinds of antigen compositions, and after the mixing of PRV and SIV antigen, the antigen composition of PRV and SIV has the effect mutually strengthening immune effect, and this exceeds the expectation of field of veterinary those of ordinary skill.
2) vaccine combination of prevention and therapy PRV with SIV infection is compared with the effect of the mono-vaccine of PRV or SIV, to in pig injecting immune process, the stress of pig body is little, therefore, vaccine combination safety of the present invention is better, can avoid repeatedly the untoward reaction that immunoprophylaxis occurs.
3) in the vaccine combination that infects of above-mentioned prevention and therapy PRV and SIV, swine flue antigen can be not only the totivirus of deactivation, can also be the subunit protein (HA albumen is the major antigen of virus) of deactivation, the composition of vaccine combination can be the swine flue antigen of PRV live virus antigen and deactivation (totivirus or subunit) and forms.
4) by using heat-resisting lyophilized protecting agent, vaccine combination effectively can be preserved at 2 ~ 8 DEG C, and more conventional freeze drying protectant, reduces cost, facilitates transport.Overcome the freeze dried vaccine used at present and be generally-15 DEG C of preservations, need cold chain preservation and transport, cost intensive and easily cause vaccine effect to decline; In addition, therefore, develop heat-resisting pseudorabies, swine flue vaccine combination, effectively can prevent the popular of pseudorabies and swine flue in swinery, can immune programme for children be simplified again, reach the effect that two diseases prevented by a pin.
5) with after resisting pstudorabies, swine flue vaccine combination immunity piglet, effectively can prevent that PRDC's is popular.
culture presevation
Swine influenza virus (H1N1 hypotype) ZJS strain (Swineinfluenzavirus (H1N1subtype) strainZJS), be separated by Pulaike Biological Engineering Co., Ltd., identify, (be called for short: CCTCC in China typical culture collection center; Address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University) carry out preservation, preservation date: on August 13rd, 2012, deposit number: CCTCCNO.V201233.
Swine influenza virus (H3N2 hypotype) WX strain (Swineinfluenzavirus (H3N2subtype) strainWX), be separated by Pulaike Biological Engineering Co., Ltd., identify, (be called for short: CCTCC in China typical culture collection center; Address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University) carry out preservation, preservation date: on August 13rd, 2012, deposit number: CCTCCNO.V201234.
Detailed description of the invention
In the present invention, PBS(0.01mM, pH7.4, prepare by described in " molecular cloning " third edition).
For making the present invention easier to understand, describe the present invention in detail below in conjunction with embodiment, these embodiments only play illustrative effect, are not limited to range of application of the present invention, NM specific experiment method in the following example, conveniently experimental technique carries out usually.
Embodiment
Embodiment 1: the preparation of resisting pstudorabies, swine flue vaccine combination
1. the preparation of PRV (Pseudorabies virus) antigen
Use rolling bottle cell culture method.By PRV (Pseudorabies virus) gene-deleted strain, (SA215 strain is TK -/ gE -/ gI -three gene delections, be disclosed in Chinese patent CN101186902, this strain is preserved in CCTCC with deposit number V200002) suitably dilute with viral dilution liquid (the DMEM culture medium of serum-free), by M.O.I=0.1 be inoculated in cover with monolayer ST cell (purchased from CCTCC, numbering GDC0060), Spin cells bottle 2 weeks gently, 37 DEG C of absorption 30min, add containing 3%(v/v) the DMEM cell maintenance medium of calf serum, put the cultivation of 37 DEG C of rotations (10 ~ 12 turns/hour).Observe 1 ~ 2 every day, Growth of Cells is good, cultivates harvestings on the 2nd ~ 5 and Cell sap, freeze thawing 3 times for 37 DEG C, virus liquid doughnut is filtered post (1.2 μm, aperture with 0.45 μm) and filters, remove cell debris, put less than-20 DEG C preservations.
