CN101376878A - Pig propagation and breathing syndrome virus stain, and inactivated vaccine prepared thereby - Google Patents
Pig propagation and breathing syndrome virus stain, and inactivated vaccine prepared thereby Download PDFInfo
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Abstract
The invention discloses a separated porcine reproductive and respiratory syndrome virus JJ strain with the microorganism collection number of CGMCC NO.2129, an inactivated vaccine prepared by the virus strain and a preparation method thereof. The porcine reproductive and respiratory syndrome virus inactivated vaccine has good immune protection effectiveness, and can effectively prevent porcine reproduction and respiratory syndrome.
Description
Technical field
The present invention relates to a kind of virus stain, relate in particular to boar breeding and breathing syndrome virus stain.The invention still further relates to inactivated vaccine for preparing by this pig breeding and breathing syndrome virus stain and preparation method thereof; Belong to the animal vaccine field.
Background technology
Pig breeding and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) be by the caused a kind of new transmissible disease of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), with sow breeding difficulty, piglet with breed the porcine respiratory disease and high mortality is main special disease.This disease broke out in the North America in 1987 first, spread in the whole world rapidly, caused serious economy loss for world's pig industry.In China, the popular and distribution of PRRS is also increasingly extensive, and harm is on the rise.Especially 2006, the eqpidemic disease that is called as " nameless high-fever " was broken out on some pig farms, China south, and the pig industry of locality has been caused huge harm and fear.The main pathogen that now confirmed to cause this unknown high fever is the PRRSV that has made a variation, and with " nameless high-fever " called after " high-pathogenicity blue ear disease ".Breaking out once more of high-pathogenicity blue ear disease beaten alarm bell to people, how to control epidemic situation fast and effectively, reduces financial loss to greatest extent and become pendulum problem demanding prompt solution in face of people.
Experience of prevention and treatment shows both at home and abroad, and vaccine immunity is anti-this disease of system and the major measure of avoiding causing the tremendous economic loss.At present, existing multiple attenuated live vaccine, inactivated vaccine and subunit vaccine come out, and have brought into play vital role in the anti-system of PRRS.But foreign study shows, the atypical and acute PRRS that is caused by emerging PRRSV strain of the U.S. and the outburst recently of other North America country can not provide effective protection with current vaccine.
For preventing that effectively high-pathogenicity blue ear disease from China and even worldwide spreading, pressing for the PRRS virus vaccines that can effectively prevent to cause described illness at present.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, and a kind of new pig breeding and breathing syndrome virus stain are provided, and can effectively prevent pig breeding and respiratory syndrome by the prepared inactivated vaccine of this strain.
The inventor from Tianjin one pig farm that high heat the takes place pig that dies of illness adopt and be separated to a strain PRRSV the pathological material of disease, detecting through RT-PCR is the PRRSV american type, institute's isolated viral cell culture is revert to animal produce PRRS specificity clinical symptom, high heat, flush, appetite stimulator, spirit is depressed, hind leg is walked lamely even paralysis, sickness rate 100%.And in the experimental animal body, be separated to this virus once more.With institute's isolating strain amplification back order-checking, gene sequencing is the result show, its genome total length is 15324bp (comprising the PolyA tail) (its gene order is seen SEQ IDNO.1), and (PRRSV is Arteriviridae Arterivirus member to be respectively 89.7%, 61.8% with PRRSV american type type strain (VR2332) and Europe class type strain (LV) complete genome sequence homology.Virus has cyst membrane, spherical in shape, and genome is the sub-thread positive chain RNA, and the about 15kb of size is divided into Europe class and american type); Comparison shows that its Nsp2 and GP5 genovariation maximum with other representative PRRSV.Sequential analysis shows that this strain is the PRRSV that variation has taken place, Nsp2 compares with America standard strain VR2332 with the GP5 gene, nucleotide homology is respectively 83.7%, 89.2%, its amino acids coding homology is respectively 77.9%, 87.9%, the present invention is with institute's isolating strain called after pig breeding and breathing syndrome virus (Porcine Reproductive and RespiratorySyndrome Virus, PRRSV) TJ strain, submit breeding of this pig and breathing syndrome virus TJ strain strain to preservation, its microbial preservation number is: CGMCC NO.2129; The preservation time: on August 9th, 2007; The classification name is: pig breeding and breathing syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus); Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Another technical problem to be solved by this invention provides the above-mentioned pig breeding of a kind of usefulness and prepares the method for inactivated vaccine with breathing syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus) TJ strain.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
Above-mentioned pig breeding of a kind of usefulness and breathing syndrome virus TJ strain prepare the method for inactivated vaccine, comprise: pig breeding of the present invention is carried out large scale culturing with breathing syndrome virus TJ strain, gather in the crops viral liquid, will make water after the viral liquid deactivation, after water and oil phase emulsification, promptly.
