CN101633909B - Attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome - Google Patents
Attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome Download PDFInfo
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Abstract
The invention provides an attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome, a formulation thereof and a preparation method of the attenuated live vaccine strain and the formulation thereof. The attenuated live vaccine strain has obvious immunity protection effect on the pig-pig infection breeding and respiratory syndrome. The vaccine formulation also has long protection period and stable storage.
Description
Technical field
The present invention relates to a kind of attenuated live vaccine that prevents pig-pig infection breeding and respiration syndrome.
Background technology
(Porcine Reproductive and Respiratory Syndrome is by PRRSV (Porcine Reproductive and RespiratorySyndromeVirus, a boar transmissible disease that PRRSV) causes PRRS) to porcine reproductive and respiratory syndrome.Clinical symptom is a characteristic with sow breeding difficulty and piglet expiratory dyspnea.Their early stage all shows as heating, drowsiness, poor appetite, burnout, expiratory dyspnea, cough etc.This disease is found in the North Carolina state (1987) in the U.S. the earliest, after this finds this disease in Canada (1998), Germany (1990), Britain (1991) in succession.1992 International Office of Epizootics (OIE) with this sick called after porcine reproductive and respiratory syndrome (PRRS).China has also reported the popular of this disease in 1996.It is two kinds of genotype that PRRSV is divided into: american type and Europe class.And antigens genotyping and gene type basically identical.There is bigger antigenic difference between the amphitypy.
The popular world's pig industry of giving of PRRS has caused enormous economic loss.Therefore prevent PRRS more and more to obtain various countries practitioner's attention.Wherein vaccine prevention has been obtained certain progress.Inactivated vaccine is the vaccine of development early, and it does not exist the poison that looses to return strong danger with virulence. but for being that main PRRSV preventive effect is not an ideal very with cellular immunization.
U.S. Boehringer Ingleheim company releases attenuated live vaccine RespPRRS/Repro first in nineteen ninety-five
TM, and get permission to be used for 3-18 Healthy Youth sow, multiparity sow, sucking piglets and the growing-finishing pig in week, duration of immunity is more than 4 months.The less toxic vaccine Prime Pac that Schering-Plough animal health company produces
RPRRS is used for sow or the replacement gilt breeding vaccination in preceding 3-6 week, has obtained preventive effect preferably.But PRRSV very easily morphs, and causes attenuated vaccine preventive effect clinically not satisfactory, also is the major cause of pig farm recurrence PRRS.
High-pathogenicity porcine reproductive and respiration syndrome had been broken out in China in 2006; The cause of disease that causes the outburst of this time eqpidemic disease is the PRRSV that variation has taken place; Comparing existence with popular PRRS strain in the past makes a variation largely; Therefore, need develop to the attenuated live vaccine that causes China PRRS popular strain, popular to prevent China PRRS's effectively.
Summary of the invention
Technical disadvantages based on prior art exists the object of the present invention is to provide and can effectively prevent PRRS in the popular geographic generation of China.For reaching the object of the invention, the contriver is located away from the PRRSV TJ strain of the porcine reproductive and respiratory syndrome virus on pig farm, Tianjin from a strain, and carries out attenuation and cultivate, thereby has obtained to meet the attenuated vaccine strain of requirement of the present invention, thereby the invention provides:
A kind of porcine reproductive and respiratory syndrome virus attenuated live vaccine TJM, its microbial preservation number is CGMCC No.3121; Classification name: porcine reproductive and respiratory syndrome virus; The preservation address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation time is on June 15th, 2009;
The present invention also provides a kind of porcine reproductive and respiratory syndrome virus attenuated live vaccine; It is characterized in that; Its Nsp2 nucleotide sequence total length is 2490bp; Compare consecutive miss 598-717 amino acids with PRRSV TJ strain (the GenBank accession number is EU860248) Nsp2 nucleotide sequence, lack 120 amino acid altogether; Compare with VR-2332 strain Nsp2 nucleotide sequence, lack the 481st, 537-565 position and 628-747 amino acids, lack 150 amino acid altogether, nucleotide sequence is shown in SEQ ID NO:1.
