CN103571797A - Hog cholera cell adaptive virus, hog cholera virus live vaccine prepared from hog cholera cell adaptive virus, and preparation method of hog cholera virus live vaccine - Google Patents
Hog cholera cell adaptive virus, hog cholera virus live vaccine prepared from hog cholera cell adaptive virus, and preparation method of hog cholera virus live vaccine Download PDFInfo
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Abstract
The invention relates to a hog cholera cell adaptive virus, a hog cholera virus live vaccine prepared from the hog cholera cell adaptive virus, and a preparation method of the hog cholera virus live vaccine. In the method, the hog cholera cell adaptive virus is used as a vaccine preparing virus seed, an original virus seed, a basic virus seed and a production seed of the hog cholera cell adaptive virus are prepared by continuous passage cell culture, in the multiplication of the production virus seed of the hog cholera cell adaptive virus in a passage cell line, a qualified virus solution as a hog cholera attenuated vaccine antigen is continuously obtained, and the hog cholera cell adaptive live vaccine is prepared by distributing, packaging, and freezing-drying; due to the adoption of the method, a rabbit passage is not required for preparing the virus seed in the preparation of the hog cholera virus seed, the animal welfare problem of killing rabbits and other animals in order to prevent the hog cholera is solved, the possibly carried exogenous factor and a latent virus-disperse risk in the preparation of the virus vaccine are reduced. When preparing the hog cholera virus live vaccine by using the method, the virus breeding time is short, the gaining time is large, the amount of the virus solution obtained from unit cell is high, the virus content is high and the production cost is low. The prepared hog cholera virus live vaccine has the preferable safety and the high immune efficacy.
Description
Technical field
The invention belongs to domestic animals biological pharmacy technical field, the Pestivirus suis living vaccine and the preparation method that relate to the cell adapted poison of a kind of swine fever, by it, are made.
Background technology
Swine fever is a kind of height contagious disease being caused by Pestivirus suis (Classical swine fever virus, CSFV), and the pig of different ages, sex and kind all can infect, and mortality ratio is up to 80%～90%.Swine fever is divided into one of category-A disease by International Animal Health tissue, and China is divided into a class disease.It is at present generally acknowledged safety and effectively vaccine in the world that Chinese scholar cultivates successful hog cholera lapinised virus seedling, and by means of the intensive inoculation of C strain vaccine and comprehensive measures for prevention and control, the countries concerned have controlled swine fever effectively, have even eliminated swine fever.In China, the typical swine fever of the popular appearance of swine fever and atypia swine fever coexist, persistent infection and inapparent infection coexists, immunological tolerance and band poison syndrome coexists etc., and therefore, the popular form that swine fever is new has proposed new challenge to the anti-disease of effecting a permanent cure of China's pig industry.
Vaccine inoculation is still the major measure of the current anti-swine fever processed of China, and within 2007, the Ministry of Agriculture has put into effect the policy of swine fever being carried out to compulsory immunization.China produces the cell that hog cholera lapinised virus cell vaccine uses at present bull testis cell primary cell and pig testis clone (ST cell).But these vaccines still need to breed swine fever spleen poison as seed culture of viruses with a large amount of rabbits in production practice, when preparing swine fever spleen poison, owing to connecing malicious rabbit individual difference, easily cause swine fever kind poison preparation technology unstable, while spleen poison complicated process of preparation, aseptic being difficult to controlled, and also has danger and the animal welfare problem of potential loose poison.
Chinese patent CN 101181637A discloses a kind of method with producing swine fever live vaccine with cell line, the method selects continuous cell line as seedling cell, and the going down to posterity and cultivating with cell to seedling, then carry out the breeding of cell seed culture of viruses and the breeding of seedling venom, finally join seedling, packing and freeze-drying and produce live vaccines of hog cholera.The method is to adopt ST clone when breeding Pestivirus suis " C strain " is produced seed culture of viruses, but when the basic seed culture of viruses of breeding, still need to use a large amount of rabbits, and the method receives when the breeding of swine fever seedling venom that the poison time is long, harvesting frequency is less, the virus liquid of unit cell results is few.In addition, the method viral level is low, and vaccine cost is high.
Therefore, the problem existing is at present that need to research and develop a kind of viral level high, the Pestivirus suis living vaccine that cost is low, and a kind ofly when prepared by swine fever seed culture of viruses, do not re-use rabbit and breed Pestivirus suis, and the viral proliferation time is short, harvesting frequency is many, the large Pestivirus suis living vaccine preparation method of viral liquid measure of unit cell results.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provide a kind of swine fever cell adapted poison, the Pestivirus suis living vaccine and the preparation method that by it, are made, the method is used Pestivirus suis to adapt to poison as preparing swine fever attenuated vaccine seed culture of viruses, through passage cell cultured continuously, prepare the cell adapted toxogen of swine fever beginning seed culture of viruses, the basic seed culture of viruses of the cell adapted poison of swine fever, the cell adapted poison of swine fever is produced seed culture of viruses, the qualified virus liquid of breeding rear results continuously by the cell adapted poison production of swine fever seed culture of viruses at continuous cell line is again as swine fever attenuated vaccine antigen, again through joining seedling, packing, freeze-drying makes the cell adapted virus live vaccine of swine fever.The method does not re-use rabbit and breeds Pestivirus suis when prepared by swine fever seed culture of viruses, and the viral proliferation time is short, harvesting frequency is many, and the viral liquid measure of unit cell results is large, and viral level is high, with low cost.
