CN105582535A - Preparation method of CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and product of CSF and PR bivalent live vaccine - Google Patents
Preparation method of CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and product of CSF and PR bivalent live vaccine Download PDFInfo
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Abstract
The invention provides a preparation method of a CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and a product of the CSF and PR bivalent live vaccine. The method comprises the following steps of culturing a CSF virus lentogen strain and a PR virus lentogen strain by a cell roller bottle or a bioreactor; harvesting obtained cell culture virus solutions; mixing the two kinds of obtained cell culture virus solutions according to a certain volume percent; adding a stabilizing agent and antibiotics into the mixed solution; and performing freeze-drying to obtain the CSF and PR bivalent live vaccine. The CSF and PR bivalent live vaccine is used for immunization; the workload of the immunization can be reduced; the immunization times can be reduced; the immunologic paralysis due to frequent immunization is avoided; the stress on swinery is correspondingly reduced; and the goal of preventing two kinds of diseases by one vaccine is achieved.
Description
Technical field
The invention belongs to veterinary biologics technical field, more specifically relate to preparation method and the goods thereof of a kind of swine fever, pseudorabies bigeminal live vaccine.
Background technology
Swine fever (CSF) and pseudoabies (PR) are two large Important Infectious Diseases of harm intensive industrialized piggery, in China swinery, extensively exist, and cause huge economic loss to pig industry. Immunity inoculation is the important measures of swine fever and the anti-system of pseudoabies. Application hog cholera lapinised virus vaccine and pseudorabies living vaccines play a significantly greater role to the anti-system of these two kinds of epidemic diseases at present. But pig is a lot of by vaccine kind, in practical operation, spininess, multiple dose, panimmunity program are difficult to reasonable arrangement, both loaded down with trivial details, time-consuming, effort, increase cost, and easily cause with Louing and plant, directly affect preventive effect. Adopt multi-joint (or multivalence) vaccine immunization, to reach the object of prevention multiple infectious disease. Meanwhile, the immune programme for children that these two kinds of epidemic diseases are taked is more approaching, and this development for swine fever and pseudorabies bigeminal live vaccine provides may. Can effectively prevent and control the generation of CSF and two kinds of epidemic diseases of PR and popular in order to reach primary immune response, alleviate the workload of immunity inoculation, reduce immune time, avoid because of the frequent immune immunological paralysis causing, corresponding reduced to swinery stress.
Summary of the invention
In view of this, the object of this invention is to provide a kind of swine fever that overcomes above-mentioned defect, pseudorabies bigeminal live vaccine and preparation method thereof.
For achieving the above object, the invention provides the preparation method of a kind of swine fever, pseudorabies bigeminal live vaccine, comprise the steps:
(1) with cell spinner bottle or bioreactor culture CSFV low virulent strain, pseudorabies virus low virulent strain;
(2) gather in the crops respectively the cell cultivation venom that described step (1) obtains;
(3) two kinds of cells that described step (2) obtains are cultivated venom and are mixed by the percent by volume of 1:2-1:1.5, and in mixed liquor, add stabilizing agent and antibiotic, obtain swine fever, pseudoabies cell live vaccine through vacuum freezedrying.
Further, wherein said bioreactor is TideCell microcarrier bioreactor, and contains microcarrier in described bioreactor, and described microcarrier is cytodex series microcarrier, and the use density of microcarrier is 2-25g/L.
Further, wherein said step (1) comprises the steps:
(1a) seedling going down to posterity and cultivating with cell: described passage cell, through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, continues to cultivate with cell growth medium, forms passage cell individual layer;
(1b) breeding of cell seed culture of viruses: described CSFV low virulent strain is seeded to the passage cell individual layer obtaining in described step (1a), continues to cultivate with cell maintenance medium; Change liquid once every results on the 4th～5, get two receipts, three and receive cell cultivation venom as production seed culture of viruses; Described pseudorabies virus low virulent strain is seeded to the passage cell individual layer obtaining in described step (1a), continues to cultivate with cell maintenance medium; After 2 ~ 3 days, harvesting is cultivated venom as production seed culture of viruses;
(1c) breeding of seedling venom:
Get the passage cell individual layer obtaining in described step (1a), discard described cell growth medium, inoculation, containing the cell maintenance medium containing CSFV obtaining in described step (1b), continues to cultivate;
Get the passage cell individual layer obtaining in described step (1a), discard described cell growth medium, inoculation, containing the cell maintenance medium containing pseudorabies virus obtaining in described step (1b), continues to cultivate.
Further, wherein said step (2) comprises the steps:
After being seeded to passage cell individual layer containing the cell maintenance medium of CSFV, cultivate, connect poison and gather in the crops and change liquid for the first time for latter 5 days, change liquid once later every results on the 4th, the venom of results is placed in-15 DEG C of following preservations; After being seeded to passage cell individual layer containing the cell maintenance medium of pseudorabies virus, cultivate, connect after poison harvesting after 2 ~ 3 days and cultivate venom, the venom of results is placed in-20 DEG C of following preservations.
Further, to cultivate temperature used be 36 DEG C ~ 37 DEG C to the cell in wherein said step (1a), (1b), (1c).
Further, the inoculum concentration while inoculation in wherein said step (1b), (1c) by volume percentage is counted the maintenance medium containing 1%～2% Cells for production seed culture of viruses.
Further, cell growth medium in wherein said step (1a) is containing 90%～92%MEM liquid, 8%～10% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell growth medium is 7.0～7.2, and wherein the unit of MEM liquid and calf serum is percent by volume.
Further, wherein said step (1b) and (1c) in cell maintenance medium containing 95%～98%MEM liquid, 2%～5% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell maintenance medium is 7.2～7.4, and wherein the unit of MEM liquid and calf serum is percent by volume.
