CN102727881A - Highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine and preparation method and application thereof - Google Patents
Highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention particularly relates to a highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine and the preparation method and the application of the highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain-porcine parvovirus disease bigeminal live vaccine. The highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine is the freeze-dried vaccine of mixed viral antigen liquid containing highly pathogenic porcine reproductive and respiratory syndrome virus and porcine parvovirus, the content of the highly pathogenic porcine reproductive and respiratory syndrome virus in per dose of the freeze-dried vaccine is 105-7 TCID50/ml, the content of the porcine parvovirus virus is 105-7 TCID50/ml, and the highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine is prepared via the following steps: preparing highly pathogenic porcine reproductive and respiratory syndrome virus liquid, preparing porcine parvovirus liquid, matching the vaccines and freeze-drying the vaccine. The bigeminal live vaccine provided by the invention has excellent immunogenicity and safety, can prevent two epidemic diseases including the highly pathogenic porcine reproductive and respiratory syndrome and the porcine parvovirus disease simultaneously, thereby preventing two diseases by one injection.
Description
Technical field
The present invention relates to the veterinary biologics technical field, be specifically related to a kind of high-pathogenicity porcine reproductive and respiration syndrome (JXA1-R strain)-porcine parvovirus bigeminal live vaccine.
Background technology
Porcine reproductive and respiratory syndrome (PRRS); Claim reproductive and respiratory syndrome again; Be a kind of height contagious disease of the pig that caused by porcine reproductive and respiratory syndrome virus (PRRSV), the pig of all ages and classes, kind and sex all can infect, but with in-pig and 1 monthly age with the susceptible of interior piglet.This disease is a principal character with sow miscarriage, stillborn fetus, weak tire, mummy tire and piglet dyspnea, septicemia, high mortality etc.Highly pathogenic PRRS is a kind of acute, the high lethal infectious disease that is caused by the porcine reproductive and respiratory syndrome virus variant, and clinical manifestation is a main feature with fever, sickness rate height and mortality rate high " three-hypers ".Summer in 2006, this disease (claiming " unknown high fever of pigs " at that time) is popular in a big way in China, makes China's pig industry suffer the economic loss of unprecedented heaviness.Can PRRSV be divided into american type and Europe class according to dna homolog property and serology difference, popular in China mainly is the american type strain.Owing to still do not have this sick specific medicament of treatment at present, therefore use specificity vaccine to become the major measure of prevention and control PRRS.Developed the vaccine of two types of inactivated vaccine and attenuated live vaccines at present both at home and abroad, it is generally acknowledged that the inactivated vaccine safety is good, and the attenuated live vaccines immune effect has been better.Still do not have improved vaccine at present and prevent this sick present stage, the pig farm that the PRRS medical history is arranged with receive the strong poison of PRRSV and infect the pig farm inoculation PRRSV attenuated live vaccines that threatens, to control should disease with to reduce economic loss significant.
Porcine parvovirus is that the breeding difficulty of the boar that caused by pig parvoviral (PPV) is sick; With infected the sow particularly negative multiparity sow of first farrowing sow and serology output stillborn fetus, monster, mummy tire, weak son, and the sow non-evident sympton is a characteristic.Pig parvoviral is a kind of very obstinate cause of disease, is distributed widely in all over the world, and swinery infects the back and is difficult to eradicate, and causes enormous economic loss to pig industry.At present; This disease is polluted very serious in China, positive rate reaches more than 90%, almost is difficult to the pig farm of finding a PPV negative; And should disease normal and breeding difficulty disease mixed infections such as pig circular ring virus 2 viral disease, pig blue-ear disease, porcine pseudorabies have become one of more common swine diseases of China at present.Prove that through blood clotting inhibition test and serum neutralization test the PPV that is separated to of various places belongs to a serotype together so far.Primary disease does not still have the efficacious therapy method at present, but because the serotype of PPV is single and immunogenicity is high, so vaccination remains generally acknowledged effective prevention and control measure, can to boar particularly the reserve boar carry out vaccination and prevent primary disease.The vaccine of at present having succeeded in developing also large scale investment use has inactivated vaccine and attenuated live vaccines.Wherein, the PPV attenuated vaccine has outstanding advantages such as immunogenicity is good, generation antibody in immunity back is fast, the length of holding time, and uses comparatively extensive.At present; The PPV attenuated vaccine strain mainly contains NADL-2 strain (cell culture adapted strain), HT strain (the low temperature passage is bred strain) and HT-SK-C strain (HT strain repeated transmission generation behind ultraviolet radiation breed strain); The existing abroad commercial prod of the attenuated live vaccines that utilizes these vaccine strains to produce, this has brought into play important function for the prevention and control of porcine parvovirus.
