CN102719407B - Porcine parvovirus BQ-C strain and application of porcine parvovirus BQ-C strain in preparation of inactivated porcine parvovirus vaccine - Google Patents
Porcine parvovirus BQ-C strain and application of porcine parvovirus BQ-C strain in preparation of inactivated porcine parvovirus vaccine Download PDFInfo
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Abstract
The invention discloses a porcine parvovirus BQ-C strain and application of the porcine parvovirus BQ-C strain to the preparation of an inactivated porcine parvovirus vaccine, and belongs to the field of biological vaccines. The porcine parvovirus BQ-C strain has the advantages of high immunogenicity, good cultural character and the like; and porcine parvovirus (BQ-C strain) are inoculated to swine testis (ST) cells, a culture is harvested and inactivated by using binary ethylenimine (BEI), and the culture and a commercial MontanideTM ISA15AVG adjuvant of SEPPIC are mixed and emulsified to form the inactivated porcine parvovirus vaccine. The experiment proves that: diseases caused by porcine parvoviruses can be prevented by immunizing primiparous healthy female sows by using the prepared inactivated porcine parvovirus vaccine; and moreover, the invention has the advantages that the vaccine is safe, an antibody can be quickly generated, the immune period is long-lasting, and the like.
Description
Technical field
The present invention relates to a kind of pig parvoviral and the application in preparation pig parvoviral inactivated vaccine thereof, particularly a strain separates the pig parvoviral BQ-C strain that obtains and the inactivated vaccine that is prepared by this virus strain voluntarily.Belong to the biovaccine field.
Background technology
Pig parvoviral (Porcine parvovirus, PPV) be one of main pathogen of causing pig breeding dysfunction, what PPV infected mainly is pregnant pig, particularly first farrowing sow is infected before pregnancy easily, cause the farrowing sow miscarriage, produce stillborn foetus, fetus mummification etc., and parent lacks clinical symptom usually; Certainly also can cause other symptoms.From Mayr in 1966 and Mahnel find and confirm that PPV exists and pathogenic since, Chinese scholars has been carried out research extensively and profoundly to it.In recent years, PPV infects and is the expansion ascendant trend, has caused enormous economic loss for global pig industry.In China, Pan Xuezhu has been separated to PPV first in nineteen eighty-three.After this, several strain PPV have been separated to successively in various places, at present, it is found that the frequent and some other virus of pig parvoviral, for example polyinfections such as the breeding of porcine circovirus 2 type and pig and respiratory system syndrome virus, cause weanling pig multisystem syndrome, dermatitis, respiratory syndrome etc., cause very big loss for the pig industry in the world.Though different isolates all belongs to same serotype, can cause the various clinical symptom, but all to cause breeding difficulty.At present, China's multiparity sow and herd boar PPV positive rate are up to more than 80%.
The present inventor was separated to a strain pig parvoviral in 2005 from the pig farm, Heilongjiang Province, was virulent strain through laboratory qualification, the strain called after PPV BQ-C strain behind the domestication clone.Utilize PPV BQ-C strain, carried out the development work of porcine parvovirus inactivated vaccines, screening cultivates good, the strong malicious BQ-C strain of the stable pig parvoviral of tiring of immunogenicity, selected the ST cell of the most suitable this virus multiplication and met inactivator, adjuvant and other conditions of making vaccine.Production technique, security, protection ratio, immune programme for children, minimum dose, antibody extended period and preservation period etc. to goods have all been carried out test determination, and have obtained a large amount of testing datas, prove that this vaccine safety is effective.On this basis, carried out middle trial production, through sampling inspection, all meet the quality standard of drafting, and pilot product proof test and potency test have been carried out, its result confirms that also this vaccine can effectively prevent the sow breeding difficulty that is caused by pig parvoviral, on the basis of laboratory test, pilot-scale production, the present invention has developed pig parvoviral (BQ-C strain) inactivated vaccine, its result confirms that also this vaccine can effectively prevent the sow breeding difficulty that is caused by pig parvoviral, has further verified every quality standard of this vaccine.
Summary of the invention
One of purpose of the present invention provides the pig parvoviral strain that a kind of immunogenicity is good, tire stable;
Two of purpose of the present invention provides the application of described pig parvoviral strain in the preparation vaccine;
Three of purpose of the present invention provides a kind of vaccine that is prepared by described pig parvoviral strain;
Four of purpose of the present invention provides described vaccine and uses in the tiny medicine of preparation prevention pig.
