CN101851609B - Porcine parvovirus L strain and use thereof in preparation of porcine parvovirus inactivated vaccines - Google Patents
Porcine parvovirus L strain and use thereof in preparation of porcine parvovirus inactivated vaccines Download PDFInfo
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- CN101851609B CN101851609B CN201010110218A CN201010110218A CN101851609B CN 101851609 B CN101851609 B CN 101851609B CN 201010110218 A CN201010110218 A CN 201010110218A CN 201010110218 A CN201010110218 A CN 201010110218A CN 101851609 B CN101851609 B CN 101851609B
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Abstract
The invention discloses a porcine parvovirus L strain and use thereof in preparation of porcine parvovirus inactivated vaccines. In the invention, microbial preservation number of the separated strain is CGMCC No. 3352. The strain is highly homologous with an NADL-2 strain and a China strain and keeps high reproduction, and the viruses are stable. Oil adjuvant is added into antigen liquid which inactivates the viruses by using binary ethylenimine to prepare the bidirectional oil-emulsion inactivated vaccines; and the prepared inactivated vaccines are applied in a vaccine inoculation experiment of 10,276 pigs (most of which are first farrowing sows), and in the experiment, an immune dosage of 2 millimeters is given to each pig through muscle injection, and each time of inactivated vaccine inoculation can obtain an immune period of over 6 months. Under the conditions that a 10 millimeter overdose vaccine is injected into a pig and that single dose inoculation is repeatedly carried out for three times, no abnormal reaction happens, which proves that the inactivated vaccines have high immunogenicity and safety.
Description
Technical field
The present invention relates to the new isolating virus stain of a strain, relate in particular to strain pig parvoviral poison L strain, the invention still further relates to the purposes of viral L strain in the preparation porcine parvovirus inactivated vaccines, belong to the prevention and control field of porcine parvovirus.
Background technology
Pig parvoviral (PPV) can cause the breeding difficulty of pig, mainly show as fetus and embryo's infection and death, and parent does not show clinical symptom usually.The negative sow of serology mainly first half of gestation through the mouth and nose infective virus, the immunoincompetent fetus of result is infected through placenta, thereby causes morbidity.Learn that on inspection in swinery all over the world, this virus is ubiquitous, it is popular to be region on most of pig farms.This is sick popular very wide, in Europe, all there is report in U.S.A, a lot of countries in Asia and Oceania.The each province and city of China are separated to pig parvoviral in succession, and seroprevalence is very high.This disease is very harmful to pig industry.Because PPV does not still have effective drug treatment at present, therefore, this sick epidemic prevention just seems even more important.
In recent years, generally acknowledge that using vaccine is the unique method of prevention PPV, raising sow breeding potential.Up to the present; More than 10 national reports such as the existing in the world U.S., Australia, Japan, Denmark, Spain, Czech, Germany, the former Soviet Union and China have been developed the PPV vaccine; Wherein have 3 less toxic vaccines except that the U.S. and Japan; All the other are inactivated vaccine, and have all become commercialization production seedling, and inactivator commonly used has AEI, BEI, formaldehyde solution and beta-propiolactone.The Mengeling of the U.S. (1979) has also reported production application a kind of PPV and pseudorabies (PrV) bigeminy seedling, effect is better.
China all generally uses the PPV inactivated vaccine, and the main inactivator of use has: Superlysoform, B2 propiolactone (B2PL), N-acetylethylenimine (AEI), divinyl imines (BEI) etc.Have test-results to show, the protection effect of the vaccine that PPV makes after with the Superlysoform deactivation is not as effective with the protection of the vaccine of making after the deactivation of divinyl imines; With the protection of the vaccine of B-propiolactone deactivation better (Pan Xuezhu etc., 1992).Immunological adjuvant also is the important factor that influences PPV inactivated vaccine immune effect.Have test-results to show, 50% white lake, maleic acetyl (EMA), profit emulsion, DDA all induce pig to produce high titer antibody respectively as the vaccine of adjuvant.But the mixture of several kinds of adjuvants is made the antibody titers all lower (Mo lito r etc., 1985) that the vaccine of adjuvant produces like the mixture of the mixture of oily adjuvant, SDS, white lake and oil-emulsion and SDS, white lake.At present domestic have two kinds of pig parvoviral inactivated vaccines to supply this disease of prevention.A kind of is pig parvoviral deactivation white lake vaccine, and another kind is a pig parvoviral inactivated oil adjuvant seedling.Use the deactivation of divinyl imines for pig parvoviral, and add the formulated two-way oil-emulsion inactivated vaccine of oily adjuvant, also do not report.