In order effectively to prevent above-mentioned 2 kinds of eqpidemic diseases, in the routine immunization program is all listed the vaccine of these 2 kinds of infectious disease by each enterprise of raising pigs greatly.Because of only being arranged at present on the market, sells single Seedling; To prevent this 2 kinds of eqpidemic diseases simultaneously; The same age bracket pig only often needs immune 2 pins; This makes the immune density of each age group pig increase, and the expense of vaccine, the manpower when annotating Seedling and to pig cause stress and the short time high density annotate Seedling and cause the phase mutual interference between each vaccine, the pig farm operator is suffered untold misery.Along with the development of China's intensive pig production industry, traditional univalent vaccine can't satisfy immune needs, and pressing for can the anti-many sick vaccine of a pin.
Summary of the invention
In order to overcome the defective of prior art; The object of the present invention is to provide a kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; Can prevent sick and these the two kinds of eqpidemic diseases of porcine parvovirus of high-pathogenicity porcine reproductive and respiration syndrome simultaneously; Manpower when avoiding annotating Seedling and to pig cause stress and the short time high density annotate Seedling and cause the phase mutual interference between each vaccine, accomplish " pin is prevented two diseases ".
Another object of the present invention is to provide the method for preparing of a kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine, step is simple, and operability is high.
Another purpose of the present invention is high-pathogenicity porcine reproductive and the application of respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine in setting up high-pathogenicity porcine reproductive and respiration syndrome-porcine parvovirus bigeminal live vaccine quality standard.
For realizing above-mentioned purpose, the technical scheme that the present invention adopted is following:
A kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; It is the freeze dried vaccine that contains the blended hybrid virus antigen liquid of high-pathogenicity porcine reproductive and breath syndrome virus and pig parvoviral, and high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in every part freeze dried vaccine
5-7TCID
50/ ml, pig parvoviral virus content is 10
5-7TCID
50/ ml.
High-pathogenicity porcine reproductive of the present invention and respiration syndrome-porcine parvovirus bigeminal live vaccine also contains freeze drying protectant.
Preferably, freeze drying protectant of the present invention is a sucrose skimmed milk freeze drying protectant, and the volume ratio of its consumption and hybrid virus antigen liquid is 1:1.Said sucrose skimmed milk freeze drying protectant is composed of the following components: sucrose 5～10g, defatted milk 100ml.
In the preferred scheme, high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in high-pathogenicity porcine reproductive of the present invention and respiration syndrome-every part freeze dried vaccine of porcine parvovirus bigeminal live vaccine
5.5-6.5TCID
50/ ml, pig parvoviral virus content is 10
5.5-6.5TCID
50/ ml.
Bigeminal live vaccine of the present invention is used to prevent high-pathogenicity porcine reproductive and respiration syndrome and porcine parvovirus.Press head that label indicates part, with sterile saline or special-purpose diluted vaccine, intramuscular injection, 1 part/head/inferior.Immunity is 2 times before the replacement gilt breeding, at interval 2～3 weeks; Multiparity insemination of sows immunity in preceding 1 month 1 time; Boar is exempted from 6 monthly age head, immunity in later per 6 months 1 time.