2. the preparation of swine influenza virus (ZJS strain and WX strain) antigen
Use rolling bottle cell culture method.By SIVH1N1 hypotype ZJS strain and H3N2 hypotype WX strain seed culture of viruses respectively by the inoculum concentration of M.O.I=0.1 be inoculated in respectively cover with monolayer mdck cell (purchased from ATCC, numbering CRL-34), 37 DEG C of absorption 30min, add containing 3%(v/v) the DMEM cell maintenance medium of calf serum, put the cultivation of 37 DEG C of rotations (10 ~ 12 turns/hour).Observe 1 ~ 2 every day, Growth of Cells is good, cultivate harvestings on the 2nd ~ 5 and Cell sap for 37 DEG C, freeze thawing 3 times, used by two-strain liquid (ZJS strain and WX strain) doughnut (1.2 μm, aperture with 0.45 μm) to filter post respectively to filter, removing cell debris, then add formalin 37 DEG C of deactivation 18h of 0.2% ~ 0.3%, put less than-20 DEG C preservations.
3. the preparation of swine influenza virus (ZJS strain and WX strain) subunit antigen
Respectively by SIV(ZJS strain and WX strain) HA gene (gene order shown in SEQNO.1 and SEQNO.2 namely in sequence table) be cloned in Baculovirus Gene group respectively, and make it be in the control of polyhedrosis gene, by by the SIV(ZJS strain of baculovirus infection insect cell expression and WX strain) HA gene.By the recombinant virus that builds with M.O.I=0.01 infected insect cell, 5 days harvesting culture fluid and cell after infection, after multigelation 3 times, 12000rpm is centrifugal, gets supernatant, puts less than-20 DEG C preservations.
4. antigenic content measures and result
PRV (Pseudorabies virus) antigenic content measures: virus liquid is done 10 times of serial dilutions, get 10 -5, 10 -6, 10 -7, 10 -8, 10 -95 dilution factors, inoculate respectively on 96 porocyte culture plates of ST cell monolayer, each dilution factor inoculates 8 holes, sets up feminine gender not connect poison contrast simultaneously, puts into 5%CO 2cultivate 48 ~ 72h for 37 DEG C in incubator, observation of cell pathological changes (CPE), calculates viral TCID according to Reed-Muench method 50, result is viral level 10 8.5tCID 50/ ml.
Swine influenza virus (ZJS strain and WX strain) antigenic content measures: 10 times of serial dilutions are done in the swine influenza virus ZJS strain before deactivation and WX strain respectively, gets 10 -5, 10 -6, 10 -7, 10 -8, 10 -95 dilution factors, inoculate respectively on 96 porocyte culture plates of mdck cell monolayer, each dilution factor inoculates 8 holes, sets up feminine gender not connect poison contrast simultaneously, puts into 5%CO 2cultivate 48 ~ 72h for 37 DEG C in incubator, observation of cell pathological changes (CPE), calculates viral TCID according to Reed-Muench method 50, result is the viral level 10 of swine flue ZJS strain 8.2tCID 50the viral level 10 of/ml, swine flue WX strain 8.3tCID 50/ ml.
Swine influenza virus (ZJS strain and WX strain) subunit antigen assay: by the laggard promoting the circulation of blood of swine influenza virus (ZJS strain and WX strain) subunit antigen 2 times dilution solidifying (HA) experiment, result is the antigenic content 18Log2 of the antigenic content 17Log2 of swine flue ZJS strain, swine flue WX strain.
5. the preparation of vaccine combination
By pseudorabies live virus and freeze drying protectant (containing 1.5% gelatin of V/V, 2.5% bovine serum albumin, 10.0% trehalose, 6.0% polyvinylpyrrolidone, 3.0% sorbitol, 3.0% sodium glutamate and 0.05% vitamin C aqueous solution) in 1:1(V/V) after ratio mixing, fully shake all, quantitative separating, namely obtains pseudorabies live virus after carrying out lyophilisation rapidly after subpackage; By swine flue (H1N1 hypotype and H3N2 hypotype) inactivation of viruses liquid/subunit antigen by 9:1(V/V) add aqueous vaccine adjuvant (the 1% carbomer aqueous solution containing W/V), quantitative separating, is prepared into swine flue inactivated vaccine.Prepare vaccine 5 kinds altogether, its concrete antigen composition and content are in table 1.
The antigen composition of the different vaccine of table 1 and content proportioning
Remarks: "-" represents in vaccine without this component.When vaccine combination uses, the swine flue inactivated vaccine dilution pseudorabies lyophilizing live-virus vaccine that aqueous adjuvants is prepared, every part pig vaccinate 2ml/ head.