For this reason, the invention provides the cell adapted poison of a kind of swine fever, is to be obtained after continuous cell line cultured continuously by fever virus lapinized Chinese Strain, and by after the intravenous inoculation healthy rabbits 1ml of its ear source, its body temperature is reactionless or only occur slight fever reaction.
According to the present invention, described adaptation poison is the cell adapted CC strain of Pestivirus suis (Classical Swine Fever Cell Adapted Virus strain CC) of fever virus lapinized Chinese Strain, its deposit number is: CCTCC NO:V201224, is preserved in Chinese Typical Representative culture collection center and (is called for short: CCTCC; Address: No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University).Preservation date: on 06 08th, 2012.
The cell adapted CC strain of Pestivirus suis described in the present invention has following characteristic:
I) stability in continuous cell line: described cell adapted poison is not less than 10 at continuous cell line 20～40 generation scope increment viral level
7.0tCID
50/ ml;
Ii) rabbit body temperature is reactive: described cell adapted poison is with 10
7.0tCID
50after the intravenous inoculation healthy rabbits 1ml of/ml ear source, its body temperature is reactionless or only occur slight fever reaction;
Iii) immunogenicity: minimum immunogenicity≤10 of described cell adapted poison to pig
-7.0/ ml, with 1 TCID
50the negative health pig of immunizing dose intramuscular inoculation hog cholera antibody, after immune two weeks, attacking 1ml swine fever crossdrift is blood poison (10
5mLD/ml) 100% protection;
Iv) security: by described cell adapted poison with 10
7.0tCID
50/ ml intramuscular injection piglet or pregnant sow 10ml injection do not cause morbidity or dead, and sow does not cause miscarriage or stillborn foetus.
The present invention further provides a kind ofly according to the preparation method of the cell adapted poison of swine fever of the present invention, it accesses at least 20 generations of continuous cell line cultured continuously by fever virus lapinized Chinese Strain, and described incubation time is 24h～72h/ generation.
The present invention also provides a kind of Pestivirus suis living vaccine making according to the cell adapted poison of swine fever of the present invention.
It is a kind of according to the preparation method of Pestivirus suis living vaccine of the present invention that the present invention also further provides, and comprising:
Steps A, prepares attenuated vaccine antigen;
Step B, adds suitable lyophilized vaccine by preparing swine fever attenuated vaccine with antigen, fully mixes rear quantitative separating, and freeze-drying, makes Pestivirus suis living vaccine;
Wherein, in steps A, the cell adapted poison of swine fever is produced to seed culture of viruses access continuous cell line and cultivate, harvested cell supernatant liquor is after the assay was approved as preparing swine fever attenuated vaccine antigen continuously.
According to the inventive method, in steps A, the cell adapted poison of swine fever is produced seed culture of viruses and is adapted to and be no more than for 1 generation in continuous cell line cultivation.Described continuous results comprise produces seed culture of viruses access passage cell by the cell adapted poison of swine fever, cultivates harvested cell supernatant liquor after 2～5 days, and adds maintenance medium and continue the cyclical operation of cultivating, and the number of times of described circulation is 8～10 times.
In one embodiment of the invention, be also included in steps A following steps before:
Step 1, prepares original seed culture of viruses: the cell adapted poison access of swine fever continuous cell line is cultivated, and harvested cell supernatant liquor is after the assay was approved as the cell adapted toxogen of swine fever beginning seed culture of viruses;
Step 2, prepares basic seed culture of viruses: the cell adapted toxogen of swine fever beginning seed culture of viruses access continuous cell line is cultivated, and harvested cell supernatant liquor is after the assay was approved as the basic seed culture of viruses of the cell adapted poison of swine fever;
Step 3, seed culture of viruses is produced in preparation: the basic seed culture of viruses access of the cell adapted poison of swine fever continuous cell line is cultivated, and harvested cell supernatant liquor is produced seed culture of viruses as the cell adapted poison of swine fever after the assay was approved.
And in step 1, the cultivation of the cell adapted poison of swine fever in continuous cell line was no more than for 3 generations.In step 2, the cultivation of seed culture of viruses of cell adapted toxogen beginning of swine fever in continuous cell line was no more than for 3 generations.In step 3, the cultivation of the basic seed culture of viruses of the cell adapted poison of swine fever in continuous cell line was no more than for 3 generations.Described incubation time is 24h～72h/ generation.
