CN102965332B - Swine testicular clone cell line and production method of classical swine fever live vaccine - Google Patents
Swine testicular clone cell line and production method of classical swine fever live vaccine Download PDFInfo
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Abstract
The invention provides a highly classical swine fever virus infective swine testicular clone cell strain ST-B2, which has a preservation number of CCTCC NO.C2011101, and a preparation method of the swine testicular clone cell. The method comprises the steps of: 1) subjecting a swine testicular cell to limited dilution, conducting cell cloning and subcell cloning, thus obtaining a subcellular clone strain; and 2) selecting a subclone strain of the swine testicular cell with a highest classical swine fever virus infection rate, i.e. the highly infective swine testicular clone cell of the classical swine fever virus. The invention also provides a method for preparation of a high titer classical swine fever virus solution and a classical swine fever live vaccine from the swine testicular clone cell. The swine testicular clone cell provided in the invention has a high classical swine fever virus infection rate, and the classical swine fever vaccine produced from the swine testicular cell line ST clone cell strain ST-B2 by a virus-carrying and virus transmission technique has high virus titer, hard exposure to BVDV (bovine viral diarrhea virus) pollution and good pureness. By the virus-carrying and virus transmission technique, a resurgent cell can undergo continuous passage to at least 15 generations. The cell resurrection and virus inoculation times can be reduced, the production process is simplified, and the production efficiency is improved.
Description
Technical field
The present invention relates to a kind of zooblast and produce method and the product of live vaccines of hog cholera, refer to especially a kind of method and product that uses pig testis cloned cell line to produce live vaccines of hog cholera.
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 category-A disease by OIE of the world, 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.
The major measure that vaccine inoculation is still China and prevents and treats at present swine fever, China produces the cell that hog cholera lapinised virus seedling uses at present bull testis cell, porcine kidney cell (IBRS-2 or PK15 clone), pig testis cell (ST cell).But the vaccine of above-mentioned cells produce for confirming in the practice of this production of vaccine, its virus titer is not high, and production technique is unstable, and particularly virus titer reduces rapidly with the increase of passage number, causes widely different between vaccine batch.
In addition, because bovine viral diarrhea virus and Pestivirus suis belong to flaviviridae pestivirus, the homology of two-strain is very high, therefore the bull testis cell or the calf serum that have polluted BVDV all can cause vaccine to pollute, and the infection rate of viral diarrhea virus (BVDV) is very high in China cows, the live vaccines of hog cholera immune swine of producing with such zooblast, often causes immuning failure, causes serious consequence.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of pig testis clone cell, and produces method and the product of live vaccines of hog cholera.
The pig testis clone cell of a kind of high infected pigs provided by the invention pestivirus, called after pig testis clone (swine testicular cell lines) clone strain ST-B2, be deposited in Chinese Typical Representative culture collection center (CCTCC), address Wuhan, China Wuhan University, preserving number is CCTCC NO:C2011101, and preservation date is on November 07th, 2011.
The infectious pig testis clone cell of the height of Pestivirus suis in the present invention, refer to the pig testis clone cell high to swine fever virus infection, refer in particular to the pig testis clone cell of the Pestivirus suis that can produce high titre, also refer to the pig testis clone cell of the live vaccines of hog cholera that can produce high-titer.
The present invention also provides a kind of preparation method of pig testis clone cell as above, comprises the following steps:
1) pig testis cell is carried out to limiting dilution and carry out cell clone and ubcellular clone, obtain ubcellular clone strain;
2) selection is the infectious pig testis clone cell of height of Pestivirus suis to the subclone strain of the highest pig testis cell of swine fever virus infection rate.
Preferably, in the preparation method of the present invention's use pig testis as above clone cell, described step 1) for using not by the pig testis cell of virus infection, after being unicellular, tryptic digestion carries out gradient dilution, respectively at 37 DEG C of 5%CO
2condition under be incubated at and in 96 porocyte culture plates, be cultured to after naked eyes visible cell clone, carry out ubcellular cultivation, obtain subclone cell strain;
Preferably, in the preparation method of the present invention's use pig testis as above clone cell, described step 2) for the strain of pig testis cell subclone is inoculated in 96 porocyte culture plates, Pigs Inoculated pestivirus is in 37 DEG C of 5%CO
2condition under cultivate 2h, use Immunoperoxidase monolayer cell test method(s) to detect and infect the cell that virus is maximum, select the subclone strain of the highest pig testis cell of swine fever virus infection rate to be the infectious pig testis clone cell of height of Pestivirus suis.