The method for preparing of a kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine, it may further comprise the steps:
1) preparation of high-pathogenicity porcine reproductive and breath syndrome virus JXA1-R strain virus liquid: with high-pathogenicity porcine reproductive and breath syndrome virus JXA1-R low virulent strain is the Marc-145 cell that 1% amount inoculation has grown up to monolayer by volume; Rotating and culturing; When cytopathy appears in 70% above cell; Results virocyte culture, freeze thawing is after centrifugal or remove by filter cell debris, frozen in below-40 ℃;
2) preparation of pig parvoviral liquid: adopt synchronous inoculation method; When the ST cell divides bottle to go down to posterity; Be that 1～2% amount is inoculated the pig parvoviral low virulent strain synchronously by volume, when being cultured to 70% above cell and cytopathy occurring, results virocyte culture; Freeze thawing is after centrifugal or remove by filter cell debris, frozen in below-20 ℃;
3) join Seedling and lyophilizing: above-mentioned steps 1), 2) two kinds of cell culture venom obtaining by lyophilizing after every part PRRSV of vaccine viral level be 10
5-7TCID
50/ ml, PPV viral level are 10
5-7TCID
50What/ml calculated two kinds of viral liquid joins the Seedling consumption, mixes according to the consumption that calculates, and adds stabilizing agent, obtains high-pathogenicity porcine reproductive and respiration syndrome-porcine parvovirus bigeminal live vaccine through lyophilisation.
The temperature of rotating and culturing is 37 ℃ in the step 1), rotary speed be 9～12 change/hour.
Temperature is 37 ℃ when cultivating step 2), and incubation time is 72～96 hours.
The present invention's used seed culture of viruses in the process of preparation bigeminal live vaccine is high-pathogenicity porcine reproductive and breath syndrome virus JXA1-R strain, identifies, takes care of and supply by China Animal Disease Control And Prevention Center.
The pig parvoviral that the present invention adopted is the CG03-R strain, identifies, takes care of and supply by China Veterinery Drug Inspection Office.
The application in setting up high-pathogenicity porcine reproductive and respiration syndrome-porcine parvovirus bigeminal live vaccine quality standard of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine.
Beneficial effect of the present invention is:
High-pathogenicity porcine reproductive of the present invention and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine are a kind of Seedlings that joins more; Each antigenic component in the vaccine all reaches the immune effect of corresponding single Seedling, and through measuring, test pig is once inoculated this vaccine; The inoculation back can produce immunity on 14th～21; Duration of immunity reached more than 4 months, sustainable 3～4 weeks of PRRSV maternal antibody that the immune sow cub of institute pig obtains, sustainable 6～7 weeks of PPV maternal antibody; Conceived 30～50 days 3 repeated inoculations of in-pig, single dose of overdose (2 times of using dosages) inoculation or overdose (10 times of using dosages) inoculation replacement gilt; All do not find any abnormal response, explain that the prepared bigeminal live vaccine of the present invention has good immunogenicity and safety; And can prevent high-pathogenicity porcine reproductive and respiration syndrome disease and these two kinds of eqpidemic diseases of porcine parvovirus simultaneously; Manpower when avoiding annotating Seedling and to pig cause stress and the short time high density annotate Seedling and cause the phase mutual interference between each vaccine, accomplish " pin is prevented two diseases ".
Embodiment below in conjunction with concrete is done further explain to the present invention.