Further, wherein said passage cell is bull testis cell, CEF, pig testis subculture cells ST, porcine kidney cell PK15 or porcine kidney cell IBRS-2.
Further, the setup parameter of wherein said bioreactor is: pH6.5-7.8,36 DEG C ~ 37 DEG C of temperature, dissolved oxygen 30-80%, mixing speed 30-100rpm.
Further, the cultivation of wherein said bioreactor adopts batch formula, stream to add or the mode of perfusion cultures, and the speed of perfusion is 0.5-5 working volume every day according to the density of cell.
In addition, the present invention also provides a kind of swine fever, pseudorabies bigeminal live vaccine, and described swine fever, pseudorabies bigeminal live vaccine make by said method.
The technique effect that the present invention realizes is as follows:
The preparation method of swine fever of the present invention, pseudorabies bigeminal live vaccine, preparation process is simple and stable, easy to operate, viral level is high, differences between batches are little, easy to control the quality, can significantly improve the advantage such as vaccine output and quality, minimizing allergic reaction. The swine fever, the pseudorabies bigeminal live vaccine security that utilize preparation method of the present invention to obtain are good, immune efficacy is high, and the strong virus attack of swine fever, pseudoabies is had to good immunization. Immunity inoculation swine fever and pseudorabies living vaccines in prior art, adopt separately immunity, spininess, multiple dose, both loaded down with trivial details, time-consuming, effort, increase cost, be difficult to again reasonable arrangement immune programme for children, and swine fever and pseudoabies be immunosuppressive disease, the unreasonable meeting of immunizing dose causes phase mutual interference, and easily cause Lou and plant, directly affect preventive effect. By swine fever, pseudorabies bigeminal live vaccine immunity inoculation, can alleviate the workload of immunity inoculation, reduce immune time, avoid the immunological paralysis that causes because of frequent immune, corresponding reduced to swinery stress, reach " pin is prevented two diseases ". And, utilize bioreactor large scale and high density to cultivate, can improve to greatest extent unit volume Growth of Cells density in bioreactor, in viral vaccine is produced, there is very large potentiality and advantage, be embodied in: (1) cell density increases greatly, and virus concentration greatly improves; (2) vaccine valence improves, and side reaction reduces; (3) reduce labour intensity, reduce production cost; (4) the production of vaccine property monitored improves; (5) ensure product quality stable homogeneous.
Brief description of the drawings
Fig. 1: the production technological process of swine fever, pseudorabies bigeminal live vaccine;
Fig. 2: hog cholera antibody variation diagram in immune swine serum;
Fig. 3: the hog cholera antibody positive rate figure of immune swine;
Fig. 4: pseudoabies antibody variation diagram in immune swine serum;
Fig. 5: the pseudoabies antibody positive rate figure of immune swine.
Detailed description of the invention
For making the present invention easier to understand, further set forth the present invention below in conjunction with specific embodiment. Should be understood that these embodiment are only for illustrating the present invention instead of for limiting the scope of the invention, in the following example, NM specific experiment method, carries out according to normal experiment method conventionally.
Be noted that following illustrating is all exemplary, be intended to the invention provides further invention. Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of conventionally understanding with the technical field of the invention personnel. Swine fever of the present invention, pseudorabies bigeminal live vaccine are produced and inspection seed culture of viruses, all purchased from Chinese veterinary microorganism culture presevation administrative center (CVCC).
The preparation of embodiment 1 swine fever, pseudorabies bigeminal live vaccine
As shown in Figure 1, the preparation method of swine fever, pseudorabies bigeminal live vaccine, technical scheme is:
(1) training method: applying biological reactor or cell spinner bottle cultured cell and breeding CSFV and pseudorabies virus.
(2) select permissive cell as seedling cell: one of bull testis cell, CEF, pig testis (ST) passage cell, pig kidney (PK15) passage cell, pig kidney (IBRS-2) passage cell.
(3) seedling going down to posterity and cultivating with cell: above-mentioned permissive cell, through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, continues to cultivate with cell growth medium, while forming good individual layer, goes down to posterity or virus inoculation for continuing;
(4) produce the breeding with seed culture of viruses:
A. live vaccines of hog cholera is produced the breeding with seed culture of viruses: with the MEM cell maintenance medium containing 3～5% cow's serums, fresh spleen seed culture of viruses is made to viral suspension, inoculate well-grown above-mentioned permissive cell individual layer, 37 DEG C are continued to cultivate; Change liquid once every results on the 4th～5, get two receipts, three and receive cell cultivation venom as production seed culture of viruses.
Cell seed culture of viruses qualification: the qualification of live vaccines of hog cholera Cells for production seed culture of viruses should meet fever virus lapinized Chinese Strain seed culture of viruses standard completely, and pig is had no side effect safely, and the every 1ml of cell seed culture of viruses is containing virus >=500,000 rabbit infective doses.
B. pseudorabies living vaccines is produced the breeding with seed culture of viruses: with the MEM cell maintenance medium containing 3～5% cow's serums, by the well-grown above-mentioned permissive cell individual layer of pseudorabies virus low virulent strain inoculation, 37 DEG C are continued to cultivate; After 2～3 days, harvesting is cultivated venom as production seed culture of viruses.
Cell seed culture of viruses qualification: the qualification of pseudorabies living vaccines Cells for production seed culture of viruses should meet pseudorabies virus low virulent strain seed culture of viruses standard completely, and pig is had no side effect safely, and the every 1ml of cell seed culture of viruses is containing virus >=108TCID50。
(5) breeding of seedling venom:
A. the breeding of live vaccines of hog cholera seedling venom: get the above-mentioned permissive cell blake bottle that forms good cell monolayer, discard cell growth medium, inoculate the toxic MEM cell maintenance medium containing 3～5% cow's serums, 37 DEG C are continued to cultivate; After connecing poison, within 5th, do to gather in the crops and change liquid for the first time, change liquid 1 time later every results on the 4th, the cell venom of results is put-15 DEG C of following preservations.