Embodiment 2: resisting pstudorabies, swine flue vaccine combination storage and transport prepared by different freeze drying protectant compare
1. material
Resisting pstudorabies, swine flue vaccine combination prepared by the heat-resisting lyophilized protecting agent optimized select V4 and the V5 vaccine in embodiment 1; Resisting pstudorabies, swine flue vaccine combination prepared by prior art arrangement (5% lactose, 10% skim milk aqueous solution containing V/V) are V6 and V7.The antigenic component of V6 and V4 is consistent, and only freeze drying protectant is different; The antigenic component of V7 and V5 is consistent, and only freeze drying protectant is different.
2. EXPERIMENTAL DESIGN
The vaccine combination (V4, V5, V6 and V7) prepared by different freeze drying protectant is placed sampling in latter 10 days in 37 DEG C and is tested; measure PRV (Pseudorabies virus) content in vaccine combination; judge that whether vaccine is qualified according to viral level; i.e. viral level decline >=0.5log; for defective; viral level decline <0.5log, for qualified.According to product quality, the quality of the more different frozen-dried protective vaccinating agent of storage and transport condition.
3. result of the test
The resisting pstudorabies adopting the heat-resisting lyophilized protecting agent optimized to prepare, swine flue vaccine combination (V4 and V5) are tested 37 DEG C of placements for latter 10 days, and vaccine product meets the requirements; The resisting pstudorabies adopting conventional formulation to prepare, swine flue vaccine combination (V6 and V7) are tested 37 DEG C of placements for latter 10 days, and vaccine product is undesirable, the results are shown in Table 2.The resisting pstudorabies adopting the heat-resisting lyophilized protecting agent optimized to prepare, swine flue vaccine combination (V4 and V5) are 2 ~ 8 DEG C of preservations; do not need cold chain; and the resisting pstudorabies adopting conventional formulation to prepare, swine flue vaccine combination (V6 and V7) need-15 DEG C of preservations; need cold chain; therefore; the resisting pstudorabies adopting the heat-resisting lyophilized protecting agent optimized to prepare, swine flue vaccine combination product quality are better than vaccine combination prepared by formula; and preserve, transport more convenient, save cost.
Vaccine combination prepared by the different freeze drying protectant of table 2 compares
Embodiment 3: after resisting pstudorabies, swine flue vaccine combination immunity piglet, PR antibody production compares
1. material
Resisting pstudorabies, swine flue vaccine combination select V4 and the V5 vaccine in embodiment 1; Pseudorabies list Seedling selects the V1 vaccine in embodiment 1.
2. animal experiment design
Select 21 age in days piglet 30, be divided into 3 groups, 10/group, the 1st group of every pig be musculi colli injection pseudorabies list Seedling V12.0ml(1 head part/head respectively), the 2nd and 3 groups of every pigs musculi collis injection resisting pstudorabies, swine flue vaccine combination V4 and V52.0ml(1 head part/heads respectively).In immunity latter 28 days, gather each group of piglet serum, adopt ELISA method to measure PRV (Pseudorabies virus) antibody titer.
PRV (Pseudorabies virus) Antibody Results criterion: colony's overall positive rate 90% ~ 100% assignment 3 points, colony's overall positive rate 70% ~ 89% assignment 2 points, colony's overall positive rate 50% ~ 69% assignment 1 point, colony's overall positive rate less than 50% assignment 0 point.Individual value for antibody 0.0 ~ 0.19 assignment 3 points of piglet, individual value for antibody 0.2 ~ 0.39 assignment 2 points of piglet, individual value for antibody 0.4 ~ 0.6 assignment 1 point of piglet, individual value for antibody more than 0.61 assignment 0 point of piglet.