Summary of the invention
One of the object of the invention provides the new isolating pig parvoviral strain of a strain;
Two of the object of the invention is that above-mentioned pig parvoviral strain is used to prepare the two-way oil emulsion inactivated vaccine of porcine parvovirus;
Three of the object of the invention provides a kind of method for preparing the two-way oil emulsion inactivated vaccine of above-mentioned porcine parvovirus;
Above-mentioned purpose of the present invention realizes through following technical scheme:
The new isolating pig parvoviral of one strain (PPV) L strain, its microbial preservation number are: CGMCC No.3352; The classification name is: pig parvoviral; The preservation time is: on October 23rd, 2009; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The separation method of pig parvoviral of the present invention (PPV) L strain comprises:
Aseptic collection is got tissues such as the liver, mesenteric lymph nodes, kidney, brain of fetal death of sow.Add sterile saline with 1: 10, grind, the preparation tissue suspension, after 3 freeze thawing repeatedly, the centrifugal 15min of 2000r/min collects supernatant, filters through 0.2ul filter membrane filter again, filtrate is put-30 ℃ of refrigerators and freezes preservation.Get on the isolating strain inoculation ST passage cell, at CO
2Cultivate under 37 ℃ of conditions, pair cell pathology effect every day (CPE) is observed.Form result shows: the cell pyknosis, and prominent upright the gathering, particle increases, the cytogamy that has, plaque appears in cell detachment.
Porcine parvovirus L strain negative staining electron microscope observations of the present invention: can be observed outward appearance and be sexangle or circle, the virus particle of no cyst membrane, diameter is about 20nm.
Porcine parvovirus L strain immunofluorescence technique check result of the present invention: sample infects the ST passage cell, and cell has pathology, in nucleus tenuigenin, and the visible green specificity fluorescent of apple that is, negative control does not have specificity fluorescent and occurs.
Viral hemoagglutination is active to be detected: gather in the crops toxic cell cultures fluid power aggegation GPRBC, have higher hemagglutination activity, and along with the increase of the incubation times that goes down to posterity, the flexibility of viral pair cell strengthens, the viral hemoagglutination valency has raising.
The sequencing result of porcine parvovirus L strain of the present invention: PPV VP2 is checked order, and sequencing result is seen accompanying drawing 4, utilizes DNA MAN software that L strain VP2 gene fragment is carried out sequential analysis, and the evolutionary tree of L strain and other a few strain PPV VP2 is analyzed, and sees attached list 1 and accompanying drawing 5.The genetic distance that shows L strain and NADL-2 strain and China strain is nearest.
With the deactivation of above-mentioned pig parvoviral strain, add adjuvant, just can obtain porcine parvovirus inactivated vaccines;
Preferably, a kind ofly utilize parvovirus strain of the present invention to prepare the method for the two-way oil emulsion inactivated vaccine of porcine parvovirus, comprising: cultivate described porcine parvovirus L strain, obtain the pig parvoviral antigen liquid; With the deactivation of pig parvoviral antigen liquid; Concentrate; Prepare oil phase and water respectively, water and oil phase are mixed back emulsification, promptly get.
The concentration of divinyl imines and action time the effect of deactivation vaccine there is the influence of highly significant; Mirror; The present invention selects for use the divinyl imines as inactivator; The using dosage of inactivator, action time, inactivating efficacy etc. are carried out a series of tests, to optimize divinyl imines deactivation pig parvoviral program.The final discovery with end level 0.02% divinyl imide liquor deactivation pig parvoviral effect to well; When with end level 0.02% divinyl imide liquor at 30 ℃, 100r/min, under the condition of gas bath vibration, the effect of 72h deactivation pig parvoviral be the best.