In order to achieve the above object, the present invention has adopted following technique means:
The present inventor was separated to a strain pig parvoviral in 2005 from the pig farm, Heilongjiang Province, was virulent strain through laboratory qualification, the strain called after PPV BQ-C strain after the domestication.PPV BQ-C of the present invention strain is to produce to separate the weak son from the pregnant sow that typical pig parvoviral infection takes place to obtain, and therefore compares the propagation titre height on cell with other strain that is separated to, and is good to the immunogenicity of pig.As the vaccine seed culture of viruses, imitating inspection is the 7th～10 generation of BQ-C strain with strong poison after pig testis continuous cell line (ST) goes down to posterity.This strain can cause that the ST cell infects more than 90%.
A kind of pig parvoviral of the present invention, for after going down to posterity at the ST cell the 7th generation virus strain, called after pig parvoviral BQ-C strain, classification called after pig parvoviral, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, and its culture presevation is numbered: CGMCC No.6091, preservation date are on May 8th, 2012.
The present invention also provides the application of described pig parvoviral in preparation pig parvoviral inactivated vaccine.
A kind of pig parvoviral inactivated vaccine of the present invention is characterized in that containing the pig parvoviral BQ-C of the present invention strain after the deactivation.
In pig parvoviral inactivated vaccine of the present invention, preferably, described pig parvoviral BQ-C strain adopts following method to carry out deactivation: the deactivation under 32 ℃ of conditions of 0.3% divinyl imines (BEI) added 0.02% Sulfothiorine and stops deactivation after 96 hours.
In the present invention, preferably, described pig parvoviral inactivated vaccine is by 15%(v/v) commercialization MontanideTM ISA15AVG adjuvant and the 85%(v/v of France match BIC Corp) the pig parvoviral with divinyl imines (BEI) deactivation (BQ-C strain) emulsification after obtain.
In the present invention, emulsifying process is the commercialization MontanideTMISA15AVG adjuvant that adopts France match BIC Corp, can be directly used in vaccine emulsification preparation after the degerming; Before the emulsification mixing is frozen in same batch of viral lyolysis of deactivation, stir with 200r/min at aseptic indoor refiner, then according to water antigen and 8.5: 1.5 volume proportion of oil phase adjuvant; The emulsification program stirred for earlier water being put into emulsor, slowly adds the oil phase adjuvant then, with 800r/min continuously stirring 30 minutes; The vaccine that production obtains belongs to the oil-in-water formulation, for stable interface and elimination bubble, obtains the optimum emulsification effect, needs packing after static 30 minutes.
Further, the present invention also provides the purposes of described inactivated vaccine in the biological products of the disease that the preparation prevention is caused by pig parvoviral.
Especially, described disease is the sow breeding difficulty that the healthy negative sow of primiparity is taken place.
Description of drawings
Fig. 1 is the fluoroscopic examination result of pig parvoviral BQ-C strain after the ST cell is cultivated different time;
Figure 1A is 0 hour fluoroscopic examination result of inoculation; Figure 1B is 8 hours fluoroscopic examination result of inoculation; Fig. 1 C is 16 hours fluoroscopic examination result of inoculation; Fig. 1 D is 24 hours fluoroscopic examination result of inoculation; Fig. 1 E is 32 hours fluoroscopic examination result of inoculation; Fig. 1 F is 40 hours fluoroscopic examination result of inoculation; Fig. 1 G is 48 hours fluoroscopic examination result of inoculation; Fig. 1 H is 56 hours fluoroscopic examination result of inoculation; Fig. 1 I is the fluoroscopic examination result of normal control cell.
Fig. 2 is the one step growth after the ST cell is infected in pig parvoviral BQ-C strain.
Embodiment
Also the present invention will be further described in conjunction with the embodiments below by experiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Separation and the culture identification of embodiment 1 pig parvoviral BQ-C strain
1 seed culture of viruses source and standard
According to the new biological product requirements of customs declaration, in conjunction with a large amount of testing data that the present invention obtains, with reference to " Chinese veterinary drug allusion quotation " (version in 2005), seedling is identified with seed culture of viruses, now try the middle seed culture of viruses standard of rules (draft) and carry out following explanation.