The inspection of seedling venom: test by existing " Chinese veterinary pharmacopoeia ", seedling should be without bacterium, mould, mycoplasma growth with venom. Cell venom has no side effect safely to pig, and each time virus-culturing fluid of receiving is measured with rabbit respectively, and every 1ml is containing virus >=500,000 rabbit infective doses.
B. the breeding of pseudorabies living vaccines seedling venom: get the above-mentioned permissive cell blake bottle that forms good cell monolayer, discard cell growth medium, inoculate the toxic MEM cell maintenance medium containing 3～5% cow's serums, 37 DEG C are continued to cultivate; After connecing poison, results born of the same parents on the 2nd～3 cultivate venom, and the venom of results is put-20 DEG C of following preservations.
The inspection of seedling venom: test by existing " Chinese veterinary pharmacopoeia ", seedling should be without bacterium, mould, mycoplasma growth with venom. Cell venom has no side effect safely to pig, and each time virus-culturing fluid of receiving is tired with raji cell assay Raji respectively, and every 1ml is containing virus >=108.0TCID50。
(6) join seedling, packing and freeze-drying: by the swine fever being up to the standards, pseudorabies virus nutrient solution, mix with the percent by volume of 1:2, be mixed in same container, add stabilizing agent, add appropriate antibiotic simultaneously, fully shake up quantitative separating; After packing, carry out rapidly getting product after vacuum freezedrying.
Product inspection
[physical behavior] this product is faint yellow Sponge Porosity agglomerate, easily departs from bottle wall, dissolves rapidly after adding dilution.
[steriling test] tests by existing " Chinese veterinary pharmacopoeia ", should be without bacterium, fungus growth.
[mycoplasma inspection] tests by existing " Chinese veterinary pharmacopoeia ", grows without mycoplasma.
[diagnostic test] vaccine does suitable dilution (1 part/ml) with PBS liquid, mixes with anti-swine fever, the pseudorabies virus specific serum of equivalent, at room temperature with 1 hour, vibrates 1-2 time therebetween. After neutralization, 4 bottles of inoculating cells (hole), every bottle of (hole) 0.1ml, observes 7 days, should be without CPE; And 2 of intravenous injection rabbit, every 1ml, thermometric is observed, and should react without body temperature.
[exogenous virus inspection] mixes 1000 times of vaccine dilutions with anti-swine fever, the pseudorabies virus specific serum of equivalent, at room temperature with 1 hour, vibrate 1-2 time therebetween. After neutralization, 2 of inoculation rabbit, every 1ml did not cause thermal response and Mortality in 120 hours. And test by existing " Chinese veterinary pharmacopoeia ", should pollute without exogenous virus.
[safety verification] vaccine does suitable dilution (10 part/ml) with PBS liquid, and intramuscular injection 7-10 age in days is without 4 of the piglets of pseudorabies virus neutralizing antibody, and every 2ml, observes 14 days, should be without abnormal response.
Meanwhile, select through the detection of neutralization test method and (before note seedling, observe 5～7 each survey morning and afternoon every day body temperature 1 time without the healthy susceptible wean pig of hog cholera antibody. Select body temperature, spirit, the normal person's use of appetite). Every batch of freeze dried vaccine sample or same batch each sub-batch sample mixed in equal amounts, the head indicating by label part is diluted to every milliliter with sterile saline and contains 6 part vaccines, 4 of intramuscular injection pigs, every 5ml (containing 30 using dosages). After note seedling, body temperature is respectively observed and surveyed to every day at the upper and lower noon 1 time, observes 21. Body temperature, spirit, appetite do not have significant change compared with before note seedling; Or hyperthermia exceedes 0.5 DEG C, but be no more than l DEG C, delay and be no more than 2 (4 temperature time); Or subtract food be no more than l day, it is qualified that vaccine can be judged to. Exceed more than normal temperature l DEG C if any l temperature of pig body, but be no more than 1.5 DEG C, delay and be no more than 2 temperature time, it is qualified that vaccine also can be judged to. Exceed above-mentioned standard if any the reaction of 1 pig; Or while there is suspicious other body temperature reaction and other anomaly, available 4 pigs are heavily examined 1 time. Still there is same reaction in the pig of inspection heavily, and vaccine should be judged to defective. Also can be 2 of pig megathermal period blood sampling involution pigs, every former blood 5m1 of the suspicious pig of intramuscular injection, thermometric is observed 16. As all reactionless, it is qualified that vaccine can be sentenced. As the 1st assay, to have confirmed vaccine dangerous, should heavily not examine.
[efficacy test]
1 swine fever part is pressed the dated head part of label, with SPSS, every part vaccine is diluted to 7500 times, and ear vein injection body weight is 2 of 1.5～3kg rabbit, every lml. After family's rabbit inoculation, respectively survey body temperature morning and afternoon 1 time, after 48 hours, surveyed body temperature 1 time every 6 hours, react and attack malicious result and carry out synthetic determination according to body temperature.
1. after rabbit inoculation vaccine, body temperature reaction normal is as follows:
Sizing 48～96 hours incubation periods of thermal response (++), temperature rise is obvious curve, has at least 3 temperature time to exceed normal temperature more than 1 DEG C, and delays 18～36 hours. As delay more than 42 hours, must attack poison, attack the rear reactionless sizing heat that is judged to of poison.
Slight fever is reacted 48～96 hours (+) incubation periods, and temperature rise is obvious curve, has at least 2 temperature time to exceed normal temperature more than 0.5 DEG C, and delays 12～36 hours.