The present invention further provides a kind of method for preparing above-mentioned vaccine strain; Said method comprises microbial preservation number is gone down to posterity in cell for the PRRSV TJ strain of CGMCC NO.2129; In per 10 generations, carried out the plaque clone purification; Obtain causing low virulent strain a little less than the virulence of said strain is caused, and obtain said vaccine strain.
In a preferred embodiment of the invention, in the above-mentioned method for preparing vaccine strain, said cell is Marc-145 cell or the relevant cell that can be used for the PRRSV growth.
In a preferred embodiment of the invention, said Marc-145 cell or the relevant cell that can be used for the PRRSV growth are cultivated according to following any one method and are obtained:
(1) in rolling bottle, cultivates Marc-145 cell or the relevant cell that can be used for the PRRSV growth, make 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 Marc-145 cell or the relevant cell density that can be used for the PRRSV growth reach 5-10 * 10
7/ ml--5-10 * 10
8/ ml.
The present invention also provides a kind of preparation that contains above-mentioned attenuated vaccine strain.
In a preferred embodiment of the invention, above-mentioned preparation contains heat-resisting lyophilized protecting agent.
The present invention further provides preparing such formulations, and it comprises:
(1) cultivates PRRSV TJM strain (microbial preservation number be CGMCC No.3121);
(2) the viral liquid of results steps (1) acquisition;
(3) preparation heat-resisting lyophilized protecting agent;
(4) said viral liquid and heat-resisting lyophilized protecting agent are mixed the back and quantitatively divide in the device peace bottle, add a cover and be placed in the Freeze Drying Equipment, through pre-freeze, drying process freeze dried vaccine.
In a preferred embodiment of the invention; In above-mentioned preparation method; PRRSV TJM strain is inoculated into Marc-145 cell or relevant with infective dose 0.01-0.5 can be used for carrying out large scale culturing on the cell of PRRSV growth; Cultivated 3-5 days, and when cytopathy reaches 70%, gathered in the crops viral liquid.
In a preferred embodiment of the invention; In above-mentioned preparation method; Said step (3) disposes heat-resisting lyophilized protecting agent as follows, and the lactalbumin hydrolysate of the sucrose of 10-30% mass volume ratio, 0.01-5% mass volume ratio L-Sodium Glutamate, 0.1-10% mass volume ratio N-Z, 10-30% mass volume ratio, the gelatin mixed dissolution of 0.1-10% mass volume ratio are after obtain said heat-resisting lyophilized protecting agent behind the autoclaving.
In a preferred embodiment of the invention, in above-mentioned preparation method, in step (4), viral liquid of volume ratio 6-8 part and the dried protective material of volume ratio 2-4 part are mixed the said preparation of preparation.
The present invention further provides the nucleotide sequence molecule, and its sequence is shown in SEQ ID NO:1.
The present invention further provides the application of above-mentioned nucleic acid molecule in vaccine discriminating of preparation porcine reproductive and respiratory syndrome virus and diagnostic reagent.
Attenuated vaccine strain provided by the present invention and related preparations are showing significant technique effect in prevention aspect the porcine reproductive and respiratory syndrome, and 5 generations virulence did not take place are returned by force behind this attenuated vaccine strain inoculation test animal, have solved virulence well and have returned strong problem; Safety testing shows that behind vaccine single dose, single dose repetition and the 10 multiple doses inoculation pig, body temperature is normal, does not have any clinical symptom, cuts open inspection observation lungs and does not have carnification, proves that the porcine reproductive and respiratory syndrome living vaccine is to pig safety; The vaccine potency test shows that vaccine of the present invention can produce the better protecting effect for the attack of strong poison, can effectively prevent porcine reproductive and respiratory syndrome; The test of vaccine immunity extended period shows that immune duration is 4 months, and can guarantee provides effective protection to pig in the duration of immunity; The test of vaccine preservation period shows that vaccine is 24 months 2～8 ℃ of preservation perives.
Figure of description
Fig. 1 is 2～8 ℃ of preservation period tests of porcine reproductive and respiratory syndrome living vaccine;
Fig. 2 is 37 ℃ of anti-aging tests of porcine reproductive and respiratory syndrome living vaccine.