1.1 seed culture of viruses source
The seed culture of viruses that manufacturing this product is used is pig parvoviral BQ-C strain, be that the inventor obtained after the morbidity pig only separates domestication voluntarily in 2005, because this strain is to produce to separate the weak son from the pregnant sow that typical pig parvoviral infection takes place to obtain, and compare the propagation titre height on cell with other strain that is separated to, immunogenicity to pig is good, so select it to be used for the production of vaccine.As the vaccine seed culture of viruses, imitating inspection is the 7th～10 generation of BQ-C strain with strong poison after pig testis continuous cell line (ST) goes down to posterity.This seed culture of viruses for after the ST cell goes down to posterity the 7th generation virus strain, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCCNo.6091, preservation date are on May 8th, 2012.
1.2 seed culture of viruses (CGMCC No.6091) standard
1.2.1 red cell agglutination valency
Get the U-shaped micro-reaction plate in 96 holes, every hole adds 25 μ l PBS(0.01mol/L, pH value 7.0～7.4), add 25 μ l antigens in first each hole of row, then antigen is carried out 2 times of serial dilutions, discard 25 μ l until the 11st hole.Every hole adds 25 μ l PBS, adds 0.6% guinea-pig red blood cell suspension, 25 μ l again, mixes with micro oscillator, puts under the room temperature and acts on 1 hour.During result of determination, with the highly diluted multiple of the antigen of 50% red cell agglutination as judging terminal point.The result: seed culture of viruses should be not less than 1:512 to the guinea-pig red blood cell agglutination titer.
1.2.2 viral level
With the DMEM nutrient solution seed culture of viruses is made continuous 10 times of serial dilutions, each extent of dilution is got 100 μ l and is added in the 96 porocyte culture plates, and each extent of dilution is done 8 repetitions, adds the ST cell suspension that digestion disperses subsequently, and every hole 100 μ l(cell contents are with 3 * 10
5/ mL), and establish normal cell and cultivate contrast, put 37 ℃, contain 5% CO
2Cultivate in the incubator, observation of cell pathology (CPE) day by day, the cell pyknosis is assembled, and particle increases, and the cytogamy that has, comes off, occurs more space and then be judged to CPE.Observed 7, and recorded cytopathic hole count, calculate the TCID of virus according to the Reed-Muench method
50The result: every milliliter of viral liquid is not less than 10
6.0TCID
50
1.2.3 virulence
The virus generation is pig parvoviral BQ-C strain the 7th～10 generation cell toxicant; Attacking the toxic agent amount is that every milliliter of viral level should be not less than 10
6.0TCID
50Program is 4 of healthy negative first farrowing sows, and gestation is difference intramuscular injection pig parvoviral BQ-C strain the 7th～10 generation cell toxicant 2ml, collunarium 2ml after 35 days, and viral level is respectively 3 * 10
6.0TCID
50Criterion is to attack poison to cut open in back 40 days extremely, and loss tire pig perhaps has 3/5 sow that breeding difficulty (stillborn foetus, mummy tire and molten tire) takes place more than 25%.
1.2.4 immunogenicity
Seed culture of viruses is inoculated the ST cell synchronously breed, gather in the crops viral liquid, after the BEI deactivation, add Montanide
TMISA 15AVG adjuvant mixing and emulsifying is made the oil-in-water-type inactivated vaccine, with 5 of healthy susceptible first farrowing sows (PPV HI antibody titer is not higher than 1:8), intramuscular injection is by these rules seedling 2ml before the breeding, after gestation 35～40 days, together with 5 of the identical contrast pigs of condition, collunarium and through each 2ml(10 of intramuscular injection pig parvoviral BQ-C strain
6.0TCID
50), attack poison and cutd open all pigs extremely in back 40 days.Control group should at least 4 breeding difficulty symptoms such as stillborn foetus, mummy tire or fetus heavily absorb occur, and immune group should at least 4 head protections.
1.2.5 pure check
The primordial seed of the pig parvoviral BQ-C strain of setting up is criticized the 10th～11 generation, the 12nd～21 generation of basic seed lot and seeding to be criticized for the 22nd～25 generation and has carried out bacterium, mould, mycoplasma check, and the 11st, 12 and 24 generation seeds culture of viruses have been carried out the check of exogenous virus, the result shows, the primordial seed that the present invention sets up is criticized, basic seed lot, seeding is criticized does not all have bacterium, mould, mycoplasma and exogenous virus pollute, and is all pure.