In 48～96 hours incubation periods of suspicious reaction (±), it is indefinite that temperature curve rises and falls, and delays less than 12 hours; Or incubation period more than 24 hours, less than 48 hours and exceed 96 hours to 120 hours and occur thermal response.
Body temperature reaction is secondary peak, and once peak meets sizing thermal response (++) or slight fever reaction (+) standard person, must attack poison. Attack after poison when reactionless, this rabbit thermal response can be judged to sizing heat or slight fever reaction.
Reactionless (-) body temperature is normal.
2. result is judged:
After note seedling, when 2 rabbit are all sizing thermal response (++), or 1 rabbit is when being sizing thermal response (++), another 1 rabbit and being slight fever reaction (+), and vaccine is judged to qualified.
After note seedling, when 1 rabbit is sizing thermal response (++) or slight fever reaction (+), another 1 rabbit is suspicious reaction (±); Or 2 rabbits are while being all slight fever reaction (+), can after note seedling, within 7th～10, attack poison (inoculate fresh spleen and drench poison or freeze-drying poison). While attacking poison, add 2 of contrast rabbits, attacking toxic agent amount is 50～100 times of emulsions. Every rabbit ear vein injection 1ml.
Attack body temperature reaction normal after poison as follows:
In 24～72 hours incubation periods of thermal response (+), temperature rise is obvious curve, exceedes normal temperature more than 1 DEG C, delays 12～36 hours.
Suspicious reaction (±) incubation period, it is indefinite that temperature curve rises and falls less than more than 24 hours or 72 hours, delays less than 12 hours or exceed 36 hours and do not decline.
Reactionless (-) body temperature is normal.
Attack after poison, when 2 contrast rabbits are all sizing thermal response (++), or 1 rabbit is sizing thermal response (++), and another 1 rabbit is slight fever reaction (+), and 2 note all reactionless (-) of seedling rabbit, vaccine is sentenced qualified.
After note seedling, be sizing heat (++) or slight fever reaction (+) if any 1 rabbit, another 1 rabbit is suspicious reaction (±) or without thermal response (-), can adopt and cut open the method for killing or adopting painstaking effort isolated viral suspicious reaction rabbit or reactionless rabbit, distinguish whether subclinical infection; Or after note seedling, 2 rabbits are all slight fever reaction, also can be to 1 rabbit isolated viral wherein. Method is: after vaccine inoculation, between 96～120 hours, rabbit is cutd open and killed, take spleen, make 50 times with physiological saline and dilute emulsions (spleen emulsion should be aseptic), or take painstaking effort (whole blood), inoculate 2 rabbit, every rabbit ear vein injection 1ml. All have 1 rabbit to occur sizing thermal response (++) 24～72 hours incubation periods, and it is qualified that vaccine can be judged to.
After note seedling, while occurring that other response situation cannot be judged, can heavily examine. Do effect inspection with rabbit, should not exceed 3 times.
2 pseudoabies parts are 10 with PBS liquid by every part vaccine dilution4、105、106Doubly, each dilution factor is respectively inoculated 4 bottles, CEF cell (hole), and every bottle of (hole) 0.1ml observes CPE, calculates TCID50. Every part viral level answers >=106.0TCID50。
[residual moisture mensuration] tests by existing " Chinese veterinary pharmacopoeia ", should meet the regulation of veterinary biologics general rule.
[vacuum mensuration] tests by existing " Chinese veterinary pharmacopoeia ", should conform with the regulations.
[effect and purposes] is for preventing swine fever, pseudoabies. Swine fever, pseudoabies duration of immunity are 1 year.
[usage and consumption]
1 usage is pressed the head part that label indicates, and diluting with PBS liquid is 1 part 1ml, intramuscular injection.
2 consumption sucking pigs are injected 1/2 part for the first time, piglet and 1 part of feeder pig injection, 2 parts of Adult Pig injection.
It is 36 DEG C ~ 37 DEG C that cell in described step (3), (4), (5) is cultivated temperature used.
Inoculum concentration while inoculation in described step (4), (5) is the maintenance medium of 1%～2% (ml/ml) Cells for production seed culture of viruses.
The formula of the cell growth medium in described step (3) is: 90%～92% (ml/ml) MEM liquid, 8%～10% (ml/ml) calf serum also add 100 ~ 200 units/ml mycillin, and pH value is adjusted into 7.0～7.2.
The formula of the cell maintenance medium in described step (4), (5) is: 95%～98% (ml/ml) MEM liquid, 2%～5% (ml/ml) calf serum also add 100 ~ 200 units/ml mycillin, and pH value is adjusted into 7.2～7.4.
Embodiment 2 swine fevers, the immunity test of pseudorabies bigeminal live vaccine to pig
1 materials and methods
1.1 material
1.1.1 swine fever, pseudorabies bigeminal live vaccine are produced by Guangdong Winsun Bio-Pharmaceutical Co., Ltd., through being up to the standards.
1.1.2 test and derive from the bi-crossbreeding of Guangdong Winsun Bio-Pharmaceutical Co., Ltd. animal used as test field from numerous autotrophy with pig, without CSFV, PRRSV, PRV, PPV, PCV-II medical history and do not inoculate 20 of the health pig of CSF vaccine, PR vaccine and PRRS vaccine. Detect through ELISA and neutralization test method, select without swine fever, pseudoabies neutralizing antibody.
1.1.3 hog cholera antibody detection kit is purchased from IDEXX company.
1.1.4 pseudoabies antibody assay kit is purchased from IDEXX company.
1.2 method
1.2.1 inoculation method
10 of the health pig of selection swine fever and pseudoabies negative antibody, immunity inoculation swine fever, pseudorabies bigeminal live vaccine, every 1.0ml(is containing 1 part).