In the present invention, described passage cell is ST clone, PK15 clone, STK clone or BT clone.Preferred described passage cell is BT clone.Particularly, the present invention is cultivating the cell adapted poison of swine fever and is preparing the cell adapted poison of the cell adapted toxogen of swine fever beginning seed culture of viruses, the basic seed culture of viruses of the cell adapted poison of swine fever and swine fever and produce in seed culture of viruses process, adopt the clone cultivation of going down to posterity, passage cell is ST clone, PK15 clone, STK clone or BT clone.Preferred described passage cell is BT clone.
The present invention also provides a kind of method of cultivating Pestivirus suis simultaneously, and it adopts the continuous cell line cultivation of going down to posterity, and wherein, described passage cell is BT clone.
In the present invention, with rabbit, vaccine potency is imitated to inspection.The head part of pressing defined dilutes 7500 times by stroke-physiological saline solution by every part vaccine, and ear vein injection body weight is 2 of 1.5～3kg rabbit, every 1ml.After family's rabbit inoculation, respectively survey body temperature morning and afternoon 1 time, after 48 hours, every 6 hours, survey body temperature 1 time, its body temperature reaction normal is as follows:
Sizing thermal response: in 48～96 hours latent period, body temperature rise is obvious curve, has at least 3 temperature time to surpass normal temperature more than 1 ℃, and delays 18～36 hours.As delay more than 42 hours, must attack poison, attack the rear reactionless sizing thermal response that is judged to of poison.
Slight fever reaction: in 48～96 hours latent period, body temperature rise is obvious curve, has at least 2 temperature time to surpass normal temperature more than 0.5 ℃, and delays 12～36 hours.
Reactionless: body temperature is normal.
In addition, 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 body temperature reaction can be judged to sizing thermal response or slight fever reaction.
The present invention uses Pestivirus suis to adapt to poison as preparing swine fever attenuated vaccine seed culture of viruses, through passage cell cultured continuously, prepare the cell adapted toxogen of swine fever beginning seed culture of viruses, the basic seed culture of viruses of the cell adapted poison of swine fever, the cell adapted poison of swine fever is produced seed culture of viruses, the qualified virus liquid of breeding rear results continuously by the cell adapted poison production of swine fever seed culture of viruses at continuous cell line is again as swine fever attenuated vaccine antigen, again through joining seedling, packing, freeze-drying makes the cell adapted virus live vaccine of swine fever, the method no longer needs to go down to posterity to prepare seed culture of viruses with rabbit when prepared by swine fever seed culture of viruses, solved as preventing swine fever to massacre the animal welfare problems such as rabbit and reducing the risk that may carry exogenous factor and potential loose poison in the preparation of spleen poison.Pestivirus suis living vaccine prepared according to the methods of the invention, the viral proliferation time is short, harvesting frequency is many, and the viral liquid measure of unit cell results is large, and viral level is high, and can reduce production of vaccine cost.The security of prepared Pestivirus suis living vaccine is good, immune efficacy is high.
culture presevation
The cell adapted CC strain of Pestivirus suis (Classical Swine Fever Cell Adapted Virus strain CC),, evaluation separated by Pulaike Biological Engineering Co., Ltd., (be called for short: CCTCC at Chinese Typical Representative culture collection center; Address: carry out preservation, preservation date No. 16, Luo Jia Shan road, Wuchang District, Wuhan City, Hubei Province Wuhan University): on 06 08th, 2012, deposit number: CCTCC NO:V 201224.
For the strong bacterial classification of acquired immunity, the freeze-drying after responsive pig body rejuvenation accreditation of each bacterial classification is preserved and preservation.
Embodiment
Below in conjunction with embodiment, describe the present invention in detail, these embodiment only play illustrative effect, are not limited to range of application of the present invention.
Embodiment
Embodiment 1: hog cholera lapinised virus adapts to the screening of cell
1. virus and cell strain
Hog cholera lapinised virus is P480 for spleen poison (China Veterinery Drug Inspection Office), and BT cell (China Veterinery Drug Inspection Office) is as adapting to virus clone.ST cell (ATCC, American type culture collection, US mode culture collection warehousing), PK15 cell (China Veterinery Drug Inspection Office), IBRS-2 cell (China Veterinery Drug Inspection Office), bull testis primary cell (self-control), SK6 cell (ATCC).
2. experimental technique
(1) hog cholera lapinised virus proliferation test in cell
1. the preparation method of bull testis cell
1 pair, the aseptic testis of getting new-born calve, be soaked in Hank ' the s liquid that contains 1000U/ml penicillin, under aseptic condition, remove packing and testis cyst membrane, then add 0.25wt% trysinization liquid to digest, after digestion, add the substratum containing serum to stop digestion, then with suction pipe, cell is dispelled.Last cell suspension filters with sterile gauze, and the cell suspension of getting after filtration carries out cell counting, and then adjusting cell density is that 500,000 cells/ml is laid in culturing bottle, is placed in 37 ℃ containing 5%CO
2incubator in cultivate.After cultivating into individual layer, be primary cell, can be used for connecing poison or go down to posterity.