Wherein, said oil phase can prepare with reference to following method: 94 parts of injection white oils, Si Ben-806 part and StAl mix for 1.5 parts, and preparation obtains oil phase;
Said water can prepare with reference to following method: add the 4ml tween-80 in the pig parvoviral antigen liquid after the 96ml deactivation, adding Thiomersalate to final concentration is 0.01wt%; Preparation obtains water;
Preferably, said water and oil phase are carried out emulsification according to 4: 1.5 mixed together.
Inactivated vaccine immunizing dose of the present invention is a pig muscle injection 2ml/ head, once inoculates inactivated vaccine, and the terrain duration of immunity can reach more than 6 months.Overdose inoculation 10ml/ head behind 3 repeated inoculations of single dose, is not all found any abnormal response, explains that the prepared inactivated vaccine of the present invention has good immunogenicity and security.
Description of drawings
Fig. 1 cytopathic effect (CPE) result.
The electron microscopic examination result of Fig. 2 virus.
Fig. 3 immunofluorescence technique check result.
The process flow sheet of Fig. 4 pig parvoviral strain of the present invention inactivated vaccine.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.Separation, cultivation, the evaluation of embodiment 1 porcine parvovirus L strain
One, selection
1. pathological material of disease collection: aseptic collection is got tissues such as the liver, mesenteric lymph nodes, kidney, brain of fetal death of sow.Add sterile saline with 1: 10, grind, the preparation tissue suspension, after 3 freeze thawing repeatedly, the centrifugal 15min of 2000r/min collects supernatant, filters through 0.2ul filter membrane filter again, filtrate is put-30 ℃ of refrigerators and freezes preservation.
2. separation and Culture: above-mentioned pathological material of disease inserted by 10% content of virus-culturing fluid do not form as yet in the cell culture of individual layer, 1h is made in 37 ℃ of senses, change add contain 2% read Ox blood serum the MEM nutrient solution, cultivated 5 for 37 ℃.The s-generation is cultivated 78h, gathers in the crops toxic nutrient solution, after 3 freeze thawing, receives poison.5 generations of continuous passage.
3. basic seed lot is set up: get original seed culture of viruses, insert by 0.5% of virus culture liquid measure and do not form as yet in the ST cell culture of individual layer, put 37 ℃ of cultivations, when pathology reaches 70%, gather in the crops toxic cell culture fluid, through 3 freeze thawing after, receive malicious.6 generations of continuous passage.
4. seeding is criticized foundation: get basic seed, insert by 0.5% of virus culture liquid measure and do not form as yet in the ST cell culture of individual layer, put 37 ℃ of cultivations, when pathology reaches 70%, gather in the crops toxic cell culture fluid, through 3 freeze thawing after, receive malicious.11 generations of continuous passage.
Final acquisition one strain has the required pig parvoviral strain of expected characteristics, and called after and called after porcine parvovirus L strain;
Get on the isolating strain inoculation ST passage cell, at CO
2Cultivate under 37 ℃ of conditions, pair cell pathology effect every day (CPE) is observed.The result shows: the cell pyknosis, and prominent upright the gathering, particle increases, the cytogamy that has, plaque appears in cell detachment.Pathology is seen Fig. 1.
Two, negative staining electron microscope is observed
After cultivating 5-7d, sick cell supernatant sucrose gradient ultracentrifugation, separating viral particles carry out negative staining electron microscope to be observed.
The result shows: can be observed outward appearance and is sexangle or circle, and the virus particle of no cyst membrane, diameter is about 20nm.See Fig. 2
Three, immunofluorescence technique inspection
Get on the isolating strain inoculation ST passage cell, at CO
2Cultivate 47h under 37 ℃ of conditions, when CPE reaches 70%, pass the s-generation, select for use s-generation culture after 3 freeze thawing, do the fluorescence antibody inspection.Get the ST cell deckglass culture of cultivating 24h, virus inoculation is cultivated through 24h again.Take out deckglass, with the PBS flushing, through acetone fixed, again with dyeing 30min in the wet box of pig parvoviral fluorescence antibody, flushing, sealing, microscopy.