Among the above-mentioned preparation method, preferred: described viral liquid prepares according to following method: cultivate the Marc-145 cell; Pig of the present invention breeding and breathing syndrome virus TJ strain through continuous passage with repeatedly be inoculated on the Marc-145 cell with infective dose (MOI) behind the plaque clone purification and cultivate, are gathered in the crops viral liquid; Preferred, with pig of the present invention breeding and breathing syndrome virus TJ strain through continuous passage with repeatedly be inoculated on the Marc-145 cell with infective dose (MOI) 0.01-0.5 behind the plaque clone purification and cultivate, cultivated 3-5 days, and when cytopathy reaches 80%, gathered in the crops viral liquid;
Preferably, described Marc-145 cell is cultivated according to following any one method and obtained: (1) cultivates the Marc-145 cell in rolling bottle, make its cell density reach 5-10 * 10
7/ ml; (2) in bio-reactor, add the suspension culture or the DNAcarrier free suspension culture of adhering to carrier, make its cell density reach 5-10 * 10
7/ ml--5-10 * 10
8/ ml; (3) with Marc-145 cell suspension culture in the lower concentration serum of serum-free or 1-5%.
Preferably, described oil phase prepares in accordance with the following methods: in 65-90 parts by volume injection white oil, add 5-15 parts by volume department classes 80 and 5-25 parts by volume tween 80, with add be stirred to transparent till, after the sterilization, standby.
Preferably, described emulsification method may further comprise the steps: water intaking is put in the emulsion tank mutually, slow rotation stirs, slowly add oil phase simultaneously, stirred 10～30 minutes with 10000r/min～15000r/min again after adding, add 10% (W/V) Thiomersalate solution before stopping stirring, making its ultimate density is 0.01% (W/V).
Preferably, described viral liquid ablation method is: adding the divinyl imines in viral liquid is 1mM-3mM to final concentration, and deactivation 4-28h under 37 ℃ of conditions neutralizes the viral liquid after the deactivation with Sulfothiorine.
The preparation-obtained inactivated vaccine of the inventive method centrifugal 15 minutes with 3000r/min, no demixing phenomenon shows the vaccine emulsify well that the inventive method is prepared.
Present domestic deactivation vaccine preparation nearly all uses formaldehyde as inactivator, the inventor is through test of many times, find adopting the BEI inactivation of viruses to prepare antigen, is safely and effectively through check, has overcome that formaldehyde is big as the inactivator side effect, the halfway shortcoming of inactivating efficacy.
The prepared vaccine of the inventive method is an oil in water vaccine, has overcome the shortcoming that the side effect of domestic water-in-oil-type vaccine is big, injection is difficult, and emulsifying effectiveness is better, also greatly reduce simultaneously cost, and easily injection, side reaction is little, and this will produce positive pushing effect to China's vaccine industry.
The present invention is when preparing inactivated vaccine with the isolating PRRSV TJ of institute strain, it is carried out continuous passage on the Marc-145 cell, in per 5 generations, carry out once viral plaque and separate, make its reduction become cell adapted poison, more resulting attenuated vaccine strain can be prepared the attenuated vaccine of breeding of prevention pig and respiratory syndrome according to the attenuated vaccine preparation method of routine.
Description of drawings
Fig. 1 PRRSV TJ strain RT-PCR detected result; M is Maker DL2000, and 1,2 is sample separation, and 3,4 is PRRSV America strain positive control.
Fig. 2 PRRS inactivation of virus kinetic curve.