3. result of the test
PR antibody test result after the pseudo-mad disease of resisting pstudorabies, swine flue vaccine combination and pig single Seedling immunity piglet is as table 3.No matter swine flue antigen is totivirus deactivation (V4) or subunit protein (V5), after vaccine combination immunity piglet, in swinery, the antibody positive rate of pseudorabies is 100%(10/10), and porcine pseudorabies list Seedling (V1) positive rate is 80%(8/10), the piglet antibody horizontal of vaccine combination immunity is higher than the piglet level of pseudorabies list Seedling immunity, by t-test check analysis, difference extremely significantly (P value 0.005) between the two, therefore, resisting pstudorabies, pseudorabies antibody after swine flue vaccine combination immunity piglet is obviously better than pseudorabies list Seedling immune effect, namely as known from Table 3, resisting pstudorabies, in swine flue vaccine combination, two kinds of antigens not only do not disturb mutually, the immune efficacy of Proantigen can be kept, and be surprised to find that the immune efficacy to PRV (Pseudorabies virus) wherein infects obtains and strengthens significantly.
Table 3 vaccine combination and pseudorabies list Seedling immunity piglet after PR antibody test result
Embodiment 4: after resisting pstudorabies, swine flue vaccine combination immunity piglet, SIV antibody production compares
1. material
Resisting pstudorabies, swine flue vaccine combination select V4 and the V5 vaccine in embodiment 1; Swine flue list Seedling selects V2 and the V3 vaccine in embodiment 1.
2. animal experiment design
Select 21 age in days piglet 40, be divided into 4 groups, 10/group, 1st and 2 groups of every pigs musculi collis injection swine flue list Seedling V2 and V32.0ml(1 head part/head respectively), the 3rd and 4 groups of every pigs musculi collis injection resisting pstudorabies, swine flue vaccine combination V4 and V52.0ml(1 head part/heads respectively).In immunity latter 28 days, gather each group of piglet serum, adopt hemagglutination inhibition test (HI) method to measure swine flue HI antibody titer.
Swine influenza virus Antibody Results criterion: colony's overall positive rate 90% ~ 100% assignment 3 points, colony's overall positive rate 70% ~ 89% assignment 2 points, colony's overall positive rate 50% ~ 69% assignment 1 point, colony's overall positive rate less than 50% assignment 0 point.The individual HI value for antibody >=8log2 assignment 3 points of piglet, the individual HI value for antibody of piglet 7 ~ 6log2 assignment 2 points, the individual HI value for antibody of piglet 5 ~ 4log2 assignment 1 point, piglet individual HI value for antibody <4log2 assignment 0 point.
3. result of the test
SIV antibody test result after resisting pstudorabies, swine flue vaccine combination and swine flue list Seedling immunity piglet is as table 4.After vaccine combination (V4 and V5) immune piglet, in swinery, the antibody positive rate of swine flue is all higher than swine flue list Seedling (V2 and V3), the piglet level that the piglet antibody horizontal of vaccine combination (V4 and V5) immunity is more immune than swine flue list Seedling (V2 and V3) is high, by t-test check analysis, difference extremely significantly (P value 0.005) between the two, therefore, resisting pstudorabies, swine flue antibody after swine flue vaccine combination immunity piglet is obviously better than swine flue list Seedling immune effect, namely as known from Table 4, resisting pstudorabies, in swine flue vaccine combination, two kinds of antigens not only do not disturb mutually, the immune efficacy of Proantigen can be kept, and be surprised to find that to obtain the immune efficacy of swine influenza virus infection wherein and strengthen significantly.
Table 4 vaccine combination and swine flue list Seedling immunity piglet after SIV antibody test result
Embodiment 5: after resisting pstudorabies, swine flue vaccine combination immunity piglet, pseudorabies virulent strain counteracting toxic substances protected effect compares
1. material
Resisting pstudorabies, swine flue vaccine combination select V4 and the V5 vaccine in embodiment 1; Pseudorabies list Seedling selects the V1 vaccine in embodiment 1.
2. animal experiment design
Select 21 age in days piglet 20, be divided into 4 groups, 5/group, 1st group of every pig musculi colli injection pseudorabies list Seedling V12.0ml(1 head part/head respectively), 2nd and 3 groups of every pigs musculi collis injection resisting pstudorabies, swine flue vaccine combination V4 and V52.0ml(1 head part/heads respectively), the 4th group is blank.After vaccine immunity 28 days, with 10 7.0pFU PRV (Pseudorabies virus) virulent strain (Fa strain) carries out challenge test, Continuous Observation 14 days, measures piglet body temperature, observes piglet clinical symptoms and record piglet death condition.