1.2.6 specificity
With DMEM liquid seed culture of viruses is diluted to 200TCID
50/ 0.1ml with the anti-pig parvoviral specific serum equivalent mixing of deactivation, puts in 37 ℃ and 1 hour, and 3 bottles (cell content is with 3 * 10 to inoculate well-grown ST cell synchronously
5/ ml), establish virus control, normal cell contrast and negative serum contrast simultaneously, put 37 ℃, contain 5% CO
2In the incubator, cultivated 5～7.Cytopathy (CPE) all should not appear in neutralization virus group and normal cell control group; CPE all should appear in virus control group and negative serum control group.
1.2.7 seed culture of viruses uses generation
The pig parvoviral BQ-C strain that original separation is obtained passing 40 generations (namely being numbered on the bacterial strain basis of CGMCC No.6091 33 generations of continuous passage in culture presevation) on the ST cell continuously, has been measured each TCID for virus
50, select the 15th, 20,25,30,35,40 generation virus make vaccine after the deactivation respectively, inoculate 5 the monthly age replacement gilt, gestation is attacked poison 35 days the time.The result shows, virus goes down to posterity at the ST cell, after the 7th generation (comprise the 7th generation virus, i.e. culture presevation is numbered the virus strain of CGMCC No.6091) each generation viral level is stable, all is not less than 10
6.0TCID
50, and HA tires all 2
9More than, reached the standard of making vaccine.Challenge test shows that the vaccine immunity pig of the 15th, 20,25,30 generations virus preparation is not having significant difference to the protectiveness of BQ-C strain attack, and fetus is healthy survival all; Attacking poison behind the vaccine immunity of the 35th and 40 generations virus preparation respectively has 2 and 1 stillborn foetus, and 4 pigs of control group all have in various degree stillborn foetus and weak tire.According to above result, produce for guaranteeing good immunogenicity and the security of seed culture of viruses, the present invention has determined the high reps of going down to posterity of virus in 27 generations, and original seed culture of viruses was 7～11 generations, the basis seed culture of viruses was the 12nd～21 generation, produced to be limited in 3 generations (the 22nd～25 generation) with the highest passage number of seed culture of viruses.
1.2.8 determining of seed culture of viruses preservation period
The wet poison of pig parvoviral BQ-C strain is kept under-20 ℃ and-70 ℃, carries out in preserving back different time sampling that HA tires and TCID
50Measure, the result show wet poison in-20 ℃ frozen 18 months, the poison valency begins obvious decline when preserving 30 months for-70 ℃; The freeze-drying poison is kept under-20 ℃ and-70 ℃, carries out in preserving the annual sampling in back that HA tires and TCID
50Measure.The result slightly descends-20 ℃ of tiring of virus of preserving down 36 months, and the HA that preserves 48 months virus tires and TCID
50Value obviously descends, and tires and TCID at-70 ℃ of HA that preserve 48 months virus down
50Do not have obvious variation, preserve decline in 60 months obviously.Consider other unfavorable factors that when virus is preserved, exist, guarantee viral preservation period reliability, so the wet malicious preservation period at-20 ℃ of pig parvoviral BQ-C strain is decided to be 12 months, be decided to be 24 months-70 ℃ preservation perives; Pig parvoviral freeze-drying poison is decided to be 36 months-20 ℃ shelf time; The freeze-drying poison is 48 months-70 ℃ shelf time.
2 production cells
2.1 cell source
The ST cell available from China (Wuhan) typical culture collection center, is preserved by Harbin Veterinary Medicine Inst., China Academy of Agriculture.
2.2 the selection of cell
The present invention has selected for use continuous cell line PK-15 and ST to carry out the propagation of virus in the culturing process of virus, the pathology time of virus on the ST cell early as a result, the propagation titre is the highest, the increment rule of pig parvoviral BQ-C strain (CGMCC No.6091) on the ST cell as shown in Figure 1, pig parvoviral BQ-C strain (CGMCC No.6091) infect behind the ST cell one step growth as shown in Figure 2.It is better that the foreign scholar also has report to select the ST cell to carry out the breeding ratio of PPV, so selected the ST cell to carry out the propagation of virus.