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 or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Separation, the acquisition of embodiment 1 PRRSV TJ strain
One pig farm that high heat the takes place pig that dies of illness is adopted and is separated to a strain virus the pathological material of disease from Tianjin, be special disease property PRRS cytopathy through 2 generations of Marc-145 cell blind passage, detect through RT-PCR, 2 samples all expand the purpose fragment that 433bp and 468bp, prove that this strain isolated is the american type (see figure 1).Institute's isolated viral cell culture is revert to animal produce PRRS specificity clinical symptom, high heat, flush, appetite stimulator, spirit is depressed, and hind leg is walked lamely even paralysis, slight expiratory dyspnea, sickness rate 100%.In the experimental animal body, be separated to this virus once more, to the order-checking of increasing of its GP5 and Nsp2 gene, the result shows that this strain is the PRRSV that variation has taken place, with this strain called after PRRSV TJ strain, the nucleotide sequence of this strain is seen SEQ ID NO:1, submit this strain to preservation, its microbial preservation number is: CGMCC NO.2129.
The purifies and separates of embodiment 2 viruses
10 times of serial dilutions are made in PRRSV TJ strain, and dilution range is 10
-2-10
-6, the 6 porocyte culture plates that cover with individual layer Marc-145 cell are inoculated in the dilution back, the 1ml/ hole, and absorption 1h abandons viral liquid, uses the MEM of serum-free to clean 2 times, adds the mixed solution of 2 * nutritive medium and sterilization agar solution, the 3ml/ hole, room temperature is cooled off and is placed on 37 ℃ of 5% CO
2Incubator is cultivated 3-4d, observes plaque, and the single plaque in the less hole of picking plaque goes down to posterity.
(1), PRRSV deactivation
(1) cell cultures: in rolling bottle, cultivate the Marc-145 cell, make its cell density reach 5-10 * 10
7/ ml;
(2) virus inoculation: being 0.01-0.5 with infective dose (MOI) is inoculated into PRRSV TJ of the present invention strain in the Marc-145 cell that step (1) cultivated, and cultivates 3-5 days, gathers in the crops viral liquid when cytopathy reaches 80%;
(3) gather in the crops to step (2) and add BEI in the viral liquid, final concentration is respectively 1mM, 2mM and 3mM, makes its thorough mixing, place 37 ℃ to carry out deactivation then, 30min behind (0h) and the adding BEI before the adding BEI, 1h, 2h, 4h, 8h, 12h, 24h, 28h collects sample (10ml/ time) respectively, and collected sample is used for virus titer to be measured, and draws the deactivation kinetic curve.Determine BEI final concentration and best inactivation time, the inactivation of virus kinetic curve is seen Fig. 2.
(2), PRRSV inactivating efficacy check
It is thorough to guarantee the PRRSV deactivation to carry out the inactivating efficacy check after the PRRSV deactivation.
Get 4 of the rolling bottles that well-grown covers with 90% individual layer Marc-145 cell, the prepared inactivation of viruses liquid 25ml of 2 bottle graft kind steps () wherein, all the other 1 bottle of negative contrasts, 1 bottle of positive contrast in addition.Cultivate 5d in 37 ℃.Observe CPE, do not have CPE as detecting bottle and negative control bottle, and typical PRRSV pathology appears in the positive control bottle, carries out the blind passage second time.Liquid in the results rolling bottle inserts after the freeze thawing in the corresponding square vase, and every bottle of 15ml cultivates 5d in 37 ℃.Observe CPE, think then that as the result is the same viral liquid deactivation is complete.
Through check, the inactivation of viruses liquid that step () is prepared, its inactivation of virus is complete.
The preparation of embodiment 4 PRRSV inactivated vaccines
The viral liquid of 2 deactivations of embodiment is further passed through following processing:
1) preparation water: get physiological saline appropriate amount part, add virus (antigen) the liquid appropriate amount of the prepared deactivation of embodiment 2, fully shake mixes it, water, standby;
2) preparation oil phase: in 65-90 parts by volume injection white oil, add class 80 of 5-15 parts by volume department and 5-25 parts by volume tween 80, with add be stirred to transparent till, 121 ℃ of autoclavings 20 minutes must oil phase, and are standby;
3) water intaking is put in the emulsion tank mutually, starts the motor slow rotation and stirs, and adds oil phase simultaneously slowly, stirred 10～30 minutes with 10000r/min～15000r/min again after adding, add 10% (W/V) Thiomersalate solution before stopping stirring, making its ultimate density is 0.01% (W/V), promptly.