The present invention also provides the preparation method of a kind of pig testis clone cell as above for the hog cholera venom of high titre, and by above-mentioned pig testis clone cell, with after trysinization, synchronous inoculation is containing the cell maintenance medium of fresh spleen poison, and 37 DEG C containing 5%CO
2in incubator, cultivate 2-3d, after cell covers with individual layer, go down to posterity according to the ratio of 1: 3, the viral supernatant liquid of simultaneously gathering in the crops passage cell can obtain the hog cholera venom of high titre.
Preferably, in the preparation method of the hog cholera venom of the present invention's high titre as above, described passage number is for can be greater than for 15 generations.
Preferably, in the preparation method of the hog cholera venom of the present invention's high titre as above, described virus titer is for being greater than 10
6.0tCID
50/ ml.
The present invention also provides a kind of use method that as above the pig testis clone cell described in 1 is produced live vaccines of hog cholera, comprises the following steps:
1) produce the preparation with seed lot virus liquid: with after trysinization, synchronous inoculation is containing the cell maintenance medium of fresh spleen poison by pig testis clone cell claimed in claim 1, and 37 DEG C containing 5%CO
2in incubator, cultivate 2-3d, after cell covers with individual layer, go down to posterity 2 times according to the ratio of 1: 3, the viral supernatant liquid of simultaneously gathering in the crops the passage cell of the s-generation, the third generation can obtain the hog cholera venom of high titre;
2) preparation of virus liquid for seedling: pig testis clone cell claimed in claim 1 covers with after individual layer, with digesting containing the pancreatin of 0.5%EDTA, adopt simultaneously inoculated method inoculation to criticize malicious cell maintenance medium containing 5% seeding, put 37 DEG C and cultivate 3-4 days containing in the incubator of 5% carbonic acid gas, after cell covers with individual layer, go down to posterity with trysinization according to the ratio of 1: 3, gather in the crops viral supernatant simultaneously, use the same method and went down to posterity at least 15 generations, in per generation, is all collected viral supernatant liquor as seedling venom;
3) seedling of virus liquid: the virus liquid of above-mentioned results is added to lyophilized vaccine, and freeze-drying, can obtain the live vaccines of hog cholera that pig testis clone cell is produced.
Preferably, the present invention's pig testis clone cell as above is produced in the method for live vaccines of hog cholera, the tiring as every part viral level >=9000 rabbit infective dose of described living vaccine, and every part is containing cell venom >=0.01ml.
Therefore, the invention provides and use pig testis clone cell as above, manufacture has obtained a kind of hog cholera venom of high titre, and the live vaccines of hog cholera of high-titer.
As seen from the above, the present invention has obtained the strain pig testis cell high to swine fever virus infection rate by a large amount of careful tests, use this pig testis clone ST clonal cell line ST-B2, to pass with poison the swine Fever Vaccine that malicious method is produced, virus titer is high, be not vulnerable to the pollution of BVDV, the pure property of vaccine is good; Be with the malicious method of poison biography to make the cell of each recovery can continue to go down to posterity and reached at least 15 generations, reduced recovery cell and connect malicious number of times, simplified production technique, and improved production efficiency.
biomaterial preservation information
Pig testis clone clone strain ST-B2, is deposited in Chinese Typical Representative culture collection center (being called for short CCTCC), address Wuhan, China Wuhan University, and preserving number is CCTCC NO:C2011101, preservation date is on November 07th, 2011.
Embodiment
The present invention's growth media formula used is: 91%v/vDMEM liquid, 9%v/v calf serum PH are adjusted into 7.1; The formula of described maintenance medium is: 98%v/vDMEM liquid, 2%v/v calf serum PH are adjusted into 7.3; Pestivirus suis is fever virus lapinized Chinese Strain, purchased from China Veterinery Drug Inspection Office, and bacterial strain deposit number CVCC AV1412.