Piglet body temperature result criterion: compose according to the natural law of piglet body temperature more than 40.5 DEG C and divide, 0 ~ 1 day assignment 0 point, 2 ~ 3 days assignment 1 point, more than 3 days assignment 2 points.
Piglet clinical symptoms result criterion: piglet, without clinical symptoms assignment 0 point, without obvious clinical symptoms assignment 1 point, occurs obvious clinical symptoms assignment 2 points.Show as spirit without obvious clinical symptoms poor, but appetite does not significantly change, in 2 ~ 3 days, recover normal; Obvious clinical symptoms shows as lethargy, loss of appetite, occurs flocking together, the significantly clinical symptoms such as rapid breathing, continues more than 3 days.
Piglet death outcome criterion: piglet survival assignment 0 point, the dead assignment 2 points of piglet.
3. result of the test
After vaccine combination (V4 and V5) immune piglet, the counteracting toxic substances of the strong poison of pseudorabies the results are shown in Table 5.As can be seen from Table 5; within after vaccine combination (V4 and V5) immune piglet 28 days, carry out counteracting toxic substances protection; can effectively for immune piglet provide protection, and pseudorabies list Seedling (V1) is all inferior to vaccine combination (V4 and V5) in the body temperature, clinical symptoms and death condition etc. of piglet.Although no significant difference (P value is respectively 0.094 and 0.119) between t-test inspection display vaccine combination (V4 and V5) and pseudorabies list Seedling (V1); but relative comparison group; pole significant difference (P value is respectively 0.000 and 0.001) is there is between vaccine combination (V4 and V5) and matched group; and between pseudorabies list Seedling (V1) and matched group, there is not significant difference (P value 0.169); therefore, the protected effect of vaccine combination (V4 and V5) is better than pseudorabies list Seedling (V1).
The counteracting toxic substances result of the strong poison of pseudorabies after table 5 vaccine combination and pseudorabies list Seedling immunity piglet
Namely from the above results, not only the immune efficacy of PRV (Pseudorabies virus) is obtained in resisting pstudorabies of the present invention, swine flue vaccine combination and strengthen significantly, and in the strong malicious counteracting toxic substances situation of PRV (Pseudorabies virus), show better immune efficacy.
Embodiment 6: after resisting pstudorabies, swine flue vaccine combination immunity piglet, swine influenza virus counteracting toxic substances protected effect compares
1. material
Resisting pstudorabies, swine flue vaccine combination select V4 and the V5 vaccine in embodiment 1; Swine flue list Seedling selects V2 and the V3 vaccine in embodiment 1.
2. animal experiment design
Select 21 age in days piglet 50, be divided into 5 groups, 10/group, 1st and 2 groups of every pigs musculi collis injection swine flue list Seedling V2 and V32.0ml(1 head part/head respectively), 3rd and 4 groups of every pigs musculi collis injection resisting pstudorabies, swine flue vaccine combination V4 and V52.0ml(1 head part/heads respectively), the 5th group is blank.After vaccine immunity 28 days, often group chose 5 piglets with 10 8.0tCID 50/ first tap hits swine flue H1N1 hypotype ZJS strain, often organizes remaining 5 piglets with 10 8.0tCID 50/ first tap hits swine flue H3N2 hypotype WX strain, Continuous Observation 14 days, measures piglet body temperature and observes piglet clinical symptoms; Within after vaccine immunity 14 days, cut open and kill piglet, measure pulmonary lesion situation.
Piglet body temperature result criterion: compose according to the natural law of piglet body temperature more than 40.5 DEG C and divide, 0 ~ 1 day assignment 0 point, 2 ~ 3 days assignment 1 point, more than 3 days assignment 2 points.
Piglet clinical symptoms result criterion: piglet, without clinical condition assignment 0 point, without obvious clinical symptoms assignment 1 point, occurs obvious clinical symptoms assignment 2 points.Show as spirit without obvious clinical symptoms poor, but appetite does not significantly change, in 2 ~ 3 days, recover normal; Obvious clinical symptoms shows as lethargy, loss of appetite, occurs flocking together, the significantly clinical symptoms such as rapid breathing, continues more than 3 days.
Piglet pulmonary lesion result criterion: piglet pulmonary is without pathological changes assignment 0 point, and there is fraction pathological changes assignment 1 point in pulmonary, and pulmonary exists large area pathological changes assignment 2 points.