The monitoring of hog cholera antibody, pseudoabies antibody
Before immunity, test pig was carried out to vena cava anterior blood sampling in latter 14 days, 21,28,35,50,65,95,125,155,185,215,245,275,305,335,365 with immunity respectively, every 3 ~ 5ml, separation of serum, adopt respectively swine fever ELISA detection kit, pseudoabies ELISA detection kit to detect corresponding antibodies, method and criterion are with reference to IDEXX-ELISA kit description. It is 0.40 positive that hog cholera antibody is greater than, and is less than or equal to 0.30 negatively, is suspicious between 0.30 to 0.40; It is 0.70 negative that pseudoabies antibody is greater than, and is less than or equal to 0.60 positively, is suspicious between 0.60 to 0.70.
3 results
The hog cholera antibody testing result (seeing Fig. 2, Fig. 3, table 1 and table 2) of 3.1 immune swines
The hog cholera antibody mean value of table 1 immune swine
The hog cholera antibody positive rate of table 2 immune swine
From Fig. 2, Fig. 3, table 1 and table 2, can find out: immune swine immunity after 7 days antibody against swine fever virus level start to rise, after 28 days, reach higher level, can maintain and reach more than 12 months, latter 365 days serum antibodies of immunity still remain positive, the immune swine fever of health pig, pseudorabies bigeminal live vaccine be describeds after the antibody against swine fever virus positive can maintain more than 12 months.
The pseudoabies antibody test result (seeing Fig. 4, Fig. 5, table 3 and table 4) of 3.2 immune swines
The pseudoabies antibody mean value of table 3 immune swine
The pseudoabies antibody positive rate of table 4 immune swine
From Fig. 4, Fig. 5, table 3 and table 4, can find out: immune swine immunity after 7 days pseudorabies virus antibody horizontal start to rise, after 28 days, reach higher level, can maintain and reach more than 12 months, latter 365 days serum antibodies of immunity still remain positive, the immune swine fever of health pig, pseudorabies bigeminal live vaccine be describeds after pseudorabies virus antibody positive can maintain more than 12 months.
In sum, this test is by immune swine fever, pseudorabies bigeminal live vaccine, immune swine different time serum antibody is detected, result shows: health pig immunity swine fever, pseudorabies bigeminal live vaccine after 7 days antibody against swine fever virus level start rising with pseudorabies virus antibody horizontal, after 28 days, reach higher level, can maintain and reach more than 12 months, latter 365 days serum antibodies of immunity still remain positive, illustrates after the immune swine fever of health pig, pseudorabies bigeminal live vaccine that antibody against swine fever virus and pseudorabies virus antibody positive can maintain more than 12 months.
The comparative experiments of embodiment 3 swine fevers, pseudorabies bigeminal live vaccine and live vaccines of hog cholera, pseudorabies living vaccines
1 material
3 batches of 1.1 test vaccine swine fevers, pseudorabies bigeminal live vaccines, lot number: CSF-PR200801, CSF-PR200802, CSF-PR200803, test with reference to existing " Chinese veterinary pharmacopoeia " method of inspection qualified;
3 batches of live vaccines of hog cholera, lot number: CSF200801, CSF200802, CSF200803, test by existing " Chinese veterinary pharmacopoeia " " live vaccines of hog cholera (cell source) " method of inspection qualified;
3 batches of pseudorabies living vaccines, lot number: PR200801, PR200802, PR200803, test by existing " Chinese veterinary pharmacopoeia " " pseudorabies living vaccines " method of inspection qualified.
1.2 test animal
1.2.1 rabbit to derive from Guangdong Winsun Bio-Pharmaceutical Co., Ltd. animal used as test field white from the large ear of New Zealand of numerous autotrophy, select the Healthy Rabbits that body weight is 1.5 ~ 3.0kg.
1.2.2 pig derives from Guangdong Winsun Bio-Pharmaceutical Co., Ltd. animal used as test field and did not inoculate the bi-crossbreeding of CSF vaccine and PR vaccine from numerous autotrophy without CSF, PR, PP, PC-II, PRRS medical history. Detect through neutralization test method, select the healthy susceptible pig of swine fever and pseudoabies neutralizing antibody feminine gender.
2 methods and result
2.1 physical behavior inspections visually observe vaccine physical behavior. Three groups of each 3 batches of vaccines are faint yellow Sponge Porosity agglomerate, easily depart from bottle wall, dissolve rapidly after adding dilution.
2.2 steriling tests are tested by existing " Chinese veterinary pharmacopoeia " annex, three groups of each 3 batches of equal asepsis growths of vaccine.
2.3 mycoplasma inspections are tested by existing " Chinese veterinary pharmacopoeia " annex, and three groups of each 3 batches of vaccines are all grown without mycoplasma.
2.4 diagnostic tests do suitable dilution by swine fever, pseudorabies bigeminal live vaccine with serum free medium, fully mix with anti-swine fever, the pseudorabies virus specific serum of equivalent, put in room temperature and 1 hour jolting therebetween 2 ~ 3 times. After neutralization, two of inoculation rabbits, every rabbit ear vein injection 1.0ml, thermometric is observed and is judged and undertaken by existing " Chinese veterinary pharmacopoeia ". Pigs Inoculated testis (ST) passage cell 6 bottles (hole) simultaneously, observation of cell pathology (CPE). Result inoculation rabbit is without thermal response, and CPE does not appear in inoculation ST cell.
2.5 exogenous virus inspections are tested by existing " Chinese veterinary pharmacopoeia " annex, and three groups of each 3 batches of vaccines all pollute without exogenous virus.