After the emulsification, get the prepared vaccine of 10ml, with 3000r/min centrifugal 15 minutes, no demixing phenomenon.
Embodiment 5 PRRS inactivated vaccine safety testings
Get 3 batches of the prepared PRRS inactivated vaccines of embodiment 4, through the negative pig of musculi colli injection 3-5 PRRS in age in week, carry out single dose respectively, a single dose repeated inoculation and an overdose inoculation, 2ml/ dosage, 15 pig/groups.Annotated behind the seedling 28 days, and observed inoculation position, have no adverse reaction.Test-results explanation PRRS inactivated vaccine of the present invention is safe to immune animal.
The safety testing result of the negative pig of table 1 vaccination PRRS
10 of the negative ablactation of the healthy PRRS of 30-35 age in days piglets are used in test altogether, and test is divided into 2 groups, every group of 5 animals.
The PRRS inactivated vaccine that first group of (immune group) immunization embodiment 4 is prepared, dosage is 2ml/ pig, 10
6.0TCID
50/ head part/2ml, the musculi colli injection;
Second group is control group, the emulsification control vaccine of inoculation 2ml Marc-145 control cells nutrient solution, 2ml/ head part.
Animal clinical response and vaccine side effect are observed in immunity back, weekly blood sampling, separation of serum, be used for virus and separate and Serum Antibody Detection; Measure body weight weekly.
Test-results:
1, two groups of experimental animal weightening finish no significant differences after the vaccine inoculation;
2, attack poison after, the morbidity of 2 pigs of immune group, the performance fervescence, appetite stimulator, flush, spirit is depressed, hind leg is walked lamely even paralysis, sickness rate 40% is acted normally for all the other 3; 5 pigs of control group all fall ill, and sickness rate is 100%.
The test-results explanation, vaccine of the present invention is for the attack of strong poison, and its protection ratio can reach 60%, can effectively prevent pig breeding and breathing syndrome virus.
The reduction of embodiment 7 PRRSV TJ strains
When preparing inactivated vaccine with the isolating PRRSV TJ of institute strain, it is carried out continuous passage on the Marc-145 cell, in per 5 generations, once adopted plaque separation and purification virus, make its reduction become cell adapted poison, again this low virulent strain is prepared attenuated vaccine according to the conventional preparation method of attenuated vaccine.
Claims (11)
1, pig breeding and breathing syndrome virus (Porcine Reproduct ive and RespiratorySyndrome Virus) TJ strain, its microbial preservation number are: CGMCC NO.2129.
2, according to the pig breeding and breathing syndrome virus TJ strain of claim 1, its special disease is: the gene order of this strain is shown in the SEQ ID NO:1.
3, a kind of method for preparing the inactivated vaccine of breeding of prevention pig and respiratory syndrome, comprise: the pig breeding of claim 1 is carried out large scale culturing with breathing syndrome virus TJ strain, gather in the crops viral liquid, will make water after the viral liquid deactivation, after water and oil phase emulsification, promptly.
4, according to the method for claim 3, its special disease is that described viral liquid prepares according to following method: cultivate the Marc-145 cell; The breeding of the pig of claim 1 and breathing syndrome virus TJ strain through continuous passage with repeatedly be inoculated into infective dose (MOI) behind the plaque clone purification and carry out large scale culturing on the Marc-145 cell, are gathered in the crops viral liquid.
5, according to the method for claim 4, its special disease is: with the breeding of the pig of claim 1 and breathing syndrome virus TJ strain through continuous passage with repeatedly be inoculated on the Marc-145 cell with infective dose (MOI) 0.01-0.5 behind the plaque clone purification and cultivate, cultivated 3-5 days, and when cytopathy reaches 80%, gathered in the crops viral liquid.
6, according to the method for claim 4, its special disease is that described Marc-145 cell is cultivated according to following any one method and obtained: (1) cultivates the Marc-145 cell in rolling bottle, make the Marc-145 cell density reach 5-10 * 10
7/ ml; (2) in bio-reactor, add the suspension culture or the DNAcarrier free suspension culture of adhering to carrier, make the Marc-145 cell density reach 5-10 * 10
7/ ml--5-10 * 10
8/ ml; (3) with Marc-145 cell suspension culture in the lower concentration serum of serum-free or 1-5%.