Embodiment
Below in conjunction with accompanying drawing the present invention is further described, but these descriptions are not construed as limiting the invention, protection scope of the present invention is as the criterion with claims.
A little less than the going down to posterity of embodiment 1PRRSV TJ strain causes
From Tianjin one high heat takes place the pig farm pig that dies of illness adopt and be separated to a strain porcine reproductive and respiratory syndrome virus (PRRSV) the pathological material of disease, with its called after PRRSV TJ strain, this strain GenBank accession number is EU860248.Submit this strain to preservation, its microbial preservation number is: CGMCC NO.2129.PRRSVTJ pnca gene group total length is 15324bp (comprising the PolyA tail); Its Nsp2 sequence is compared with America standard strain VR-2332; The 481st of TJ strain Nsp2 sequence deletion and 532-560 amino acids, the two nucleotide homology is 83.7%, its amino acids coding homology is 77.9%.This test-results shows that this strain isolated is the PRRSV that variation has taken place, and pig is had highly pathogenic.With PRRSV TJ strain through the continuous passage of Marc-145 cell; In per 10 generations, carried out the plaque clone purification one time; Reach F92 for the time, it is carried out complete genome sequence amplification and Study on Pathogenicity, prove the PRRSV TJ strain that reaches F92; To experimental animal safety, its called after PRRSV TJM strain is used for living vaccine research.
Embodiment 2PRRSV TJM strain virulence is returned strong test
PRRSV TJM strain causes weak by popular hyperpyrexia disease poison (PRRSV TJ strain) and identifies and preserve, and whether this research further this vaccine virus of check exists virulence to return by force.The 1st virulence returned and forced the negative piglet with PRRSV TJM strain virus inoculation healthy PRRS in 4 age in week, musculi colli injection, 2ml/ pig, 10
5.7TCID
50/ ml establishes simultaneously and does not inoculate the negative control pig.Return strong test to the 5th virulence the 2nd time preceding 1 animal is the mixing of PRRSV positive serum, as the pig of animal inoculation pvaccination thing inoculation next time.All adopt the inoculation of musculi colli injection system, establish at every turn and do not inoculate pig as negative control.Continuous 14 days fixed point determination test animal rectal temperatures behind each virus inoculation, clinical observation 14 days, the serum-virus of blood sampling detection every other day is used for next virulence and returns the inoculum that goes down to posterity by force.The 5th virulence returned behind strong experimental observation to the virus inoculation 21 days.From the 2nd on-test, the virus inoculation thing can not be by the vitro culture amplification of going down to posterity.Body temperature is normal behind continuous 5 experimental animal virus inoculations, does not show any clinical onset.The result shows, porcine reproductive and respiratory syndrome virus TJM strain returns susceptible animal, returns strong test through 5 times and all do not cause the experimental animal morbidity, explains behind this strain inoculation test animal that 5 generations virulence did not take place return by force, can be used for vaccine research.
Embodiment 3PRRSV TJM strain Genetic Variation Analysis
PRRSV TJ strain, is obtained PRRSV TJM strain a little less than causing in the continuous passage of Marc-145 cell; PRRSV TJM strain Nsp2 gene is carried out sequencing; And itself and PRRSV TJ strain and classical american type VR-2332 strain carried out gene order relatively; The result shows: PRRSV TJM strain Nsp2 sequence total length 2490bp, be respectively 98.6% and 82.9% with PRRSV TJ strain and american type standard strain VR-2332 nucleotide sequence homology, and amino acid sequence homology is respectively 97.6% and 75.8%; Compare consecutive miss 598-717 amino acids with PRRSV TJ strain, lack 120 amino acid altogether; Compare with the VR-2332 strain, lack the 481st, 537-565 position and 628-747 amino acids, lack 150 amino acid altogether.PRRSV TJM strain Nsp2 nucleotide sequence is shown in SEQ ID NO:1.
The preparation of embodiment 4 porcine reproductive and respiratory syndrome living vaccines
1) seedling prepares with viral liquid: PRRSV TJM strain is inoculated in the Marc-145 cell that covers with good individual layer with MOI:0.01-0.5; Add and to keep liquid, put in 37 ℃ of Rotary Machines and cultivate, gather in the crops viral liquid when above when CPE reaches 70%; Viral level (TCID is measured in freeze thawing 2 times
50), steriling test, mycoplasma check and exogenous virus check are carried out in sampling simultaneously.