2.6 safety verifications are selected (before note seedling, to observe 5 each survey morning and afternoon every day body temperature 1 time through the detection of neutralization test method without hog cholera antibody with without the healthy susceptible piglets of 10 ages in days of pseudoabies antibody. Select spirit, appetite, the normal person's use of body temperature). Each 3 batches of three groups of vaccines are diluted to every milliliter containing 4 part vaccines with phosphate buffer (PBS), 5 of intramuscular injection piglets respectively, every 5.0ml (containing 20 parts). After note seedling, respectively observe morning and afternoon every day and survey body temperature 1 time, observing 21. Spirit, appetite, body temperature do not have significant change compared with before note seedling, and without swine fever and pseudoabies clinical symptoms. Detailed results is in table 5.
The comparison of table 5 safety verification result
As known from Table 5, carry out safety verification by existing " Chinese veterinary pharmacopoeia ", 3 batches of swine fevers, pseudorabies bigeminal live vaccine and 3 batches of live vaccines of hog cholera and 3 batches of pseudorabies living vaccines are to all safety of pig.
Efficacy test
2.7.1 the effect comparison of swine fever, pseudorabies bigeminal live vaccine and live vaccines of hog cholera
2.7.1.1 imitate inspection with rabbit
2.7.1.1.1 by every part swine fever, 100 times of phosphate buffer (PBS) dilutions for pseudorabies bigeminal live vaccine, mix with the anti-pseudorabies virus specific serum of equivalent, at room temperature with 1 hour, jolting therebetween 2 ~ 3 times. After neutralization, be 2000 times, 4000 times, 7500 times, 10000 times by every part vaccine dilution, each dilution factor is injected respectively 2 of rabbits, every rabbit ear vein injection 1.0ml, and thermometric is observed and is judged and undertaken by existing " Chinese veterinary pharmacopoeia ". Detailed results is in table 6.
2.7.1.1.2 by every part for live vaccines of hog cholera phosphate buffer (PBS) dilution be 2000 times, 4000 times, 7500 times, 10000 times, each dilution factor is injected respectively 2 of rabbits, every rabbit ear vein injection 1.0ml, thermometric is observed and is judged and undertaken by existing " Chinese veterinary pharmacopoeia ". Detailed results is in table 6.
Table 6 is imitated the comparison of inspection result with rabbit
Note: 1.++ is sizing thermal response ,+be slight fever reaction, ± be suspicious reaction ,-be reactionless;
2.(+) for attacking malicious rear thermal response, (±), for attacking the rear suspicious reaction of poison, (-) is rear reactionless for attacking poison.
As known from Table 6, the swine fever effect of the 3 batches of swine fevers, pseudorabies bigeminal live vaccine can reach 10,000RID/ head part; And in 3 batches of live vaccines of hog cholera (cell source) 2 batches can only reach 2000RID/ head part, wherein 1 batch can reach 4000RID/ head part. Result shows, aspect CSFV content, swine fever, pseudorabies bigeminal live vaccine are obviously high than live vaccines of hog cholera.
2.7.1.2 imitate inspection with pig
2.7.1.2.1 by every part swine fever, for pseudorabies bigeminal live vaccine, phosphate buffer (PBS) is diluted to 150 times, mix with the anti-pseudorabies virus specific serum of equivalent, at room temperature with 1 hour, jolting therebetween 2 ~ 3 times. After neutralization, be 300 times, 3000 times by the dilution of every part vaccine, intramuscular injection is without 5 of the healthy susceptible pigs of swine fever neutralizing antibody respectively for each dilution factor, and every l.0ml. After immunity 14 days, together with 5 of contrast pigs, the strong arsenic bloom door of injection swine fever is blood poison 1.0ml/ head (105MLD), observe 16. Immune swine is strong living all, 5 all morbidities of contrast pig, attack in the poison observation period all dead. Detailed results is in table 3.
2.7.1.2.2 by every part for live vaccines of hog cholera phosphate buffer (PBS) dilution be 300 times, 3000 times, each dilution factor respectively intramuscular injection without each 5 of the healthy susceptible pig of swine fever neutralizing antibody, every 1.0ml. After immunity inoculation 14 days, together with 5 of the identical contrast pigs of condition, the strong arsenic bloom door of injection swine fever is blood poison 1.0ml/ head (105MLD), observe 16. 1/300 part immune swine is strong living all, 1/3000 part immune swine and each 5 all morbidities of contrast pig, attack in the poison observation period all dead. Detailed results is in table 7.
Table 7 is imitated inspection result comparison (attacking swine fever poison by force) with pig
As known from Table 7, the 3 batches of swine fevers, pseudorabies bigeminal live vaccine, with the pig of 1/3000 part immunity, all can be resisted strong virus attack; And 3 batches of live vaccines of hog cholera only could be resisted strong virus attack with the pig of 1/300 part immunity, and with the pig of 1/3000 part immunity, after swine fever strong virus attack, all pigs all demonstrate typical swine fever clinical symptoms, attack in the poison observation period all dead. Result shows, the immune efficacy of swine fever, pseudorabies bigeminal live vaccine is obviously high than live vaccines of hog cholera.
2.7.2 the effect comparison of swine fever, pseudorabies bigeminal live vaccine and pseudorabies living vaccines
2.7.2.1 use pig testis (ST) cell to imitate inspection
2.7.2.1.1 by every part swine fever, 100 times of serum free medium dilutions for pseudorabies bigeminal live vaccine, mix with the swine fever virus resistant specific serum of equivalent, at room temperature with 1 hour, jolting therebetween 2 ~ 3 times. After neutralization, be 10 by every part vaccine dilution5、106、107Doubly, each dilution factor is Pigs Inoculated testis (ST) cell 6 bottles (hole) respectively, and every bottle (hole) 0.1ml supplements maintenance medium 0.9ml, and making it total amount is 1.0ml. Observation of cell pathology (CPE), calculates TCID50. Detailed results is in table 8.