2.3 the foundation of clone
Seedling is selected the ST cell for use with clone.The scope that goes down to posterity control is in 40～55 generations, and the present invention has set up master cell bank, basic cell bank and working cardial cell storehouse.Wherein master cell bank is divided into and adorns 55 bottles; 370 bottles of basis cell bank packing; The working cardial cell storehouse is divided into has adorned 350 bottles.Cell to each generation is all pressed " Chinese veterinary drug allusion quotation " appendix and is tested, and should not have the pollution of bacterium, mould, mycoplasma and exogenous virus.
2.4 the evaluation of clone
Test according to relevant in existing " Chinese veterinary drug allusion quotation " attached sheet " producing, check the cell standard of using " regulation, all conformance with standard.Specifically see " certified test report of ST clone ".
The preparation of embodiment 2 inactivated vaccines
Preparation technology's flow process of pig parvoviral inactivated vaccine (BQ-C strain) is according to the commercialized vaccine preparation technology of maturation preparation at present.From 5 crowdes of vaccine quality inspection results of the 3 batches of vaccines and the pilot-scale production of prepared in laboratory, with the vaccine product steady quality of this technical process preparation, imitate the inspection result and meet the quality standard of formulating fully.
The preparation of 1 viral liquid
In the viral proliferation process, several factors can influence the propagation of cell and virus.PPV copies the archaeal dna polymerase that needs host cell, and optimum has vigorous multiplication capacity and is being in the mitotic division cell internal breeding in period, therefore adopts when carrying out the PPV breeding and cell inoculation culture simultaneously; Copying of virus in the serum-concentration remarkably influenced in the nutrient solution, and through a series of comparison tests, selecting the cell culture fluid serum-concentration for use is 10%, and keeping the liquid serum-concentration is 2%, can breed stable, the viral liquid of high titre; In addition to the pH value of nutrient solution, cell concn, virus inoculation dosage, receive poison time etc. and compare test, cell maintenance medium pH value control cell concn selection 1 * 10 between 7.2～7.4, when connecing poison when the result shows viral proliferation
5Individual/ml～5 * 10
5It is 1%(v/v that individual/ml, cell connects the toxic agent amount) viral level is 10
6.0TCID
50Above kind poison, the viral best harvest time of/ml is 72～96 hours, all can obtain the viral liquid of high viral level.
(trade(brand)name is 1640 to select cell culture fluid in the present embodiment for use; available from Gibco company) in serum-concentration be that 10%, pH value is controlled 7.3, (trade(brand)name is MEM to keep liquid; available from Shanghai Hu Feng bio tech ltd) in serum-concentration be 2%, the ST cell concn is 3 * 10 when connecing poison
5Individual/ml, according to 1%(v/v) cell connect the toxic agent amount to insert viral level be 10
6.0TCID
50Pig parvoviral BQ-C strain the 7th generation virus strain (culture presevation is numbered CGMCC No.6091) of/ml, viral harvest time are 72 hours.
2 inactivation technologies
Selecting final concentration is that 0.1%, 0.2%, 0.3%, 0.4% formaldehyde solution and final concentration are that 0.1%, 0.2%, 0.3%, 0.4% divinyl imines (BEI) carries out the deactivation comparison test to pig parvoviral, test-results shows, 0.3% BEI can be with the pig parvoviral complete inactivation after under 32 ℃ of conditions 96 hours, and test-results shows that the cells injury of BEI is less; PPV antigen adds 0.3% formaldehyde solution, also can reach the purpose of complete inactivation virus in 48 hours through 37 ℃ of deactivations; But formaldehyde has strong and stimulating, if residual free formaldehyde enters animal body in the vaccine, can produce untoward reaction.Thereby final definite BEI deactivation pig parvoviral under 32 ℃ of conditions with 0.3% added 0.02% Sulfothiorine and stops deactivation after 96 hours.
3 emulsifying process
Adopt the commercialization Montanide of France match BIC Corp
TMThe ISA15AVG adjuvant can be directly used in vaccine emulsification preparation after the degerming; Before the emulsification mixing is frozen in same batch of viral lyolysis of deactivation, stir with 200r/min at aseptic indoor refiner, then according to water antigen and 8.5: 1.5 volume proportion of oil phase adjuvant; The emulsification program stirred for earlier water being put into emulsor, slowly adds the oil phase adjuvant then, with 800r/min continuously stirring 30 minutes; The vaccine that production obtains belongs to the oil-in-water formulation, for stable interface and elimination bubble, obtains the optimum emulsification effect, needs packing after static 30 minutes.