Embodiments of the invention can be divided into following aspect:
1. the clone of the ST cell that responsive homogeneous growth performance is good
(1) screening of female ST cell; (2) foundation that CSFV infects ST cell detection method is with stable; (3) cultivation of single ST cell subsets; (4) screening of positive ST cell clone; (5) subclone of positive ST cell; (6) CSFV produces the mensuration of malicious valency on ST positive colony cell; (7) repeated pruning of ST positive colony cell.
2. the cultivation of clone cell and band poison cell pass poison
(1) clone cell, with after trysinization, is inoculated toxic cell maintenance medium; (2) after cell grows up to individual layer, went down to posterity by 1: 3, simultaneously harvested cell supernatant, second, third supernatant of results is criticized poison as seeding in generation; (3) the harvested cell supernatant that uses the same method, as vaccine venom, band poison passes poison and can go down to posterity more than at least 15 generations
For making the present invention easier to understand, will further set forth specific embodiments of the invention below.As special declaration not, general experimental technique of the present invention can use the conventional test method in this area to carry out.
Cell growth medium in the embodiment of the present invention is:
91%v/vDMEM liquid, 9%v/v calf bovine serum PH is adjusted into 7.1;
Cell maintenance medium in the embodiment of the present invention is:
98%v/vDMEM liquid, 2%v/v calf bovine serum PH is adjusted into 7.3.
The screening of embodiment 1:ST clone cell and utilize the method for this cells produce live vaccines of hog cholera
One, the screening of female ST cell
1. the pure property inspection of cell: without mycoplasma contamination
2. exogenous virus detects and describes exogenous virus detection method as an example of bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) example.
Utilize round pcr, ST cell is carried out to bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) and detect.Result ST cell should not carry BVDV.Detection method: the individual layer ST cell with trysinization in T25 cell bottle growth, harvested cell, the centrifugal 10min of 1000rpm, abandoning supernatant, washes one time centrifugal collecting cell precipitation with PBS.With reference to the TRIzol specification sheets of Invitrogen, extract cell total rna.The cDNA that the RNA extracting carries out obtaining after reverse transcription is as pcr template, adopt BVDV Auele Specific Primer, carry out pcr amplification, amplified production carries out agarose gel electrophoresis analysis, amplify object band person and carry BVDV, do not amplify object band person and do not carry BVDV.
Use the same method and carry out RNA viruses: Pestivirus suis (Classical Swine fever Virus, CSFV), the detection of porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV).
Detection for DNA virus pig circular ring virus (Porcine Circovirus, PCV) and PRV (Pseudorabies virus) (Pseudorabies Virus) also adopts the method for PCR to carry out, and extracts after cell DNA, directly carries out pcr amplification.
Detected result shows: BVDV, CSFV, PRRSV, PCV, PRV are all negative.
Two, the cultivation of single ST cell and subgroup thereof
Individual layer ST cell in logarithmic phase is become to individual cells with trysinization, disperse as far as possible, and carry out cell counting, then adopt limiting dilution assay, according to the density of 8 cell/ml, the amount of the cell growth medium of every hole 100 μ l adds in 96 porocyte culture plates, and the hole of containing individual cells is carried out after mark, 96 orifice plates are placed in containing 5% CO2, in 37 DEG C of cell culture incubators, cultivate until produce naked eyes visible cell clone.In this process, can, according to Growth of Cells situation, change liquid to clone cell.In the time that the Growth of Cells in hole reaches individual layer, digested, be placed in 24 porocyte culture plates, in containing 5%CO
2, in 37 DEG C of cell culture incubators, continue to cultivate.When cell covers with behind 24 holes, after being digested, be placed in 6 orifice plates and continue to cultivate, if possible, clone's cell is carried out to frozen conservation, with for subsequent use.Conditioned medium is collected rear 10000rpm, and after centrifugal 10min, 0.45 μ m membrane filtration is removed cell debris.
Three, the screening of high infective ST cell clone
By the ST cell subsets of enlarged culturing on 6 orifice plates, with carrying out cell counting after trysinization, according to 2 × 10
4every 96 holes of individual cell/, inoculate 96 porocyte culture plates, repeat 1 hole, after inoculation 24h, connect malicious CSFV virus liquid, with the poison amount that the connects virus inoculation of MOI=0.1, every hole adds 200 μ L virus stock solution useds, hatch after 1h for 37 DEG C, discard virus liquid, add cell maintenance medium, in 5%CO2, in 37 DEG C of incubators, cultivate after 2d, by Immunoperoxidase monolayer cell test detection cell infection virus situation, the hole that positive cell number is many is the ST cell subsets that CSFV infection rate is high, is the infective ST clone cell of required height.Then by its enlarged culturing on 6 orifice plates, with trysinization, and carry out the 2nd time or 3 time clonings, every time cloning all needs to do the infectious detection of CSFV, to reach the object of the high infective ST clone cell of possessing of purifying.