The specific embodiment
Embodiment 1
The method for preparing of a kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine, it may further comprise the steps:
The preparation of high-pathogenicity porcine reproductive and breath syndrome virus JXA1-R strain virus liquid: produce and use seed culture of viruses to be high-pathogenicity porcine reproductive and breath syndrome virus JXA1-R strain, choose the Marc-145 cell (the cell rolling bottle that has grown up to monolayer) of requirement, discard growth-promoting media; Produce with seed culture of viruses virus by 1% (V/V) inoculation; Add the DMEM cell culture fluid contain 2% hyclone, the cytopathy (CPE) that is caused by virus is examined under a microscope in 37 ℃ of rotations (9～12 change/hour) cultivation; When CPE reaches 70% culture of harvesting when above; Freeze thawing 1 time, through centrifugal or remove by filter cell debris, frozen in below-40 ℃; By " Chinese veterinary drug allusion quotation " appendix vaccine antigen is carried out steriling test and viral level mensuration, through measuring high-pathogenicity porcine reproductive and breath syndrome virus content>=10 in every ml cells liquid
7TCID
50/ ml;
The preparation of pig parvoviral liquid: select pig parvoviral CG03-R strain seed culture of viruses for use, adopt synchronous inoculation method, when the ST cell divides bottle to go down to posterity, inoculate production kind of poison synchronously, put 37 ℃ and cultivated 72～96 hours by the amount of 1% (V/V).Examine under a microscope the CPE that causes by virus after connecing poison, when CPE reaches 70% culture of harvesting when above, freeze thawing 1 time, through centrifugal or remove by filter cell debris, frozen in below-20 ℃; By " Chinese veterinary drug allusion quotation " appendix vaccine antigen is carried out steriling test and viral level mensuration, through measuring pig parvoviral content>=10 in every ml cells culture
7TCID
50/ ml, toxic viral liquid answer>=1: 256 to 1% GPRBC blood clotting valency;
Join Seedling and lyophilizing: get two kinds of viral liquid that are up to the standards respectively, be no less than 10 by every part PRRSV after the lyophilizing and PPV viral level
5TCID
50The amount of/ml, that calculates two kinds of compositions joins the Seedling consumption, places mix homogeneously in the same sterilization container, is to join Seedling and use hybrid virus antigen; Hybrid virus antigen is added 5% sucrose skimmed milk freeze drying protectant by 1: 1 volume ratio, fully shake up, quantitatively carry out lyophilization immediately by conventional method after the packing, every part PRRSV of vaccine viral level is 10 after the lyophilizing
5-7TCID
50/ ml, PPV viral level are 10
5-7TCID
50/ ml.
Embodiment 2
A kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; Be that high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in every part freeze dried vaccine according to the freeze dried vaccine that contains the blended hybrid virus antigen liquid of high-pathogenicity porcine reproductive and breath syndrome virus and pig parvoviral of the method preparation of embodiment 1
6.0TCID
50/ ml, pig parvoviral virus content is 10
5.8TCID
50/ ml.
Embodiment 3
A kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; Be that high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in every part freeze dried vaccine according to the freeze dried vaccine that contains the blended hybrid virus antigen liquid of high-pathogenicity porcine reproductive and breath syndrome virus and pig parvoviral of the method preparation of embodiment 1
6.2TCID
50/ ml, pig parvoviral virus content is 10
5.7TCID
50/ ml.
Embodiment 4
A kind of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; Be that high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in every part freeze dried vaccine according to the freeze dried vaccine that contains the blended hybrid virus antigen liquid of high-pathogenicity porcine reproductive and breath syndrome virus and pig parvoviral of the method preparation of embodiment 1
6.1TCID
50/ ml, pig parvoviral virus content is 10
5.8TCID
50/ ml.