4 inspection of semifinished product quality standards
4.1 steriling test
Test by existing " Chinese veterinary drug allusion quotation " appendix, answer asepsis growth.
4.2 viral level is measured
With the DMEM nutrient solution seed culture of viruses is made continuous 10 times of serial dilutions, each extent of dilution is got 100 μ l and is added in the 96 porocyte culture plates, and each extent of dilution is done 8 repetitions, adds the ST cell suspension that digestion disperses subsequently, and every hole 100 μ l(cell contents are with 3 * 10
5Be advisable about/ml), and establish normal cell and cultivate contrast, put 37 ℃, contain 5% CO
2Cultivate in the incubator, observation of cell pathology (CPE) day by day, the cell pyknosis is assembled, and particle increases, and the cytogamy that has, comes off, occurs more space and then be judged to infection.Record cytopathic hole count, calculate the TCID of virus according to the Reed-Muench method
50Every milliliter of viral liquid of result is not less than 10
6.0TCID
50
4.3 deactivation check
Get the viral liquid after the deactivation, 3 bottles (cell content is with 3 * 10 to be inoculated in the ST cell in viral liquid and 1: 10 ratio of growth media
5/ ml), cultivate to observe 5 for 37 ℃, to no pathology person 2 generations of blind passage again, establish virus control simultaneously.The acellular pathology of result.3 batches of work in-process of Laboratory Production are all qualified.
5 about the inspection after construction quality standard
5.1 safety verification standard
In order to guarantee the security of vaccine, the safety testing that the present invention has successively carried out " to the safety testing of small white mouse ", " to the safety testing of the inoculation of piglet single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation ", " to the safety testing of the inoculation of replacement gilt single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation ", the inoculation of pregnant sow single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) are inoculated 5 batches of vaccines of prepared in laboratory ".Test-results shows: after each immune animal single dose inoculation, single dose repeated inoculation, the disposable heavy dose of inoculation, a pig state is all good, and the drinking-water of searching for food is normal, no abnormal reaction; Breeding function to pregnant sow after the vaccine inoculation does not have obvious influence yet.More than test proves that all vaccine is safe.Sum up as long as do not occur phenomenons such as local redness, ulcer, erosion and neuratorphy, the minimizing of searching for food after the vaccination in 21 days by experimental observation the present invention, untoward reaction with regard to not occurring causing because of vaccine inoculation just can prove the security of vaccine later on.Therefore, regulation in tentative rules (draft): with healthy susceptible piglet (the not high 1:8 of HI antibody titer) 2 of 10～20kg, each deep intramuscular injection vaccine 4ml, other gets 2 identical pigs of condition and does not inoculate in contrast, observes continuously 21.Any part or the systemic adverse reactions that are caused by vaccination do not appear the observation period planted agent.
5.2 the formulation of efficacy test standard
5.2.1 poison protection correlation test is attacked in antibody titer and immunity
1 batch of pig parvoviral inactivated vaccine 0701 that safety testing is qualified, according to various dose 0.5ml/ head part, 1ml/ head part, 2ml/ head part, the healthy replacement gilt (PPV HI antibody is not higher than 8) at respectively immune 3～5 monthly ages of 4ml/ head part, and with 10
6.0TCID
50/ ml PPV BQ-C strain strong malicious 4ml of the 10th generation attacks, and attacks the dependency of poison protection to determine HI antibody titers and immunity.Test-results shows, when serum HI antibody titer when being not less than 1:64, the sows farrowing surviving rate reaches 97.5%, wherein a porkling miscarriage is arranged in one group, is 0 but PCR detects piglet virus separation rate, does not also have the pathogen infection that other cause the sow miscarriage.Above result shows under test conditions, when serum HI antibody titer when being not less than 1:64, can effectively stop virus vertically to pass to fetus through parent.Thereby during the check of definite vaccine potency, after inspecting standard be immune 28 days, its serum HI antibody titer was not less than 1:64.