The viral liquid that is up to the standards is as antigen for vaccine liquid, subsequent use;
2) heat-resisting lyophilized protecting agent preparation: 10-30% sucrose (mass volume ratio), 0.01-5%L-Sodium Glutamate (mass volume ratio), 0.1-10%N-Z (mass volume ratio), 10-30% lactalbumin hydrolysate (mass volume ratio), 0.1-10% gelatin (mass volume ratio) mixed dissolution are after autoclaving is subsequent use.
3) the viral liquid of 5-8 part (volume ratio) and 2-5 part (volume ratio) lyophilized vaccine are mixed the back and quantitatively divide in the device peace bottle, add a cover and be placed in the Freeze Drying Equipment, through pre-freeze, drying process freeze dried vaccine.Carry out steriling test, safety examination and efficacy test behind the vaccine freeze-drying.
3 batches of vaccines to prepared in laboratory carry out safety testing, and are specific as follows:
Get 3 batches of porcine reproductive and respiratory syndrome living vaccines of prepared in laboratory, through the negative pig of musculi colli injection 4-5 PRRS in age in week, carry out single dose respectively, single dose repeated inoculation and 10 times of overdose inoculations, 10
5.0TCID
50/ ml/ dosage, 15 pig/groups are established 5 not vaccination pigs simultaneously as contrast.After the vaccine inoculation, the determination test animal rectal temperature of fixing a point continuous 14 day every day is observed clinical symptom, experimental animal is cutd open inspection in 21 days after the vaccine inoculation to observe lungs and have or not pathology.Test-results shows that behind vaccine single dose, single dose repetition and the 10 multiple doses inoculation pig, body temperature is normal, does not have any clinical symptom, cuts open inspection observation lungs and does not have carnification.Proof porcine reproductive and respiratory syndrome living vaccine is to pig safety.
Get 3 batches of porcine reproductive and respiratory syndrome living vaccines of prepared in laboratory, inoculate the negative ablactation of healthy PRRS in 3-5 age in week piglet respectively, test is divided into 4 groups, every group of 5 animals.
First group of PRRS living vaccine to 3 batches of prepared in laboratory of the 3rd group (immune group) difference immunization, dosage is 1 a part/pig, 10
5.0TCID
50/ head part/ml, the musculi colli injection;
The 4th group is control group, inoculation 1ml Marc-145 control cells nutrient solution.
Animal clinical response and vaccine spinoff are observed in immunity back, weekly blood sampling, separation of serum, be used for Serum Antibody Detection; Measure body weight weekly.
(tire is 10 with strong poison in the PRRSV TJ strain check of 50 times of dilutions of immunity back treated animal 4 Thursday use
5.8TCID
50/ ml) to attack, challenge dose is a 2ml virus liquid/pig, intranasal vaccination, 1ml/ nostril.Behind the strong poison of animal inoculation pvaccination, observe clinical symptom every day, comprise appetite, the mental status etc.; Measure rectal temperature every day; Per 2 days blood sampling separation of serum are used for viral separation determination; Test in attacking poison and finished in back 21 days.
Test-results:
1, experimental animal body temperature is normal after the vaccine inoculation, does not see any clinical symptom, vaccine inoculation group and control group experimental animal weightening finish no significant difference;
2, attack poison after, vaccine inoculation group protection ratio can reach 4/5, the morbidity of 5/5 pig of control group, 2/5 pig death.
The test-results explanation, vaccine of the present invention can produce the better protecting effect for the attack of strong poison, can effectively prevent porcine reproductive and respiratory syndrome virus.