The result shows: sample infects the ST passage cell, and cell has pathology, in nucleus tenuigenin, and the visible green specificity fluorescent of apple that is, negative control does not have specificity fluorescent and occurs.See Fig. 3.
(4) viral hemoagglutination is active detects
Get 96 hole micro-reaction plates (U type), toxic cell culture fluid is done 2 times of serial dilutions, add and contain 4HAU pig parvoviral hemagglutinin liquid with the PBS of pH 7.2; And establish PBS and virus control, fully after the vibration, put 4 ℃; 18h; Add 0.6% GPRBC suspension again, put room temperature 1h, result of determination when the red corpuscle in the control wells is remarkable button-type.
The result shows: gather in the crops toxic cell cultures fluid power aggegation GPRBC, have higher hemagglutination activity, and along with the increase of the incubation times that goes down to posterity, the flexibility of viral pair cell strengthens, the viral hemoagglutination valency has raising.See table 1:
(5) order-checking of virus detects
Complete genome sequence according to pig parvoviral NADL-2 strain designs a pair of primer voluntarily, and VP2 gene fragment (2808bp-4407bp) is used to increase.Upstream primer is P1:5 ' CAATGAGTGAAAATGTGGAA 3 ', and downstream primer is P2:5 ' TTTGCTGTGAATGTTAGTGT 3 '.PPV is extracted DNA, and the VP2 gene fragment is carried out pcr amplification, order-checking.
The result shows: utilize DNA MAN software that L strain VP2 gene fragment is carried out sequential analysis, the gene fragment of the 1600bp that sequencing result has obtained conforming to expected results is comprising 2 Nucleotide of PPV VP1 gene and 1598 Nucleotide of VP2 gene.Sequencing result is seen SEQ ID NO:1.
The homology comparative result of PPV L strain VP2 gene fragment and other PPV strain is seen table 2.
The homology of table 2 PPV L strain VP2 gene fragment and other PPV strain relatively
The evolutionary tree of the L strain of the present invention's virus and other a few strain PPV VP2 is analyzed, and shows that the genetic distance of L strain and NADL-2 strain and China strain is nearest.
The preparation of embodiment 2 porcine parvovirus deactivation vaccines
1. seedling is with the preparation of viral liquid: the seeding seed culture of viruses inserts by 0.5% of virus culture liquid measure and does not form as yet in the ST cell culture of individual layer, puts 37 ℃ of rotating and culturing, when pathology reaches 70%, gathers in the crops toxic cell culture fluid, through 3 freeze thawing after, receives malicious.
2. deactivation: in viral liquid, add the divinyl imide liquor by 0.02% of viral liquid total amount, fully after the vibration, deactivation 72h on 30 ℃, 100r/min shaking table adds 1% hypo solution then, stops deactivation.And make steriling test.
3. concentrate: get the viral liquid after the deactivation,, filter with 0.2um filter membrane filter then through the 13000r/min continuously centrifuged.Viral liquid behind the micro-filtration is with 5 times of 50KD filter membrane filter ultrafiltration and concentration.
4. the preparation of oil adjuvant killed vaccine: according to the ratio preparation oil phase of 1.5 parts of 94 parts of injection white oils, Si Ben-806 part and StAls; Add the ratio preparation water that 4ml tween-80 and 0.01% adds Thiomersalate according to 96ml antigen; Water and oil phase carry out emulsification according to the 4:1.5 ratio, process the single-phase seedling of water-in-oil.
The process optimization test of Test Example 1 pig parvoviral strain inactivated vaccine of the present invention
Select for use the divinyl imines as inactivator, the using dosage of inactivator, action time, inactivating efficacy etc. are carried out a series of tests, to optimize divinyl imines deactivation pig parvoviral program.
(1) divinyl imines using dosage and concentration are confirmed
Set 1%, 0.2%, 0.05%, 0.02% isocyatic divinyl imines, at 30 ℃, 100r/min, under the condition of gas bath vibration, deactivation 48h does inspection of inactivation of viruses liquid cell culture infectious virus and mice infection test after the deactivation.Confirm with end level 0.02% divinyl imide liquor deactivation pig parvoviral best results.