5.2.2 minimum immune dosage and the test of minimum using dosage
3 batches of pig parvoviral inactivated vaccines 0701,0702 and 0703 that safety testing is qualified, according to various dose 0.5ml/ head part, 1ml/ head part, 1.5ml/ head part, the healthy replacement gilt (PPV HI antibody is not higher than 8) at respectively immune 3～5 monthly ages of 2ml/ head part, carry out the HI antibody test in immunity blood sampling in back 28 days.The result shows, with 1ml/ doses immune swine after 28 days HI antibody titer mean value be not less than 1:64, and the above dosage immune swine of 1.5ml/ head part after 28 days the HI antibody titer all be not less than 1:64.According to the HI antibody titer with attack poison protection correlation test result, determine to reach 1:64 when above at back 28 days pig parvoviral HI antibody titers of immunity, can protect the attack of the strong poison of farrowing sow opposing pig parvoviral fully.Consider the reduction factor of tiring that may exist in preservation, transportation and the use of vaccine.The present invention is defined as 1.5ml/ head part with the minimum immune dosage of pig parvoviral inactivated vaccine (BQ-C strain), and using dosage is defined as 2.0ml/ head part.
5.2.3 immune guinea pig and immune swine HI antibody correlation test
3 batches of pig parvoviral inactivated vaccines 0701,0702 and 0703 difference immune guinea pig and pig with Laboratory Production carry out the HI antibody titer and detect, and analyze the HI antibody dependency of cavy and pig.The result shows that when 0.5ml/ of cavy immunity, 2ml/ of pig immunity, its serum HI antibody titer all is not less than 1:64 after 28 days, and the two has tangible parallel relation.The test-results proof can replace target animals (pig) to carry out efficacy test with the laboratory animal cavy.
Therefore, according to above result, the present invention is decided to be the efficacy test standard of this product:
With 5 of the adult cavys (PPV HI antibody titer is not higher than 1:8) of body weight 350g～400g, each intramuscular injection vaccine 0.5ml, after 28 days, together with 4 of contrast cavys, PPV HI antibody titer is measured in blood sampling in the lump.Contrast cavy HI antibody titer should not be higher than 1:8, and immune guinea pig should have 4 at least and antibody response occur, and the HI antibody titer should be not less than 1:64.
With healthy susceptible pig (PPV HI antibody titer is not higher than 1:8) 5 at 3 monthly ages, each deep intramuscular injection vaccine 2ml, after 28 days, together with 4 of contrast pigs, PPV HI antibody titer is measured in blood sampling in the lump.Contrast pig HI antibody titer should not be higher than 8, annotate the seedling pig and antibody response should all occur, and the HI antibody titer should be not less than 1:64.
6 antibody growth and decline rules and immune duration test
3 batches of pig parvoviral inactivated vaccines 0701 that safety testing is qualified, 0702 and 0703 are immune health replacement gilt, multiparity pig and boar respectively.For further grasping immune efficacy and immune duration, formulate rational immune programme for children, guarantee that immune swinery keeps high and lasting antibody horizontal carries out antibody test to the immune swine different time, to grasp its antibody growth and decline rule.
Test-results shows; 3～5 monthly ages first farrowing sow immunity 2ml/ head part; booster immunization once after 2～3 weeks; the multiparity sow is in previous month immune 2ml/ head part of breeding, and its serum HI antibody is not less than 1:64 after 28 days, and antibody peak, immunity back is in 2～6 months; thereafter antibody horizontal descends gradually; still reached 1:64 to 9 months, for guaranteeing the immune protective effect of vaccine, determine that its immune duration is 7 months.
Determining of 7 immune programme for children
According to the time length of piglet maternal antibody, initial immunity should be at 5～6 monthly ages, because at this moment maternal antibody has dropped to lower level (the HI antibody titer is lower than 1:64).And the replacement gilt breeding time was generally for 6 monthly ages, suffered strong virus attack when immunity also just in time can be avoided sow in gestation when 5～6 monthly ages like this.For the lasting existence of multiparity sow owing to its internal antibody; pig parvoviral does not often constitute a threat to it; so formerly generally do not carry out immunity; but in recent years because the complicacy of clinical onset; tend to infection that increases the weight of pig circular ring virus etc. as pig parvoviral; so the present invention advocates the multiparity sow is carried out immunity before gestation, can protect pregnant sow to avoid the attack of the strong poison of pig parvoviral in the whole Gestation period like this.