The method of the cultivation of embodiment 2ST clone cell and production Pestivirus suis
The infective ST clonal cell line of the height ST-B2 (preserving number CCTCC C2011101) obtaining is with after trysinization, synchronous inoculation seeding is criticized poison, in 37 DEG C of incubators, cultivate 3-4d, ratio according to 1: 3 (volume ratio) goes down to posterity, gather in the crops supernatant simultaneously and carry out freezing preservation as seedling venom, went down to posterity at least 15 generations according to same method, gather in the crops the viral supernatant in per generation as seedling venom.
1, the mensuration of CSFV virus titer on positive ST clonal cell line ST-B2
Band poison is passed to the malicious method CSFV virus that the obtains multiplication culture continuously on high infections clone cell ST-B2 that goes down to posterity, adopt indirect immunofluorescence method to measure the titre of the CSFV virus liquid of each generation of results, specific as follows: by the ST-B2 cell trysinization in logarithmic phase, every hole 200 μ L are taped against in 96 orifice plates, after covering with individual layer, according to 10
-1-10
-8dilution CSFV virus liquid, and add in 96 orifice plates 6 holes of each extent of dilution, after 3-4 days, discard substratum, PBS washes twice, with ice methyl alcohol fixed cell 10 minutes, then add 1%BSA and seal 30 minutes, PBS washes after twice, add the primary antibodie of having diluted, hatch after 2h for 37 DEG C, PBS washes 5 times, add diluted two anti-, hatch 1h for 37 DEG C, PBS washes 5 times, then under inverted microscope, observe the fluorescence in each hole, utilize Reed-Muench Liang Shi method to calculate viral titre.The virus titer of producing continuously 3 batches is respectively: 20101101 batches of virus titers are 10
7.0tCID
50/ ml, 20101102 batches of virus titers are 10
6.92tCID
50/ ml, 20101103 batches of virus titers are 10
7.27tCID
50/ ml.
2, the repeated pruning of positive ST clonal cell line ST-B2
Positive ST clonal cell line ST-B2 is carried out to 30 generations of continuous passage, and cell growth state is good, and every 5 generations, cell is inoculated to CSFV virus, measures CSFV titre, and virus titer is stabilized in 10
6.5tCID
50/ ml left and right, chose for 10 generations and uses seed lot cell using interior clone cell as producing.
3, produce the preparation with seed lot poison
ST-B2 cell covers with after individual layer, after digesting containing the pancreatin of 0.5%EDTA, take simultaneously inoculated method to contain the cell maintenance medium of fresh spleen poison with MOI=0.1 inoculum size, put 37 DEG C and cultivate 2-3 days containing in the incubator of 5% carbonic acid gas, after cell covers with individual layer, according to going down to posterity by the ratio according to 1: 3 after trysinization, gather in the crops viral supernatant simultaneously, the viral supernatant of second, third generation of results is criticized poison as seeding.
4, the preparation of cell venom for seedling
ST-B2 cell covers with after individual layer, with digesting containing the pancreatin of 0.5%EDTA, adopt simultaneously inoculated method inoculation to criticize malicious cell maintenance medium containing 5%v/v seeding, put 37 DEG C and cultivate 3-4 days containing in the incubator of 5% carbonic acid gas, after cell covers with individual layer, go down to posterity with trysinization according to the ratio of 1: 3, gather in the crops viral supernatant simultaneously, use the same method and went down to posterity at least 15 generations, in per generation, is all collected viral supernatant as seedling venom, is stored in-70 DEG C of refrigerators.
5, the preparation of live vaccines of hog cholera and efficacy test
By existing " People's Republic of China's veterinary drug allusion quotation ", seedling is tested with venom, result is without bacterium, mould, mycoplasma growth.Cell venom has no side effect safely to pig, and 20101101,20101102,20101103 batches of virus titers are respectively 10
7.0tCID
50/ ml, 10
6.92tCID
50/ ml, 10
7.27tCID
50/ ml.The inferior virus liquid of each receipts adds lyophilized vaccine; live vaccines of hog cholera is made in freeze-drying, and vaccine batch is 20101101,20101102,20101103, measures respectively with rabbit; every part viral level >=9000 rabbit infective dose, every part is containing cell venom >=0.01ml.