2. prepare 6 kinds of cell monolayer cells
Cell, with after the trysinization containing 0.25wt% or 0.5wt%EDTA (ethylenediamine tetraacetic acid (EDTA)), is assigned to new Tissue Culture Flask with the ratio of 1: 2～1: 4, adds cell growth medium (containing the MEM substratum of 5wt% foetal calf serum), at 37 ℃, contains 5%CO
2incubator in cultivate 2～3, cell forms individual layer.
3. get hog cholera lapinised virus spleen poison, by 0.3%～0.5% (v/v) access, covered with 6 kinds of cells of individual layer, at 37 ℃, contain 5%CO
2incubator cultivate 48～72 days directly frozen, after freeze thawing 2 times, carry out respectively subculture for the second time, subculture is 20 times continuously.The every subculture 5 of virus substitutes the mensuration that identical ST cell carries out viral level, and relatively the use ability of Pestivirus suis after the continuous subculture of different cells, determines the suitable clone of the breeding cell adapted poison of swine fever.
(2) immunization is measured viral level indirectly
Get virus liquid to be measured and do ten times of serial dilutions, access 96 porocyte culture plates, then with suction pipe, splash into postdigestive ST cell, every hole 2.0 ten thousand cells, are then placed in Tissue Culture Plate 37 ℃ and cultivate 3, carry out fluorescent dye judgement.This porocyte of i.e. judgement that occurs fluorescence infects, and by R-M method, calculates TCID
50.
3. experimental result
The competence for added value measurement result of hog cholera lapinised virus in 6 kinds of cells is in Table 1.
Table 1 hog cholera lapinised virus is in the difference of different cell increment viral levels
| The different generations of Pestivirus suis | P5 | P10 | P15 | P20 |
| BT cell | 3.5 | 5.5 | 6.5 | 7.2 |
| ST cell | 3.0 | 5.1 | 6.4 | 6.5 |
| PK15 cell | 2.5 | 5.0 | 5.8 | 6.2 |
| IBRS-2 cell | 2.0 | 4.5 | 4.8 | 5.2 |
| SK6 cell | 2.3 | 3.2 | 3.5 | 3.8 |
| Bull testis primary cell | 3.0 | 4.5 | 5.5 | 6.0 |
According to experimental result, show that BT cell competence for added value is the strongest, measuring, each generation viral level is the highest, so the present invention adopts BT cell to prepare the cell adapted malicious vaccine of swine fever.
Embodiment 2: the cell adapted malicious domestication process of hog cholera lapinised virus
1. experiment material
(1) virus and cell strain
Hog cholera lapinised virus is P480 for spleen poison (China Veterinery Drug Inspection Office), and BT cell (China Veterinery Drug Inspection Office) is as adapting to virus clone.
(2) pig is used in test
Test is raised family scattered, be there is no the responsive pig of health that Pestivirus suis contacts with swine Fever Vaccine with the pig rural area of all originating.Before using, by a pig blood drawing, through neutralization test method, detect, swine fever neutralizing antibody is negative.By PCR method, detect swine fever antigen, porcine circovirus 2 type and pig breeding dysfunction and breathing syndrome virus is all negative.
2. preparation method
(1) prepare BT monolayer cell
Cell, with after the trysinization containing 0.25wt% or 0.5wt%EDTA, is assigned to new Tissue Culture Flask with the ratio of 1: 2～1: 4, adds cell growth medium (containing the MEM substratum of 5wt% foetal calf serum), at 37 ℃, contains 5%CO
2incubator in cultivate 2～3, cell forms individual layer.
(2) get hog cholera lapinised virus spleen poison, the nose of an ox first osteocyte system (BT cell) that has covered with individual layer by 0.3%～0.5% (v/v) access, contains 5%CO at 37 ℃
2incubator cultivate 3～5 days directly frozen, after freeze thawing 2 times ,-20 ℃ save backup.
(3) cell toxicant of getting the 1st generation of preserving in step 2 has covered with the BT cell of individual layer by 0.3%～0.5% (v/v) access, at 37 ℃, contain 5%CO
2incubator cultivate 3～5 days directly frozen, after freeze thawing 2 times, 20 ℃ save backup.Cell toxicant as 2nd generation.So allow virus adapt to 30 generations continuously.
(4) indirect immunization is measured the method for viral level
Get virus liquid to be measured and do ten times of serial dilutions, access 96 porocyte culture plates, then with suction pipe, splash into postdigestive ST cell, every hole 2.0 ten thousand cells, then allow Tissue Culture Plate be placed in 37 ℃ and cultivate 3, carry out fluorescent dye judgement.Occur fluorescence and judge that this porocyte infects, by R-M method calculating TCID
50.