The test of embodiment 7 porcine reproductive and respiratory syndrome living vaccine immune durations
The 3 batches of goods in laboratory are diluted to 10 with PBS
5.0TCID
50/ ml.The negative ablactation of musculi colli inoculation healthy PRRS in 4～5 ages in week piglets respectively, 1ml/ pig carries out the mensuration of duration of immunity, extracts immune group respectively in 2 months, 4 months and 6 months behind the vaccine immunity and the control group experimental animal carries out challenge test.The result shows: attacked poison in 2 months behind the vaccine immunity, 3 batches of vaccine inoculation group experimental animals all do not have death, and protection ratio can reach 4/5.Attacked poison in 4 months, 3 batches of vaccine inoculation group experimental animals all do not have death, and protection ratio can reach 4/5.Attacked poison in 6 months, 3 batches of vaccine inoculation group experimental animals all do not have death, and protection ratio can reach more than 3/5.Therefore immune duration is decided to be 4 months, can guarantee provides effective protection to pig in the duration of immunity.
The test of embodiment 8 porcine reproductive and respiratory syndrome living vaccine preservation perives
((080106,080125 and 080201) (antigenic content is respectively 10 to laboratory products with 3 batches
5.8TCID
50/ ml/ head part, 10
5.7TCID
50/ ml/ head part, 10
5.8TCID
50/ ml/ head part) in 2～8 ℃ preserve 3,6,9,12,18,24,30 and 40 months respectively sampling carry out proterties, vacuum tightness, moisture content, potency test (Fig. 1) and 37 ℃ of anti-aging tests (Fig. 2).The result: it still is the white loose agglomerate that 3 batches of goods are preserved 24 months proterties at 2～8 ℃, dissolving rapidly behind the adding diluent; Average residual moisture is in claimed range; Vacuum tightness detects and is white or purple aura; Antigenic content is respectively 10
5.2TCID
50/ ml/ head part, 10
5.2TCID
50/ ml/ head part, 10
5.3TCID
50/ ml/ head part; Place antigenic content on the 14th for 37 ℃ and be respectively 10
5.2TCID
50/ ml/ head part, 10
5.2TCID
50/ ml/ head part, 10
5.1TCID
50/ ml/ head part still is higher than freeze dried vaccine requirement (10
5.0TCID
50/ ml/ head part), thus vaccine be 24 months 2～8 ℃ of preservation perives.
Claims (6)
1. porcine reproductive and respiratory syndrome virus attenuated live vaccine TJM, its microbial preservation number is CGMCC No.3121.
2. preparation that contains the said vaccine strain of claim 1.
3. preparation according to claim 2 is characterized in that it also contains heat-resisting lyophilized protecting agent.
4. method for preparing the said preparation of claim 3 comprises:
(1) cultivates the said porcine reproductive and respiratory syndrome virus attenuated live vaccine of claim 1 TJM;
(2) the viral liquid of results steps (1) acquisition;
(3) preparation heat-resisting lyophilized protecting agent;
(4) said viral liquid and heat-resisting lyophilized protecting agent are mixed the back and quantitatively divide in the device peace bottle, add a cover and be placed in the Freeze Drying Equipment, through pre-freeze, drying process freeze dried vaccine.
5. method according to claim 4; It is characterized in that: porcine reproductive and respiratory syndrome virus attenuated live vaccine TJM is inoculated on the Marc-145 cell with infective dose 0.01-0.5 cultivates; Cultivated 3-5 days, and when cytopathy reaches 70%, gathered in the crops viral liquid.
6. preparation method according to claim 4 is characterized in that: in step (4), viral liquid of volume ratio 6-8 part and the heat-resisting lyophilized protecting agent of volume ratio 2-4 part are mixed the said preparation of preparation.
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Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101920010A (en) * | 2010-06-29 | 2010-12-22 | 西北农林科技大学 | Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof |
| UA108902C2 (en) * | 2010-11-10 | 2015-06-25 | NORTH AMERICAN REPRODUCTIVE AND RESPIRATORY PIG SYNDROME (PRRS) VIRUS | |
| EP2457583A1 (en) | 2010-11-26 | 2012-05-30 | Consejo Superior De Investigaciones Cientificas | Nucleic acids encoding PRRSV GP5-ecto and M protein |
| CN102002482B (en) * | 2010-12-08 | 2013-05-22 | 成都天邦生物制品有限公司 | Method for producing PRRS (Porcine Reproductive and Respiratory Syndrome) viruses |
| US8728487B2 (en) | 2011-01-20 | 2014-05-20 | Hua Wu | Attenuated live vaccine for prevention of porcine reproductive and respiratory syndrome |
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