Application implementation example 1
The foundation of high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine quality standard, carry out according to following steps:
1 character, steriling test, mycoplasma check, exogenous virus check, residual moisture mensuration and vacuum are measured: test by " Chinese veterinary drug allusion quotation " appendix, should meet relevant provisions;
2 diagnostic tests: vaccine is done suitable dilution (0.01 part/ml) with serum-free medium; Getting 0.1ml mixes with equivalent porcine reproductive and respiratory syndrome virus, pig parvoviral specificity positive serum; Establish virus control group and normal cell matched group simultaneously, 37 ℃ of effects were inoculated Marc-145 cell culture fluid and ST cell culture fluid respectively after 1 hour; Observed 5, and should not produce cytopathy;
3 vaccine safeties test: press label and indicate head part; Vaccine is diluted to every 2ml contains 10 parts; Age in intramuscular injection 3～4 week, PRRSV antigen, negative antibody and negative (the HI antibody titer is not higher than 1: the 8) piglet of PPV antigen-antibody were 5; Every 2ml, every day, the part and the systemic adverse reactions that are caused by vaccine should all not appear in thermometric and observing 21;
4 efficacy tests: following method is appointed and is selected one of which
1) uses cellular assay
It is 0.002 part/0.1ml that the finished product vaccine is used the serum-free medium dilution; Be respectively charged in two test tubes; Every pipe 0.5ml, the 1st pipe adds the pig parvoviral specificity positive serum of equivalent, and the 2nd pipe adds the porcine reproductive and respiratory syndrome virus specificity positive serum of equivalent.37 ℃ act on 1 hour, and this moment, viral level was 0.001 part/0.1ml.First pipe continues 10 times of serial dilutions, gets 10
-1, 10
-2, 10
-33 dilution factors are inoculated in the 96 porocyte plates that grown up to monolayer Marc-145 cell respectively, and each dilution factor is inoculated 6 holes, and every hole 0.1ml cultivated 3～5 for 37 ℃, calculated TCID
50, every part>=10
5TCID
50, it is qualified that Porcine reproductive and respiratory syndrome partly is judged to.Second pipe continues 10 times of serial dilutions, gets 10
-1, 10
-2, 10
-33 dilution factors are inoculated ST cell 96 orifice plates respectively synchronously, and each dilution factor is inoculated 6 holes, and every hole 0.1ml cultivates for 37 ℃ and observed 5, calculates TCID
50, every part>=10
5TCID
50, it is qualified that pig parvoviral partly is judged to;
2) check with pig:
The Porcine reproductive and respiratory syndrome part: with 4～6 age in week 10 of PRRSV antigen, negative antibody pigs; 1 part of 5 intramuscular injection vaccines wherein, 5 as contrast, isolated rearing under the equal conditions in addition; After 28 days, all pig muscle injection checks are with the virus-culturing fluid (>=10 of strong malicious NVDC JXA1 strain
4.5TCID
50/ ml) 3ml, every day thermometric and observing 21.5 of pigs of contrast all should fall ill, and at least 2 death, and 5 of immune swines should at least 4 head protections;
Pig parvoviral part: with 10 of negative (the HI antibody titer is not higher than 1: 8) pigs of 4～6 PPV antigen-antibodies in age in week, wherein 1 part of 5 deep intramuscular injection vaccines, 5 conduct contrasts in addition; Isolated rearing under the equal conditions, after 28 days, all pig blood samplings; Separation of serum is measured HI antibody.At least 4 antibody positives of immune swine, its HI antibody titer geometrical mean should be not less than 1: 64, and contrast pig HI antibody titer all is not higher than 1: 8.
Accomplish the foundation of high-pathogenicity porcine reproductive and respiration syndrome-porcine parvovirus bigeminal live vaccine quality standard through above-mentioned steps 1-4.
Index determining:
According to above-mentioned quality standard, 3 batches of vaccines that the embodiment of the invention is prepared carry out product inspection, and are all up to specification, and it is following that wherein several Key Quality Indicator are measured results:
1 vaccine safety test: conceived 30～50 days in-pig of overdose (2 times of using dosages) inoculation; All test pig mental status are good; Appetite is normal, and childbirth is observed in no abnormal reaction always; Clinical symptoms such as no miscarriage, premature labor, stillborn fetus, weak son performances all can not be isolated porcine reproductive and respiratory syndrome virus and pig parvoviral from the blood of its whole fetuses that produce or internal organs; 3 repeated inoculations of single dose or overdose (10 times of using dosages) inoculation replacement gilt or breeding boar are not all found any abnormal response, show that vaccine safety is reliable.