3. result of the test
After vaccine combination (V4 and V5) immune piglet, the counteracting toxic substances of swine flue H1N1 hypotype ZJS strain the results are shown in Table 6.As can be seen from Table 6; within after vaccine combination (V4 and V5) immune piglet 28 days, carry out the protection of swine flue H1N1 hypotype counteracting toxic substances; can effectively for immune piglet provide protection, and swine flue list Seedling (V2 and V3) is all inferior to vaccine combination (V4 and V5) in the body temperature, clinical symptoms and pulmonary lesion etc. of piglet.By t-test inspection display; notable difference (P value is respectively 0.011 and 0.031) is there is between vaccine combination (V4) and swine flue list Seedling (V2 and V3); also notable difference (P value is respectively 0.017 and 0.044) is there is between vaccine combination (V5) and swine flue list Seedling (V2 and V3); therefore, the protected effect of vaccine combination (V4 and V5) is better than swine flue list Seedling (V2 and V3).
The counteracting toxic substances result of swine flue H1N1 hypotype ZJS strain after table 6 vaccine combination and swine flue list Seedling immunity piglet
After vaccine combination (V4 and V5) immune piglet, the counteracting toxic substances of swine flue H3N2 hypotype WX strain the results are shown in Table 7.As can be seen from Table 7; within after vaccine combination (V4 and V5) immune piglet 28 days, carry out the protection of swine flue H3N2 hypotype counteracting toxic substances; can effectively for immune piglet provide protection, and swine flue list Seedling (V2 and V3) is all inferior to vaccine combination (V4 and V5) in the body temperature, clinical symptoms and pulmonary lesion etc. of piglet.By t-test inspection display; notable difference (P value is respectively 0.007 and 0.035) is there is between vaccine combination (V4) and swine flue list Seedling (V2 and V3); also notable difference (P value is respectively 0.021 and 0.039) is there is between vaccine combination (V5) and swine flue list Seedling (V2 and V3); therefore, the protected effect of vaccine combination (V4 and V5) is better than swine flue list Seedling (V2 and V3).
The counteracting toxic substances result of swine flue H3N2 hypotype WX strain after table 7 vaccine combination and swine flue list Seedling immunity piglet
Namely from the above results, not only the immune efficacy of swine influenza virus is obtained in resisting pstudorabies of the present invention, swine flue vaccine combination and strengthen significantly, and in the strong malicious counteracting toxic substances situation of swine influenza virus, show better immune efficacy.
Embodiment 7: resisting pstudorabies, swine flue vaccine combination and the effectiveness comparison being used alone two kinds of vaccines (pseudorabies disease live-vaccine and swine flue inactivated vaccine) immune piglet
1. material
Resisting pstudorabies, swine flue vaccine combination select V4 and the V5 vaccine in embodiment 1; Pseudorabies list Seedling selects the V1 vaccine in embodiment 1; Swine flue list Seedling selects V2 and the V3 vaccine in embodiment 1.
2. animal experiment design
Select 21 age in days piglet 50, be divided into 5 groups, 10/group, 1st group of every pig musculi colli injection pseudorabies list Seedling V12.0ml(1 head part/head respectively) and swine flue list Seedling V22.0ml(1 head part/head), 2nd group of every pig musculi colli injection pseudorabies list Seedling V12.0ml(1 head part/head respectively) and swine flue list Seedling V32.0ml(1 head part/head), 3rd and 4 groups of every pigs musculi collis injection resisting pstudorabies, swine flue vaccine combination V4 and V52.0ml(1 head part/heads respectively), the 5th group is blank.Day by day piglet clinical symptoms and injection site reaction is observed after vaccine immunity.
The convenience criterion of vaccine: every piggy injection 1 pin assignment 0 point, every piggy injection 2 pin assignment 1 point.
The criterion of piglet clinical safety: without any reaction assignment 0 point after piglet vaccination, occur untoward reaction assignment 1 point after piglet vaccination, untoward reaction comprises the situations such as convulsions, vomiting, tic, death.
The criterion of piglet inoculation local response: after piglet vaccination, local is without any reaction assignment 0 point, and after piglet vaccination, reaction assignment 1 point appears in local, and local response comprises the situations such as rubescent, swelling, abscess.