(5) the immune immunogenicity determining method of cell adapted poison to pig
Get the cell adapted poison of swine fever and with physiological saline, do 10 respectively
-4.0, 10
-4.5, 10
-5.0, 10
-5.5, 10
-6.0, 10
-6.5, 10
-7.0, 10
-7.5dilution, after dilution, difference intramuscular injection is without the healthy susceptible pig of swine fever antigen and antibody, 4 of each extent of dilution, every 1.0ml.After 10～14 days, together with 4 of the identical contrast pigs of condition, injection swine fever crossdrift is blood poison 1.0ml/ head (10
5mLD), observe 16, and then judge the Minimal Protective dosage that can allow pig protect completely.
(6) cell adapted virulence returns strong test
Get the P40 cell adapted poison in generation, its viral level of indirect immunofluorescence, then allows its viral level be adjusted into 10
7.0tCID
50/ ml, 4 of intramuscular injection swine fever antigen and negative antibody pigs, every 1ml.Observe state of health and the body temperature response situation of pig every day, immunity was cutd open and is killed lymph sample and the tonsilla of getting respectively every pig after 7 days, after even after grinding, with physiological saline, be mixed with 10 times of emulsions, then distinguish each 1 of intramuscular injection swine fever antigen and negative antibody health pig, so continuously return 7 generations of susceptible pig, and then judge whether the cell adapted poison of swine fever continuous blind passage in health pig body strengthens the virulence of pig.
Result shows, cell adapted poison returns 7 generations of susceptible pig continuously, and temperature of pig body is all reactionless, does not occur the symptom of swine fever, cut open inspection after histoorgan without observing and histologic lesion, show that the cell adapted poison of swine fever is safe within 40 generations.
(7) Pestivirus suis adaptation starts to measure every 5 methods that substitute by (4) indirect immunofluorescence method mensuration viral level and in by 2.5 the Minimal Protective dosage that pig is protected completely.The results are shown in Table 2:
The cell adapted malicious viral level of the different generation swine fevers of table 2 and minimum immune dosage are measured
Cell adapted to surveying viral level and pig by indirect immunofluorescence after 20 generations, to measure minimum immune dosage result all more stable at BT for hog cholera lapinised virus.Simultaneously also safer according to the cell adapted poison in 40 generations of result swine fever of (6), return continuously 7 generations of susceptible pig can not return by force.Therefore, 20～30 generations cell use poison can be used as seed culture of viruses and virus liquid for seedling.Take the cell adapted CC strain of the cell adapted 20 generation Pestivirus suis called after Pestivirus suis of BT, and be preserved in Chinese Typical Representative culture collection center, preserving number is: V201224.
Embodiment 3: the different generations of the cell adapted CC strain of Pestivirus suis cause the mensuration of rabbit body temperature reaction
1. test materials
(1) virus: the cell adapted poison of swine fever (P20 generation, P30 generation, P40 generation), hog cholera lapinised virus (P480, China Veterinery Drug Inspection Office);
(2) cell: nose of an ox first osteocyte (BT cell);
(3) experimental animal: 10 3～4 monthly age large ear rabbits (Henan Province's Experimental Animal Center).
2. test method
(1) mensuration of laboratory animal hog cholera antibody
By indirect hemagglutination, suppress test kit (Lanzhou veterinary institute) and measure rabbit serum hog cholera antibody titre, hog cholera antibody titre is less than 1: 2 for animal subject.
(2) indirect immunofluorescence assay method
BT cell trysinization by logarithmic phase, makes cell dilution to 2.0 * 10 after digestion
5/ ml, every hole 100 μ L are taped against in 96 orifice plates, synchronously access 10
-3～10
-6dilution hog cholera venom, every hole 100ml, 8 holes of each extent of dilution.37 ℃ of 5%CO
2incubator was cultivated after 3～4 days, discard substratum, PBS washes twice, uses ice acetone fixed cell 10 minutes, then add the primary antibodie (swine fever positive antibody) of having diluted, hatch after 2h for 37 ℃, PBS washes 5 times, adds diluted two anti-, hatch 1h for 37 ℃, PBS washes 5 times, then under inverted microscope, observes the fluorescence in each hole, utilizes Reed-Muench Liang Shi method to calculate viral titre.
(3) get P20 generation, P30 generation and P40 generation cell adapted poison use respectively indirect immunofluorescence (IFA) to measure Pestivirus suis content.
(4) the cell adapted viral disease venom of 3 generation swine fever is made respectively to 10 times of serial dilutions with sterile saline, then the virus liquid ear vein of getting after dilution is injected 2 of body weight 1.5～3kg rabbits, every 1ml, after inoculation, respectively survey body temperature morning and afternoon 1 time, after 48h, every 6h, survey body temperature 1 time, according to the response situation of body temperature with attack malicious result and comprehensively judge.Use cell adapted front P480 hog cholera lapinised virus as positive control, physiological saline is as negative control simultaneously.