As follows by the method for rabbit effect inspection vaccine potency:
(1) the inferior sterile saline dilution 3 × 10 for virus liquid of every receipts
5doubly, 4 × 10
5doubly, 5 × 10
5doubly, 6 × 10
5doubly, 7 × 10
5doubly, 8 × 10
5doubly, 9 × 10
5doubly, 10
6doubly, 2 of ear vein injection body weight 1.5～3.0kg rabbit, every rabbit ear vein injection 1ml.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 vaccination, body temperature reaction normal is as follows:
Sizing 48～96 hours latent period of thermal response (++), body 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 (+) latent period, and body 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 latent period of suspicious reaction (±), it is indefinite that temperature curve rises and falls, and delays less than 12 hours; Or latent 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 latent period of thermal response (+), body temperature rise is obvious curve, exceedes normal temperature more than 1 DEG C, delays 12～36 hours.
Suspicious reaction (±) latent 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 inapparent infection; Or after note seedling, 2 rabbits are all slight fever reaction, also can be to 1 rabbit isolated viral wherein.Method is: after vaccination, 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 latent period, 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.Through inspection, three inferior prepared living vaccines of receipts are all qualified, and concrete outcome is 20101101 batches of vaccines 1/ (3 × 10
5), 1/ (4 × 10
5), 1/ (5 × 10
5), 1/ (6 × 10
5), 1/ (7 × 10
5), 1/ (8 × 10
5) each 2 of 6 extent of dilution ear vein injection rabbit, every 1ml, know that according to body temperature reaction presenting 2 rabbit is all sizing thermal response (++), or 1 rabbit is when being sizing thermal response (++), another 1 rabbit and being slight fever reaction (+), vaccine is judged to qualified.And 1/ (9 × 10
5), 1/10
6two extent of dilution ear veins are injected each 2 of rabbit, every 1ml, according to body temperature, reaction knows that 1 rabbit is sizing thermal response (++), another 1 rabbit is suspicious reaction (±), after note seedling, within 7th～10, attacking poison (inoculates fresh spleen and drenches poison, be that fever virus lapinized Chinese Strain passes through rabbit body subculture, gather the spleen of qualitative thermal response rabbit as kind of a poison).While attacking poison, add 2 of contrast rabbits, attacking toxic agent amount is 100 times of emulsions.Every rabbit ear vein injection 1ml.Attack after poison, 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.20101102 batches of vaccines 1/ (3 × 10
5), 1/ (4 × 10
5), 1/ (5 × 10
5), 1/ (6 × 10
5), 1/ (7 × 10
5), 1/ (8 × 10
5), 1/ (9 × 10
5) each 2 of 7 extent of dilution ear vein injection rabbit, every 1ml, know that according to body temperature reaction presenting 2 rabbit is all sizing thermal response (++), or 1 rabbit is when being sizing thermal response (++), another 1 rabbit and being slight fever reaction (+), vaccine is judged to qualified.And 1/10
62 of extent of dilution ear vein injection rabbit, every 1ml, according to body temperature, reaction knows that 1 rabbit is sizing thermal response (++), another 1 rabbit is suspicious reaction (±), after note seedling, within 7th～10, attacking poison (inoculates fresh spleen and drenches poison, be that fever virus lapinized Chinese Strain passes through rabbit body subculture, gather the spleen of qualitative thermal response rabbit as kind of a poison).While attacking poison, add 2 of contrast rabbits, attacking toxic agent amount is 100 times of emulsions.Every rabbit ear vein injection 1ml.Attack after poison, 2 contrast rabbits are all sizing thermal response (++), and 2 note all reactionless (-) of seedling rabbit, vaccine is sentenced qualified.20101103 batches of vaccines 1/ (3 × 10
5), 1/ (4 × 10
5), 1/ (5 × 10
5), 1/ (7 × 10
5), 1/ (8 × 10
5) each 2 of 5 extent of dilution ear vein injection rabbit, every 1ml, know that according to body temperature reaction presenting 2 rabbit is all sizing thermal response (++), or 1 rabbit is when being sizing thermal response (++), another 1 rabbit and being slight fever reaction (+), vaccine is judged to qualified.And 1/ (6 × 10
5), 1/ (9 × 10
5), 1/10
63 extent of dilution ear veins are injected each 2 of rabbit, every 1ml, according to body temperature, reaction knows that 1 rabbit is sizing thermal response (++), another 1 rabbit is suspicious reaction (±), after note seedling, within 7th～10, attacking poison (inoculates fresh spleen and drenches poison, be that fever virus lapinized Chinese Strain passes through rabbit body subculture, gather the spleen of qualitative thermal response rabbit as kind of a poison).While attacking poison, add 2 of contrast rabbits, attacking toxic agent amount is 100 times of emulsions.Every rabbit ear vein injection 1ml.Attack after poison, 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.In detail in table 1.