(2) the divinyl imines is confirmed action time
0.02% divinyl imines; At 30 ℃, 100r/min is under the condition of gas bath vibration; Do 7,10,15,24,48,60,72 respectively, the deactivation of different time such as 80h, do inspection of inactivation of viruses liquid cell culture infectious virus and mice infection test after the deactivation.Confirm that with end level 0.02% divinyl imide liquor at 30 ℃, 100r/min under the condition of gas bath vibration, does 72h deactivation pig parvoviral best results.
(3) divinyl imines toxicity test
Set 1%, 0.2%, 0.02% isocyatic divinyl imide liquor, injection 18-22g small white mouse is observed 10d, record death toll and survival number.Confirm that 0.2% divinyl imines injection 0.2ml and 0.02% divinyl imines 0.4ml are safe dose.
The immune effect test of Test Example 2 porcine parvovirus deactivation vaccines of the present invention
According to 3 batches of vaccines of preparing method's continuous production of embodiment 2,10276 pigs (most is first farrowing sow) have been carried out the vaccine inoculation experiment.With the immunizing dose is pig muscle injection 2ml/ head, once inoculates inactivated vaccine, and the terrain duration of immunity can reach more than 6 months, explains that this vaccine has good immunogenicity.
To use dose inoculation 2ml/ head, overdose inoculation 10ml/ head is not found any abnormal response.Get the vaccine inoculation replacement gilt of trial-production, pregnant pig and do not see that immunity has any influence to reproductive function, breeding, gestation, farrowing are all normal.The proof vaccine safety is good.
< 110>Harbin Pharmaceutical Group's biovaccine Ltd
< 120>porcine parvovirus L strain and the purposes in the preparation porcine parvovirus inactivated vaccines
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Claims (4)
1. the isolating porcine parvovirus L strain of a strain, its microbial preservation number are: CGMCC No.3352.
2. the described porcine parvovirus L strain of claim 1 prevents the purposes in the pig parvoviral disease vaccine in preparation.
3. inactivated vaccine that prevents porcine parvovirus contains the antigen liquid and the adjuvant of the described porcine parvovirus L strain of claim 1 of significant quantity.
4. a method for preparing the inactivated vaccine of the said prevention porcine parvovirus of claim 3 comprises: cultivate the described porcine parvovirus L strain of claim 1, obtain the pig parvoviral antigen liquid; With end level 0.02wt% divinyl imide liquor at 30 ℃, 100r/min, under the condition of gas bath vibration, deactivation pig parvoviral antigen liquid 72h is with the deactivation of pig parvoviral antigen liquid; Concentrate; Prepare oil phase and water respectively, water and oil phase are mixed back emulsification according to the volume ratio of 4:1.5, promptly get;
Said oil phase prepares according to following method: 94 parts of injection white oils, Si Ben-806 part and StAl mix for 1.5 parts, and preparation obtains oil phase; Said water prepares according to following method: add the 4ml tween-80 in the pig parvoviral antigen liquid after the 96ml deactivation, adding Thiomersalate to final concentration is 0.01wt%; Preparation obtains water.
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| CN102091329B (en) * | 2011-01-30 | 2013-08-07 | 哈药集团生物疫苗有限公司 | Preparation method of inactivated porcine parvovirus vaccine and product thereof |
| CN102344912B (en) * | 2011-09-20 | 2013-08-14 | 哈药集团生物疫苗有限公司 | Porcine pseudorabies virus strain and porcine pseudorabies inactivated vaccine prepared by using same |
| CN103436497B (en) * | 2013-06-14 | 2015-09-02 | 河南农业大学 | A kind of pig parvoviral strain |
| CN105521488A (en) * | 2015-12-26 | 2016-04-27 | 哈药集团生物疫苗有限公司 | Porcine parvovirus-porcine pseudorabies combined inactivated vaccine, and preparation method and application thereof |
| CN107802830A (en) * | 2017-10-19 | 2018-03-16 | 吉林农业科技学院 | A kind of pig parvoviral disease vaccine emulsification method based on mineral oil adjuvant |
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