3. experimental result
(1) tested experimental rabbit is all less than 1: 2 with indirect hemagglutination inhibition test kit measurement swine fever coagulation antibody, can be for further experiment.
(2) the cell adapted viral disease poison assay of 3 generations of swine fever and different extension rate cause rabbit body temperature response situation in Table 3.
The different generation cell toxicants of table 3 cause the mensuration of rabbit body temperature reaction
Note: " ++ " represents that rabbit presents sizing thermal response; "+" represents that rabbit body temperature presents slight fever reaction; "-" represents that rabbit body temperature is reactionless.
According to the cell adapted CC strain of the different generation Pestivirus suis of experimental result to rabbit body temperature response capacity all a little less than, after 3 the cell adapted toxogen liquid inoculation of generation rabbit, only there is slight fever reaction or reactionless in its body temperature, this and original hog cholera lapinised virus have essential difference, and former P480 still can cause that for 100,000 times of hog cholera lapinised virus dilutions sizing thermal response or slight fever reaction appear in rabbit body temperature.
Embodiment 4: the preparation of the cell adapted CC strain of Pestivirus suis attenuated vaccine
1. virus and cell strain
Strain for the preparation of the cell adapted poison of swine fever is the later Pestivirus suis of P20 generation that embodiment 1 adapts to.(through test, this cell adapted poison has good immunogenicity and security, and can produce higher malicious valency in cell kind, and the present embodiment selects the cell adapted malicious CC strain in P20 generation to be preserved in Chinese Typical Representative culture collection center, and preserving number is: V201224.)。This virus strain is there is to the nose of an ox first osteocyte (BT cell) of good sensitivity, as seedling clone, use and infect and a large amount of Pestivirus suis.Attack poison seed culture of viruses.Tiring with strong poison is that classical swine fever virus Shimen is blood poison (China Veterinery Drug Inspection Office), and attacking toxic agent amount is that 1.0ml/ head is (containing 10
5.0mLD (medium lethal dose))
2. preparation method and test-results
(1) plant poison preparation
1. prepare BT monolayer cell: cell is with after the trysinization containing 0.25wt% or 0.5wt%EDTA (ethylenediamine tetraacetic acid (EDTA)), with the ratio of 1: 2～1: 4, assign to new Tissue Culture Flask, add cell growth medium (containing the MEM substratum of 5wt% foetal calf serum), at 37 ℃, contain 5%CO
2incubator in cultivate 2～3, cell forms individual layer.
2. get pig cell and adapt to the malicious BT cell that has covered with individual layer by 0.3%～0.5% (v/v) access of poison, at 37 ℃, contain 5%CO
2incubator cultivate 48～72 days directly frozen, after freeze thawing 2 times ,-20 ℃ of preservations are as kind of a poison.
Kind malicious check: plant the malicious method of inspection and test by the relevant regulations of the 15th, 19,20 pages of the veterinary drug allusion quotation > > of < < People's Republic of China (PRC) version appendix in 2005.The inventive method preparation kind of poison meets the requirements completely, and pig is had no side effect safely, without bacterium, mould, mycoplasma and exogenous virus, pollutes.By indirect immunofluorescence, measure tiring as every 1.0ml is containing virus>=10 of virus
7.0tCID
50.
(2) preparation of virus liquid for seedling
1. prepare BT monolayer cell: cell, with after the trysinization containing 0.25wt% or 0.5wt%EDTA, is assigned to new Tissue Culture Flask with the ratio of 1: 2～1: 4, add cell growth medium (containing the MEM substratum of 5wt% foetal calf serum), at 37 ℃, contain 5%CO
2incubator in cultivate 2～3, cell forms individual layer.
2. get pig cell and adapt to basic seed culture of viruses and covered with the BT cell of individual layer by 0.3%～0.5% (v/v) access, at 37 ℃ containing 5%CO
2it is directly frozen that incubator cultivation is got supernatant liquor on 48th～72, and add maintenance medium and continue to cultivate, and every 48h results once, gathers in the crops continuously 10 times later, and the virus liquid of results is placed in-20 ℃ of preservations.
The check of virus liquid for seedling: seedling is tested by the relevant regulations of 15,19,20 pages of the veterinary drug allusion quotation > > of < < People's Republic of China (PRC) version appendix in 2005 by the method for inspection of virus liquid.The inventive method is prepared virus liquid and is met the requirements completely, and pig is had no side effect safely, without bacterium, mould, mycoplasma and exogenous virus, pollutes.By indirect immunofluorescence (IFA), measure tiring of virus, the virus liquid content of 10 results is in Table 4.
The variation that the cell adapted poison of table 4 swine fever is gathered in the crops viral level continuously
It is for 9 times that every 1.0ml is containing virus>=10 that the virus liquid of gathering in the crops for 10 times as a result has
7.0tCID
50.The virus liquid of getting front 9 results mixes rear standby.