3. result of the test
Result of the test is as table 8, visible Combined vaccine (V4 and V5) is without side reaction, and two kinds of single Seedlings use (V1+V2 and V1+V3) simultaneously, but the piglet of 3/10 and 2/10 is had to occur that side reaction occurs, Symptoms is vomiting, twitch, lassitude, and the piggy injection topical manifestations of 2/10 and 2/10 is rubescent, swelling, abscess.
The comparative test result of table 8 vaccine combination and single Seedling coupling
From dosage of inoculation comparatively speaking the present embodiment shows, vaccine combination plays 1 pin, altogether 2.0ml, and two kinds of single Seedling couplings need play 2 pins, altogether 4.0ml, and apparent effect is that vaccine combination use is more convenient, time saving and energy saving; In addition, inventor is also surprised to find that, use the safety of the vaccine combination of two kinds of antigens on the contrary less to the local excitation of pig body, use after side effect also less, namely resisting pstudorabies of the present invention, swine flue vaccine combination are safer.And single Seedling coupling has the test pig of 3/10 and 2/10 to have untoward reaction, because the dosage of injection compares, vaccine combination will increase by 1 times; Comprehensively state it, vaccine combination is not only easy to use, and safer.
Embodiment 8: the Effect disquisition of control PRDC after resisting pstudorabies, swine flue vaccine combination immunity piglet
1. material
Resisting pstudorabies, swine flue vaccine combination select V4 and the V5 vaccine in embodiment 1.
2. animal experiment design
Select 21 age in days piglet 75, be divided into 3 groups, 25/group, the 1st and 2 groups of every pigs musculi collis injection resisting pstudorabies, swine flue vaccine combination V4 and V52.0ml(1 head part/heads respectively), the 3rd group is blank.Day by day observing piglet clinical symptoms in 30 days after vaccine immunity, there is quantity and the order of severity of respiratory symptoms such as coughing, breathe in record piglet.
The criterion of piglet clinical symptoms: piglet No respiratory signs assignment 0 point, there is respiratory symptom assignment 1 point in piglet, the dead assignment 2 points of piglet.
3. result of the test
Result of the test is as table 9, after visible Combined vaccine (V4 and V5) immune piglet, 2 or 3 piglets are only had to occur PRDC, and there is PRDC in matched group 14 piglets, separately there is 1 death, therefore, after resisting pstudorabies, swine flue vaccine combination immunity piglet, effectively can prevent that PRDC's is popular.Vaccine combination described in explanation can be applied to prevention and/or control in immune piglet PRDC disease medicament.
Occur that PRDC situation is added up after table 9 vaccine combination immunity piglet
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (3)

1. a vaccine combination for anti-PRV and SIV infection, it is made up of at least one PRV antigen and at least one SIV antigen;
Described PRV antigen is PRV totivirus, and it is porcine pseudorabies virus gene delection SA215 strain;
Described SIV antigen comprises complete swine flue totivirus or contains by the subunit of the gene constructed SIVHA albumen of SIVHA;
Described complete swine flue totivirus comprises H1N1 hypotype totivirus antigen and H3N2 hypotype totivirus antigen simultaneously;
Described SIVHA albumen comprises the HA albumen of H1N1 hypotype and the HA albumen of H3N2 hypotype simultaneously;
Described H1N1 hypotype antigen is swine influenza virus H1N1 hypotype ZJS strain, and deposit number is: CCTCCNO.V201233;
Described H3N2 hypotype antigen is swine influenza virus H3N2 hypotype WX strain, and deposit number is: CCTCCNO.V201234.
2. vaccine combination according to claim 1, is characterized in that,
In described vaccine combination, described PRV antigenic content is>=5.0 × 10 5.0tCID 50/ head part;
Before described H1N1 hypotype totivirus antigens inactive>=10 7.0tCID 50/ head part;
HA-HI test >=the 11Log2 of described H1N1 subtype HA protein;
HA-HI test >=the 11Log2 of described H3N2 subtype HA protein.
3. vaccine combination according to claim 1 and 2 is for the preparation of prevention and/or the application that controls in the medicine of immune piglet PRDC disease.
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