2 vaccine immunities are renderd a service
1) to 20 of primiparity replacement gilts (PRRSV antigen, negative antibody and PPV HI antibody≤1: 8); Wherein 10 in 1 part of the previous month immune vaccine of the present invention of breeding; Booster immunization is 1 time after 2～3 weeks; Each all intramuscular injection, 10 any unavoidably vaccines are as contrast in addition, isolated rearing under the equal conditions; In gestation 30～50 days the time, with above-mentioned immune swine and non-ly exempt to contrast pig each is divided into 2 groups at random, 5 every group, one group of intramuscular injection check is with the virus-culturing fluid (>=10 of strong malicious NVDC JXA1 strain
4.5TCID
50/ ml) 3ml, another group intramuscular injection pig parvoviral PPV7909 standard virulent strain 2ml (contains 10
5TCID
50/ ml), observe to childbirth; Non-ly exempt to contrast all morbidities behind the pig counteracting toxic substances PRRSV, and 2 death, 4 breeding difficulty symptoms such as miscarriage, stillborn fetus, mummy tire or fetus heavily absorb occur behind the counteracting toxic substances PPV, and equal 5/5 protection of immune swine.Pig that immune swine is farrowed birth back can detect PRRSV antibody and PPV HI antibody at the 3rd day, peaked in the 10th～14 day, and lasting decline of 3～4 week back PRRSV antibody, 6～7 week back PPV HI antibody also begin to continue decline.
2) the multiparity sow is in previous month this vaccine of dosage immunity with 1 part/head of breeding, and immune effect is the same.
3) get 6 monthly ages, 10 of healthy susceptible boars (PRRSV antigen, negative antibody and PPV HI antibody≤1: 8), wherein 5 with 1 part/this vaccine of dosage intramuscular injection, and 5 any unavoidably vaccines are as contrast in addition, isolated rearing under the equal conditions.All pigs blood sampling in 28 days, separation of serum is measured PRRSV antibody horizontal and PPV HI antibody horizontal in the serum; It is non-that to exempt to contrast pig PRRSV antibody all negative; PPV HI antibody all≤1: 8, immune swine PRRSV antibody is all positive, PPV HI tires all>=1: 128.
4) get 15 of healthy susceptible pigs in 6 ages in week (PRRSV antigen, negative antibody and PPV HI antibody≤1: 8), wherein 10 with 1 part/this vaccine of dosage intramuscular injection, and 5 any unavoidably vaccines are as contrast in addition, isolated rearing under the equal conditions.The blood sampling regularly of inoculation back, separation of serum is measured PRRSV antibody horizontal and PPV HI antibody horizontal in the serum.Can detect PRRSV antibody and PPV HI antibody on the 7th day after the immune group immunity as a result, raise gradually after 7 days, immunity was set up fully in 14～21 days; Peaked in 28～42 days; This antibody can be maintained to 120～150 days, began later on to descend, and the great majority beginning is negative after 180 days; But not exempt to contrast pig PRRSV antibody and PPV HI antibody is negative always.
The test of 3 vaccine storage lives
The finished product vaccine put under-15 ℃ of conditions preserve, regularly take out some bottles, observe its character and change, and measure the TCID of PRRSV and PPV respectively by the 3.4.1 method
50With monitoring vaccine virus changes of contents situation, establish corresponding single Seedling matched group simultaneously.3 batches of vaccines were preserved 24 months under-15 ℃ of conditions as a result, and its character does not all change; Still can the reach quality standards requirement of (draft) of viral level, to measure the result suitable with the storage life of corresponding single Seedling, sees table 1～table 2 for details.