(3) join seedling, packing and freeze-drying: by the virus liquid being up to the standards, be mixed in same container, add according to a certain percentage suitable stablizer, add appropriate microbiotic simultaneously, fully shake up, quantitative separating, carries out rapidly after packing getting product after vacuum freezedrying.
Inspection after construction: finished product is tested by the relevant regulations of the 15th, 19,20 pages of the veterinary drug allusion quotation > > of < < People's Republic of China (PRC) version appendix in 2005.The inventive method is prepared virus liquid and is met the requirements completely, and pig is had no side effect safely, without bacterium, mould, mycoplasma and exogenous virus, pollutes.By indirect immunofluorescence, measure tiring as every part is containing virus>=1.5 * 10 of finished product
4tCID
50; With 1/15000 part immune swine of every part dilution of pig check, can produce 100% and attack poison protection.
Embodiment 5: the live vaccines of hog cholera that the present invention prepares and the comparison of existing same based article
1. test materials
(1) vaccine
Method by " embodiment 2 " is prepared 3 batches of the cell adapted virus live vaccines of swine fever, lot number: examination BT110301, examination BT110302 and examination BT110303.3 batches of Guangdong Winsun Bio-Pharmaceutical Co., Ltd.'s live vaccines of hog cholera (ST clone), lot number 201101,201102 and 201103.
(2) test is all originated with pig
The responsive pig of health that rural area is raised family scattered, do not had Pestivirus suis to contact with swine Fever Vaccine.Before using, by a pig blood drawing, through neutralization test method, detect, swine fever neutralizing antibody is negative.By PCR method, detect swine fever antigen, porcine circovirus 2 type and pig breeding dysfunction and breathing syndrome virus is all negative.
2. method and result
(1) physical behavior check
Visual inspection vaccine physical behavior.The homemade 3 batches of vaccines of the present invention are oyster white Sponge Porosity agglomerate, are easy to bottle wall and depart from, and dissolve rapidly after adding diluent.
(2) steriling test
By the 15th page of the veterinary drug allusion quotation > > of the < < People's Republic of China (PRC) (version in 2005) appendix, test, homemade 3 batches of vaccines are asepsis growth.
(3) mycoplasma check
By the 20th page of (version in 2005) appendix, test, homemade 3 batches of vaccines are all grown without mycoplasma.
(4) exogenous virus check
By the 20th page of (version in 2005) appendix, test, homemade 3 batches of vaccines are mycoplasma growth.
(5) safety examination
3 batches of freeze dried vaccine samples are diluted to every milliliter containing 10 part vaccines by the dated head part of label with sterile saline, 4 of intramuscular injection pigs, every 5ml (containing 50 using dosages).Note after 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 and before notes seedling, compare and there is no considerable change; Or fervescence surpasses 0.5 ℃, but be no more than 1 ℃, delay and be no more than 2 (4 temperature time); Or subtract food be no more than 1, it is qualified that vaccine can be judged to.If any 1 temperature of pig body, surpass normal temperature more than 1 ℃, but be no more than 1.5 ℃, delay and be no more than 2 temperature time, it is qualified that vaccine also can be judged to.Reaction if any 1 pig surpasses above-mentioned standard; Or while there is suspicious other body temperature reaction and other unusual phenomenon, 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 pliotherm period blood sampling involution pigs, every former blood 5ml 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.The results are shown in Table 5.
The comparison of table 5 safety verification result
According to result, show that 3 batches of vaccines that the present invention manufactures experimently have higher security, after 50 part immune animals of 3 batches of vaccines, body temperature reaction is all no more than 1 ℃ of normal temperature.And batch be that 201102 control vaccines have 2 pigs to occur that body temperature reaction surpasses normal body temperature more than 1.5 ℃ during with 50 parts immunity.Therefore, the cell adapted poison of swine fever has better security.
(6) efficacy test
Every cell adapted malicious vaccine of part swine fever (BT clone) and commercialization live vaccines of hog cholera (ST clone) are diluted respectively to 3000 times, and intramuscular injection is without the healthy susceptible pig of swine fever antigen and antibody, every 1.0ml respectively.After 10～14 days, together with the contrast that condition is identical, note 4, injection swine fever crossdrift is blood poison 1.0ml/ head (10
5mLD), observe 16.The results are shown in Table 6.
The comparison of table 6 efficacy test result
(7) residual moisture is measured
By the 31st page of the veterinary drug allusion quotation > > of the < < People's Republic of China (PRC) (version in 2005) appendix, test, homemade 3 batches of vaccines are up to specification.
(8) vacuum tightness is measured
By the 31st page of the veterinary drug allusion quotation > > of the < < People's Republic of China (PRC) (version in 2005) appendix, test, homemade 3 batches of vaccines are all up to specification.