Breeding time, the antibody growth and decline rule after the replacement gilt immunity, duration of immunity according to replacement gilt, combination simultaneously is the practical situation of pig parvoviral infection clinically, the present invention has determined that the immune programme for children of pig parvoviral inactivated vaccine (BQ-C strain) is: 5～6 monthly ages of first farrowing sow immunity 1 time, and booster immunization is 1 time after 2～4 weeks; The multiparity sow is in preceding 3～4 week immunity of breeding 1 time; Twice of the annual immunity of boar.Each immunity is the 2ml/ head.
The preservation period of 8 vaccines
The present invention has carried out experimental study to 3 batches of inactivated vaccines (0701,0702,0703) preservation period of prepared in laboratory, 3 batches of vaccines were preserved 9,12,15,18 months under 2～8 ℃ of conditions, taken a sample respectively in each time period its proterties, security and immune efficacy are detected.The result shows that 3 batches of vaccines are preserved 18 months proterties considerable change is not taken place under 2～8 ℃ of conditions, and steriling test and security all meet the requirement of quality standard.The 0701 batch of vaccine preserves that a cavy HI antibody titer to be arranged in 18 months behind the sample immune guinea pig be 1:32 in the efficacy test; 0701 and 0702 batch of vaccine preservation all had a pig HI antibody titer in 18 months behind the sample immune swine be 1:32, but the average HI antibody horizontal of immune swine is 1:64, consider vaccine loss of tiring of causing in transportation and use, the present invention was decided to be under 2～8 ℃ of conditions preservation 15 months with the preservation period of vaccine.
9 with the immune efficacy of domestic similar commercial seedling relatively
20071228) and the B(lot number with 3 batches of vaccines 0701 of laboratory trial-production, 0702 and 0703 and commercially available vaccine A(lot number:: 20071205); distinguish healthy replacement gilt of immune 350g～400g healthy guinea pig and 5～6 monthly ages; its serum HI antibody titer all is not less than 1:64 after 28 days; reach and attack poison protection requirement; the vaccine HI antibody mean value of prepared in laboratory is a little more than commercially available A vaccine, apparently higher than commercially available B vaccine.
Respectively before immunity, 3,6,9,12 months venous blood collection separation of serum in immunity back, detection antibody production.The result shows: in back 6 months of immunity, and vaccine and commercially available A and the B vaccine immunity pig of laboratory trial-production, the HI antibody titer all more than 1:64, all can reach and attack poison protection requirement.And commercially available B vaccine immunity pig HI antibody descends obviously after 6 months.
The above only is the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and can carry out many changes to it in the spirit and scope that claim of the present invention limits, revise, even the equivalence change, but all will fall within the scope of protection of the present invention.
Claims (6)
1. pig parvoviral (Porcine parvovirus), called after pig parvoviral BQ-C strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC No.6091.
2. the application of the described pig parvoviral of claim 1 in preparation pig parvoviral inactivated vaccine.
3. pig parvoviral inactivated vaccine is characterized in that containing the described pig parvoviral of claim 1 after the deactivation.
4. inactivated vaccine as claimed in claim 3 is characterized in that described deactivation refers to divinyl imines deactivation under 32 ℃ of conditions of the described pig parvoviral employing 0.3% of claim 1 was added 0.02% Sulfothiorine and stops deactivation after 96 hours.
5. claim 3 or the 4 described inactivated vaccines application in the biological products of the disease that the preparation prevention is caused by pig parvoviral.
6. application as claimed in claim 5 is characterized in that described disease is the sow breeding difficulty that the healthy negative sow of primiparity is taken place.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311690A (en) * | 1998-07-06 | 2001-09-05 | 梅瑞尔公司 | Porcine circovirus and parvovirus vaccine |
| CN1704119A (en) * | 2004-05-28 | 2005-12-07 | 上海佳牧生物制品有限公司 | Oil emulsion inactivated vaccine for parvoviral diseases of pigs |
| CN101380470B (en) * | 2008-10-15 | 2011-06-08 | 北京中海生物科技有限公司 | Pig parvovirus live vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311690A (en) * | 1998-07-06 | 2001-09-05 | 梅瑞尔公司 | Porcine circovirus and parvovirus vaccine |
| CN1704119A (en) * | 2004-05-28 | 2005-12-07 | 上海佳牧生物制品有限公司 | Oil emulsion inactivated vaccine for parvoviral diseases of pigs |
| CN101380470B (en) * | 2008-10-15 | 2011-06-08 | 北京中海生物科技有限公司 | Pig parvovirus live vaccine |
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