2.7.2.1.2 be 10 by every part pseudorabies living vaccines with serum free medium dilution4、105、106Doubly, each dilution factor is Pigs Inoculated testis (ST) cell 6 bottles (hole) respectively, and every bottle (hole) 0.1ml supplements maintenance medium 0.9ml, and making it total amount is 1.0ml. Observation of cell pathology (CPE), calculates TCID50. Detailed results is in table 8.
Table 8 is imitated the comparison of inspection result with cell
As known from Table 4, the 3 batches of swine fevers, for pseudorabies bigeminal live vaccine, pig testis (ST) cell is imitated inspection viral level and can be reached 106.4More than TCID50/ head part; And 3 batches of pig testis for pseudorabies living vaccines (ST) cell is imitated inspection, viral level is the highest can only reach 105.8TCID50/ head part. Result shows, aspect pseudorabies virus content, swine fever, pseudorabies bigeminal live vaccine are obviously high than pseudorabies living vaccines.
2.7.2.2 imitate inspection with pig
2.7.2.2.1 by every part swine fever, for pseudorabies bigeminal live vaccine, phosphate buffer (PBS) is diluted to 100 times, mix with the swine fever virus resistant specific serum of equivalent, at room temperature with 1 hour, jolting therebetween 2 ~ 3 times. After neutralization, it is 1000 times, 10000 times by every part vaccine dilution, each dilution factor respectively intramuscular injection 7 ~ 10 ages in days without 5 of the healthy susceptible piglets of pseudoabies neutralizing antibody, every 1.0ml, after 7 days together with 5 of the identical contrast piglets of condition, every strong malicious 1.0ml (10 of intramuscular injection pseudoabies3LD50), observe 14. Immune swine is strong living all, 5 all morbidities of contrast pig, attack in the poison observation period all dead. Detailed results is in table 9. .
2.7.2.2.2 by every part for pseudorabies living vaccines phosphate buffer (PBS) dilution be 1000 times, 10000 times, each dilution factor respectively intramuscular injection 7 ~ 10 ages in days without 5 of the piglets of pseudoabies neutralizing antibody, every 1.0ml, after 7 days together with 5 of the identical contrast piglets of condition, every strong malicious 1.0ml (10 of intramuscular injection pseudoabies3LD50), observe 14. 3 batches of swine fevers, pseudorabies bigeminal live vaccine immune swines are all protected; and 3 batches of pseudorabies living vaccines are only all protected with 1/1000 part immune swine; with the part protection of 1/10000 part immune swine, 5 all morbidities of contrast pig, attack in the poison observation period all dead. Detailed results is in table 9.
Table 9 is imitated inspection result comparison (attacking pseudoabies poison by force) with pig
As known from Table 9, the 3 batches of swine fevers, pseudorabies bigeminal live vaccine, with the pig of 1/10000 part immunity, all can be resisted pseudoabies strong virus attack. 3 batches of pseudorabies living vaccines can all be resisted pseudoabies strong virus attack with the pig of 1/1000 part immunity, and attacking after the strong poison of pseudoabies with the pig of 1/10000 part immunity, part pig presents typical pseudoabies clinical symptoms, attack poison the observation period in Mortality. Result shows, aspect vaccine immunity effect, swine fever, pseudorabies bigeminal live vaccine are obviously high than pseudorabies living vaccines.
2.8 residual moistures are measured and are tested by existing " Chinese veterinary pharmacopoeia " annex, and three groups of each 3 batches of residual moistures of vaccine are all less than 4%, all meet the regulation of veterinary biologics general rule.
2.9 vacuums are measured and are tested by existing " Chinese veterinary pharmacopoeia " annex, and three groups of each 3 batches of vacuums of vaccine all conform with the regulations.
Summary: this test compares research to the quality inspection such as safety and the effect project of swine fever, pseudorabies bigeminal live vaccine and live vaccines of hog cholera, pseudorabies living vaccines. Result shows, aspect viral level and immune efficacy, swine fever, pseudorabies bigeminal live vaccine all have obvious advantage than live vaccines of hog cholera and pseudorabies living vaccines.
3 conclusions
This test, with reference to existing " Chinese veterinary pharmacopoeia " method of inspection, compares research to the quality inspection such as safety and the effect project of swine fever, pseudorabies bigeminal live vaccine and live vaccines of hog cholera and pseudorabies living vaccines. Result shows, aspect physical behavior inspection, the inspection of pure property, diagnostic test, safety verification, residual moisture inspection and vacuum inspection, three groups of vaccines each 3 batches all qualified and meet the quality standard of existing " Chinese veterinary pharmacopoeia ". Aspect viral level, swine fever, pseudorabies bigeminal live vaccine are all high than live vaccines of hog cholera and pseudorabies living vaccines viral level separately; Aspect Immunization protection, swine fever, pseudorabies bigeminal live vaccine are all high than live vaccines of hog cholera and pseudorabies living vaccines Immunization protective rate separately. Therefore, swine fever, pseudorabies bigeminal live vaccine have very large advantage than live vaccines of hog cholera and pseudorabies living vaccines, not only anti-two diseases of a pin, and ratio good immune effect separately.
Embodiment 4 immune swine Immunization Protections
The present invention is used for preventing swine fever and pseudoabies; there is good immune effect; immunity is without the health pig of antibody against swine fever virus and pseudorabies virus antibody; its immune protective rate can reach 100%; all high than live vaccines of hog cholera (cell source) and pseudorabies living vaccines Immunization protective rate separately, in table 10.
The comparison of table 10 immune swine Immunization protection effect
The above, be only the preferred embodiments of the present invention, should be understood that; for the those of ordinary skill in this technology; not departing under the prerequisite of core technology feature of the present invention, can also make some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.