7, according to the method for claim 3, its special disease is that described oil phase prepares in accordance with the following methods: in 65-90 parts by volume injection white oil, add 5-15 parts by volume department classes 80 and 5-25 parts by volume tween 80, with add be stirred to transparent till, after the sterilization, standby.
8, according to the method for claim 3, its special disease is, described emulsification may further comprise the steps: water intaking is put in the emulsion tank mutually, slow rotation stirs, slowly add oil phase simultaneously, stirred 10～30 minutes with 10000r/min～15000r/min after adding again, add 10% Thiomersalate solution before stopping stirring, making its ultimate density is 0.01%.
9, according to the method for claim 3, its special disease is that described viral liquid ablation method is: adding the divinyl imines in viral liquid is 1mM-3mM to its final concentration, deactivation 4-28h under 37 ℃ of conditions; Neutralize with Sulfothiorine again after the deactivation of virus liquid.
10, a kind of attenuated vaccine that prevents pig breeding and respiratory syndrome, its special disease is, it prepares in accordance with the following methods: the breeding of the pig of claim 1 and breathing syndrome virus (Porcine Reproductive andRespiratory Syndrome Virus) TJ strain are gone down to posterity at Marc-145 or relevant cell obtains attenuated vaccine strain a little less than causing, and the method for this attenuated vaccine strain according to the conventional attenuated vaccine of preparation prepared.
11, the inactivated vaccine of pig breeding and breathing syndrome virus, its special disease is: by the preparation-obtained product of any one method of claim 3-10.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038942A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing porcine reproductive and respiratory syndrome (PRRS) vaccines by utilizing bioreactor |
| CN102107003A (en) * | 2011-01-05 | 2011-06-29 | 重庆大学 | Porcine reproductive and respiratory syndrome Virosome vaccine and preparation method thereof |
| CN101612395B (en) * | 2008-06-24 | 2012-02-08 | 扬州优邦生物制药有限公司 | Method for producing blue-ear disease vaccine by culturing sensitive cell |
| CN102757493A (en) * | 2012-07-11 | 2012-10-31 | 北京健翔和牧生物科技有限公司 | Method for preparing chicken pox virus antibody |
| CN104593335A (en) * | 2015-03-05 | 2015-05-06 | 成都天邦生物制品有限公司 | Method for producing propagation and respiratory tract syndrome CH-1R strain viruses for pigs by seroculturing Marc-145 cells |
| CN106943591A (en) * | 2016-01-06 | 2017-07-14 | 北京健翔和牧生物科技有限公司 | Porcine reproductive and respiratory syndrome virus nanoparticles vaccine and its production and use |
| CN108300703A (en) * | 2018-02-07 | 2018-07-20 | 吉林农业大学 | Deer source bovine viral diarrhoea inactivated vaccine and preparation method thereof |
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| CN102038942A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing porcine reproductive and respiratory syndrome (PRRS) vaccines by utilizing bioreactor |
| CN102107003A (en) * | 2011-01-05 | 2011-06-29 | 重庆大学 | Porcine reproductive and respiratory syndrome Virosome vaccine and preparation method thereof |
| CN102757493A (en) * | 2012-07-11 | 2012-10-31 | 北京健翔和牧生物科技有限公司 | Method for preparing chicken pox virus antibody |
| CN104593335A (en) * | 2015-03-05 | 2015-05-06 | 成都天邦生物制品有限公司 | Method for producing propagation and respiratory tract syndrome CH-1R strain viruses for pigs by seroculturing Marc-145 cells |
| CN104593335B (en) * | 2015-03-05 | 2017-10-03 | 成都天邦生物制品有限公司 | The low cells of serum free culture system Marc 145 production pig breeding and the method for respiratory syndrome CH 1R strain virus |
| CN106943591A (en) * | 2016-01-06 | 2017-07-14 | 北京健翔和牧生物科技有限公司 | Porcine reproductive and respiratory syndrome virus nanoparticles vaccine and its production and use |
| CN108300703A (en) * | 2018-02-07 | 2018-07-20 | 吉林农业大学 | Deer source bovine viral diarrhoea inactivated vaccine and preparation method thereof |
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