3. brief summary
With reference to the veterinary drug allusion quotation > > of the < < People's Republic of China (PRC) (version in 2005) live vaccines of hog cholera (cell source) inspection after construction method, check; result shows: with 1/3000 part immune swine of 3 batches of live vaccines of hog cholera of BT cells produce, can produce 100% protection; and compare the protection better effects if of pig with commercial ST cell source swine Fever Vaccine, 3 batches of vaccine immunities are attacked poison on the 16th and are not all occurred body temperature reaction.The live vaccines of hog cholera that the present invention produces in addition carries out safety verification with 50 multiple doses, all qualified, and commercialized vaccine is while carrying out safety verification with 50 multiple doses, and obvious body temperature reaction appears in some batch.The cell adapted malicious vaccine of swine fever that the present invention's trial-production is described has better security.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. the cell adapted poison of swine fever, is to be obtained after continuous cell line cultured continuously by fever virus lapinized Chinese Strain, and by after the intravenous inoculation healthy rabbits 1ml of its ear source, its body temperature is reactionless or only occur slight fever reaction.
2. adaptation according to claim 1 is malicious, it is characterized in that: described adaptation poison is the cell adapted CC strain of Pestivirus suis of fever virus lapinized Chinese Strain (Classical Swine Fever Cell Adapted Virus strain CC), its deposit number is: CCTCC NO:V201224, is preserved in Chinese Typical Representative culture collection center.
3. according to a preparation method for the cell adapted poison of swine fever described in claim 1 or 2, it is characterized in that: fever virus lapinized Chinese Strain is accessed at least 20 generations of continuous cell line cultured continuously, and described incubation time is 24h～72h/ generation.
4. the Pestivirus suis living vaccine making according to the cell adapted poison of swine fever described in claim 1 or 2.
5. a preparation method for Pestivirus suis living vaccine according to claim 4, comprising:
Steps A, prepares attenuated vaccine antigen;
Step B, adds suitable lyophilized vaccine by preparing swine fever attenuated vaccine with antigen, fully mixes rear quantitative separating, and freeze-drying, makes Pestivirus suis living vaccine;
It is characterized in that: in steps A, the cell adapted poison of swine fever is produced to seed culture of viruses access continuous cell line and cultivate, harvested cell supernatant liquor is after the assay was approved as preparing swine fever attenuated vaccine antigen continuously.
6. method according to claim 5, is characterized in that:
In steps A, the cell adapted poison of swine fever is produced seed culture of viruses and is adapted to and be no more than for 1 generation in continuous cell line cultivation;
Described continuous results comprise produces seed culture of viruses access passage cell by the cell adapted poison of swine fever, cultivates harvested cell supernatant liquor after 2～5 days, and adds maintenance medium and continue the cyclical operation of cultivating, and the number of times of described circulation is 8～10 times.
7. method according to claim 6, is characterized in that: be also included in the following steps before steps A:
Step 1, prepares original seed culture of viruses: the cell adapted poison access of swine fever continuous cell line is cultivated, and harvested cell supernatant liquor is after the assay was approved as the cell adapted toxogen of swine fever beginning seed culture of viruses;
Step 2, prepares basic seed culture of viruses: the cell adapted toxogen of swine fever beginning seed culture of viruses access continuous cell line is cultivated, and harvested cell supernatant liquor is after the assay was approved as the basic seed culture of viruses of the cell adapted poison of swine fever;
Step 3, seed culture of viruses is produced in preparation: the basic seed culture of viruses access of the cell adapted poison of swine fever continuous cell line is cultivated, and harvested cell supernatant liquor is produced seed culture of viruses as the cell adapted poison of swine fever after the assay was approved.
8. method according to claim 7, is characterized in that:
In step 1, the cultivation of the cell adapted poison of swine fever in continuous cell line was no more than for 3 generations;
In step 2, the cultivation of seed culture of viruses of cell adapted toxogen beginning of swine fever in continuous cell line was no more than for 3 generations;
In step 3, the cultivation of the basic seed culture of viruses of the cell adapted poison of swine fever in continuous cell line was no more than for 3 generations;
Described incubation time is 24h～72h/ generation.
9. according to the method described in any one in claim 3,5～8, it is characterized in that: described passage cell is ST clone, PK15 clone, STK clone or BT clone.
10. cultivate a method for Pestivirus suis, it adopts the continuous cell line cultivation of going down to posterity, and it is characterized in that: described passage cell is BT clone.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181637A (en) * | 2007-11-30 | 2008-05-21 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
| CN101926991A (en) * | 2010-01-28 | 2010-12-29 | 洛阳普莱柯生物工程有限公司 | Classical swine fever virus vaccine and production method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101181637A (en) * | 2007-11-30 | 2008-05-21 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
| CN101926991A (en) * | 2010-01-28 | 2010-12-29 | 洛阳普莱柯生物工程有限公司 | Classical swine fever virus vaccine and production method thereof |
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