Claims (12)
1. a preparation method for swine fever, pseudorabies bigeminal live vaccine, is characterized in that, comprises the steps:
(1) with cell spinner bottle or bioreactor culture CSFV low virulent strain, pseudorabies virus low virulent strain;
(2) gather in the crops respectively the cell cultivation venom that described step (1) obtains;
(3) two kinds of cells that described step (2) obtains are cultivated venom and are mixed by the percent by volume of 1:2-1:1.5, and in mixed liquor, add stabilizing agent and antibiotic, obtain swine fever, pseudoabies cell live vaccine through vacuum freezedrying.
2. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 1, it is characterized in that, described bioreactor is TideCell microcarrier bioreactor, and contain microcarrier in described bioreactor, described microcarrier is cytodex series microcarrier, and the use density of microcarrier is 2-25g/L.
3. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 1, is characterized in that, described step (1) comprises the steps:
(1a) seedling going down to posterity and cultivating with cell: described passage cell, through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, continues to cultivate with cell growth medium, forms passage cell individual layer;
(1b) breeding of cell seed culture of viruses: described CSFV low virulent strain is seeded to the passage cell individual layer obtaining in described step (1a), continues to cultivate with cell maintenance medium; Change liquid once every results on the 4th～5, get two receipts, three and receive cell cultivation venom as production seed culture of viruses; Described pseudorabies virus low virulent strain is seeded to the passage cell individual layer obtaining in described step (1a), continues to cultivate with cell maintenance medium; After 2 ~ 3 days, harvesting is cultivated venom as production seed culture of viruses;
(1c) breeding of seedling venom:
Get the passage cell individual layer obtaining in described step (1a), discard described cell growth medium, inoculation, containing the cell maintenance medium containing pseudorabies virus obtaining in described step (1b), continues to cultivate.
4. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 1, is characterized in that, described step (2) comprises the steps:
After being seeded to passage cell individual layer containing the cell maintenance medium of CSFV, cultivate, connect poison and gather in the crops and change liquid for the first time for latter 5 days, change liquid once later every results on the 4th, the venom of results is placed in-15 DEG C of following preservations; After being seeded to passage cell individual layer containing the cell maintenance medium of pseudorabies virus, cultivate, connect after poison harvesting after 2 ~ 3 days and cultivate venom, the venom of results is placed in-20 DEG C of following preservations.
5. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 3, is characterized in that, it is 36 DEG C ~ 37 DEG C that the cell in described step (1a), (1b), (1c) is cultivated temperature used.
6. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 3, it is characterized in that, the inoculum concentration while inoculation in described step (1b), (1c) by volume percentage is counted the maintenance medium containing 1%～2% Cells for production seed culture of viruses.
7. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 3, it is characterized in that, cell growth medium in described step (1a) is containing 90%～92%MEM liquid, 8%～10% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell growth medium is 7.0～7.2, and wherein the unit of MEM liquid and calf serum is percent by volume.
8. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 3, it is characterized in that, described step (1b) and (1c) in cell maintenance medium containing 95%～98%MEM liquid, 2%～5% calf serum and 100 ~ 200 units/ml mycillin, the pH value of described cell maintenance medium is 7.2～7.4, and wherein the unit of MEM liquid and calf serum is percent by volume.
9. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 1, it is characterized in that, described passage cell is bull testis cell, CEF, pig testis subculture cells ST, porcine kidney cell PK15 or porcine kidney cell IBRS-2.
10. the preparation method of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 1, is characterized in that, the setup parameter of described bioreactor is: pH6.5-7.8,36 DEG C ~ 37 DEG C of temperature, dissolved oxygen 30-80%, mixing speed 30-100rpm.
11. preparation methods of swine fever, pseudorabies bigeminal live vaccine as claimed in claim 1, it is characterized in that, the cultivation of described bioreactor adopts batch formula, stream to add or the mode of perfusion cultures, and the speed of perfusion is 0.5-5 working volume every day according to the density of cell.
12. 1 kinds of swine fevers, pseudorabies bigeminal live vaccine, is characterized in that, described swine fever, pseudorabies bigeminal live vaccine make by the arbitrary described method of claim 1-11.
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| CN109453386A (en) * | 2018-12-26 | 2019-03-12 | 瑞普（保定）生物药业有限公司 | A kind of freeze drying protectant, pseudorabies disease live-vaccine and preparation method thereof |
| CN111035756A (en) * | 2019-12-23 | 2020-04-21 | 武汉科前生物股份有限公司 | Porcine pseudorabies virus and porcine epidemic diarrhea virus bivalent inactivated vaccine and preparation method thereof |
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| CN101695572A (en) * | 2009-10-26 | 2010-04-21 | 广东永顺生物制药有限公司 | Method for producing pseudorabies attenuated vaccine by using bioreactor and pseudorabies attenuated vaccine product |
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| CN101695572A (en) * | 2009-10-26 | 2010-04-21 | 广东永顺生物制药有限公司 | Method for producing pseudorabies attenuated vaccine by using bioreactor and pseudorabies attenuated vaccine product |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106318899A (en) * | 2016-08-30 | 2017-01-11 | 中国兽医药品监察所 | Construction and application of bovine testis passage cell strain |
| CN106318899B (en) * | 2016-08-30 | 2019-05-28 | 中国兽医药品监察所 | The foundation and its application of one plant of bull testis passage cell strain |
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| CN109453386B (en) * | 2018-12-26 | 2022-01-25 | 瑞普（保定）生物药业有限公司 | Freeze-drying protective agent, porcine pseudorabies live vaccine and preparation method thereof |
| CN111035756A (en) * | 2019-12-23 | 2020-04-21 | 武汉科前生物股份有限公司 | Porcine pseudorabies virus and porcine epidemic diarrhea virus bivalent inactivated vaccine and preparation method thereof |
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