Table 1 vaccine is preserved 12,18,24 months PRRSV viral levels and is measured TCID as a result under-15 ℃ of conditions
50/ head part
| The embodiment numbering | Tire in the basis | 12 months | 18 months | 24 months |
| 2 | 10 6.0 | 10 6.1 | 10 5.6 | 10 5.1 |
| 3 | 10 6.2 | 10 6.2 | 10 5.7 | 10 5.2 |
| 4 | 10 6.1 | 10 6.1 | 10 5.5 | 10 5.0 |
| PRRSV live vaccine matched group | 10 6.0 | 10 6.0 | 10 5.6 | 10 5.0 |
Table 2 vaccine is preserved 12,18,24 months PPV viral levels and is measured TCID as a result under-15 ℃ of conditions
50/ head part
| The embodiment numbering | Tire in the basis | 12 months | 18 months | 24 months |
| 2 | 10 5.8 | 10 5.5 | 10 5.3 | 10 5.1 |
| 3 | 10 5.7 | 10 5.6 | 10 5.4 | 10 5.2 |
| 4 | 10 5.8 | 10 5.5 | 10 5.2 | 10 5.1 |
| PPV live vaccine matched group | 10 5.8 | 10 5.6 | 10 5.4 | 10 5.1 |
4, field test
Field test is carried out on 3 batches of high-pathogenicity porcine reproductives that the embodiment of the invention is prepared and respiration syndrome-porcine parvovirus bigeminal live vaccine subordinate's of Wen Shi group in Guangdong pig farm; The test pig kind comprises multiparity sow, replacement gilt, breeding boar; Through observation to the searching for food of swinery after the immunity, drinking-water, body temperature situation; And regular antibody test, the vaccine evaluation immune effect.The result shows, mine massively food, drinking-water, body temperature etc. of immune swine are all no abnormal; The multiparity sow is in breeding preceding 1 month with immune this vaccine of 1 part/dosage only; Exempt from the back and can produce strong immunity on 14th～21; Duration of immunity is 4 months, sustainable 3～4 weeks of the PRRSV maternal antibody of the piglet that it is given birth to, sustainable 6～7 weeks of PPV maternal antibody; Replacement gilt preceding 1 month this vaccine of immunity of breeding, 2～3 week back booster immunizations 1 time, each 1 part, the immune effect of generation is with the multiparity sow; Adult boar immunity in per 6 months 1 time, each 1 part, each duration of immunity also can reach more than 4 months.Through the tracking to the whole production phase, immune swinery does not all have the generation of high-pathogenicity porcine reproductive and respiration syndrome and porcine parvovirus.Show high-pathogenicity porcine reproductive that the present invention prepares and respiration syndrome-porcine parvovirus bigeminal live vaccine to immune swine safety, effectively.
The foregoing description is merely the preferred embodiments of the present invention; Can not limit the present invention's scope required for protection with this; The variation and the replacement of any unsubstantiality that those skilled in the art are done on basis of the present invention all belong to the present invention's scope required for protection.
Claims (9)
1. a high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; It is characterized in that: it is the freeze dried vaccine that contains high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain virus and the blended hybrid virus antigen liquid of pig parvoviral, and high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in every part freeze dried vaccine
5-7TCID
50/ ml, pig parvoviral virus content is 10
5-7TCID
50/ ml.
2. high-pathogenicity porcine reproductive according to claim 1 and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine, it is characterized in that: it also contains freeze drying protectant.
3. high-pathogenicity porcine reproductive according to claim 2 and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine; It is characterized in that: said freeze drying protectant is a sucrose skimmed milk freeze drying protectant, and the volume ratio of its consumption and hybrid virus antigen liquid is 1:1.
4. high-pathogenicity porcine reproductive according to claim 3 and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine is characterized in that: said sucrose skimmed milk freeze drying protectant is composed of the following components: sucrose 5～10g, defatted milk 100ml.
5. high-pathogenicity porcine reproductive according to claim 1 and 2 and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine is characterized in that: high-pathogenicity porcine reproductive and breath syndrome virus content are 10 in every part freeze dried vaccine
5.5-6.5TCID
50/ ml, pig parvoviral virus content is 10
5.5-6.5TCID
50/ ml.
6. the method for preparing of a high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine, it is characterized in that: it may further comprise the steps
1) preparation of high-pathogenicity porcine reproductive and breath syndrome virus liquid: with high-pathogenicity porcine reproductive and breath syndrome virus JXA1-R low virulent strain is the Marc-145 cell that 1% amount inoculation has grown up to monolayer by volume; Rotating and culturing; When cytopathy appears in 70% above cell; Results virocyte culture, freeze thawing is after centrifugal or remove by filter cell debris, frozen in below-40 ℃;
2) preparation of pig parvoviral liquid: adopt synchronous inoculation method; When the ST cell divides bottle to go down to posterity; Be that 1～2% amount is inoculated the pig parvoviral low virulent strain synchronously by volume, when being cultured to 70% above cell and cytopathy occurring, results virocyte culture; Freeze thawing is after centrifugal or remove by filter cell debris, frozen in below-20 ℃;
3) join Seedling and lyophilizing: above-mentioned steps 1), 2) two kinds of cell culture venom obtaining by lyophilizing after every part PRRSV of vaccine viral level be 10
5-7TCID
50/ ml, PPV viral level are 10
5-7TCID
50What/ml calculated two kinds of viral liquid joins the Seedling consumption, mixes according to the consumption that calculates, and adds stabilizing agent, obtains high-pathogenicity porcine reproductive and respiration syndrome-porcine parvovirus bigeminal live vaccine through lyophilisation.
7. the method for preparing of high-pathogenicity porcine reproductive according to claim 6 and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine is characterized in that: the temperature of rotating and culturing is 37 ℃ in the step 1), rotary speed be 9～12 change/hour.
8. the method for preparing of high-pathogenicity porcine reproductive according to claim 6 and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine is characterized in that: step 2) in when cultivating temperature be 37 ℃, incubation time is 72～96 hours.
9. a high-pathogenicity porcine reproductive and the respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine application in high-pathogenicity porcine reproductive and respiration syndrome JXA1-R strain-porcine parvovirus bigeminal live vaccine quality standard are set up.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102295484A CN102727881A (en) | 2012-07-04 | 2012-07-04 | Highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine and preparation method and application thereof |
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Cited By (4)
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| CN104630157A (en) * | 2015-03-05 | 2015-05-20 | 成都天邦生物制品有限公司 | Method for producing porcine reproductive and respiratory syndrome JXA1-R strain virus by using low serum cultured Marc-145 cells |
| CN104849463A (en) * | 2014-11-18 | 2015-08-19 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis bivalent vaccine potency test method |
| WO2018083156A1 (en) | 2016-11-03 | 2018-05-11 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine against porcine parvovirus and porcine reproductive and respiratory syndrome virus and methods of production thereof |
| US10485866B2 (en) | 2016-11-03 | 2019-11-26 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine against porcine parvovirus |
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| CN104849463A (en) * | 2014-11-18 | 2015-08-19 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis bivalent vaccine potency test method |
| CN104849463B (en) * | 2014-11-18 | 2017-02-22 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis bivalent vaccine potency test method |
| CN104630157A (en) * | 2015-03-05 | 2015-05-20 | 成都天邦生物制品有限公司 | Method for producing porcine reproductive and respiratory syndrome JXA1-R strain virus by using low serum cultured Marc-145 cells |
| WO2018083156A1 (en) | 2016-11-03 | 2018-05-11 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine against porcine parvovirus and porcine reproductive and respiratory syndrome virus and methods of production thereof |
| US10485866B2 (en) | 2016-11-03 | 2019-11-26 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine against porcine parvovirus |
| US10660952B2 (en) | 2016-11-03 | 2020-05-26 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine against porcine parvovirus and porcine reproductive and respiratory syndrome virus and methods of production thereof |
| US10799578B2 (en) | 2016-11-03 | 2020-10-13 | Boehringer Ingelheim Vetmedica Gmbh | Vaccine against porcine parvovirus |
| TWI788309B (en) * | 2016-11-03 | 2023-01-01 | 德商百靈佳殷格翰維美迪加股份有限公司 | Vaccine against porcine parvovirus and porcine reproductive and respiratory syndrome virus and methods of production thereof |
| US11730806B2 (en) | 2016-11-03 | 2023-08-22 | Boehringer Ingelheim Vetmedica Gmbh | Methods of manufacturing an immunogenic composition comprising a recombinant protein |
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