The result of table 1 rabbit effect inspection vaccine potency
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 amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. the infectious pig testis clone cell of the height of Pestivirus suis, is characterized in that, preserving number is CCTCC NO:C2011101.
2. the pig testis clone cell described in a right to use requirement 1 is prepared the method for the hog cholera venom of high titre, it is characterized in that, by pig testis clone cell claimed in claim 1, with after trysinization, synchronous inoculation is containing the cell maintenance medium of fresh spleen poison, and 37 DEG C containing 5%CO
2in incubator, cultivate 2-3d, after cell covers with individual layer, go down to posterity according to the ratio of 1:3, the viral supernatant liquid of simultaneously gathering in the crops passage cell can obtain the hog cholera venom of high titre.
3. the preparation method of the hog cholera venom of high titre according to claim 2, is characterized in that, described passage number is for being equal to or greater than for 15 generations.
4. the preparation method of the hog cholera venom of high titre according to claim 2, is characterized in that, described virus titer is for being greater than 10
6.0tCID
50/ ml.
5. the application of pig testis clone cell as claimed in claim 1 in production Pestivirus suis.
6. right to use requires the method that the pig testis clone cell described in 1 is produced live vaccines of hog cholera, it is characterized in that, comprises the following steps:
1) produce the preparation with seed lot virus liquid: with after trysinization, synchronous inoculation is containing the cell maintenance medium of fresh spleen poison by pig testis clone cell claimed in claim 1, and 37 DEG C containing 5%CO
2in incubator, cultivate 2-3d, after cell covers with individual layer, go down to posterity 2 times according to the ratio of 1:3, the viral supernatant liquid of simultaneously gathering in the crops the passage cell of the s-generation, the third generation can obtain the hog cholera venom of high titre;
2) preparation of virus liquid for seedling: pig testis clone cell claimed in claim 1 covers with after individual layer, with digesting containing the pancreatin of 0.5%EDTA, adopt simultaneously inoculated method inoculation to criticize malicious cell maintenance medium containing 5% seeding, put 37 DEG C and cultivate 3-4 days containing in the incubator of 5% carbonic acid gas, after cell covers with individual layer, go down to posterity with trysinization according to the ratio of 1:3, gather in the crops viral supernatant simultaneously, use the same method and went down to posterity at least 15 generations, in per generation, is all collected viral supernatant liquor as seedling venom;
3) seedling of virus liquid: the virus liquid of above-mentioned results is added to lyophilized vaccine, and freeze-drying, can obtain the live vaccines of hog cholera that pig testis clone cell is produced.
7. pig testis clone cell according to claim 6 is produced the method for live vaccines of hog cholera, it is characterized in that, and the tiring as every part viral level >=9000 rabbit infective dose of described living vaccine, every part is containing cell venom >=0.01ml.
8. pig testis clone cell as claimed in claim 1 is in the application of producing in live vaccines of hog cholera.
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| CN104099292A (en) * | 2014-07-11 | 2014-10-15 | 陕西溯源农业发展有限公司 | Selective breeding method for EST sensitive cell line containing classical swine fever virus (CSFV) C strain |
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| CN104278009B (en) * | 2014-09-12 | 2017-11-17 | 江苏省农业科学院 | Serum free medium and application thereof and a kind of cultural method of CSFV |
| CN104940922B (en) * | 2015-07-02 | 2018-08-21 | 瑞普（保定）生物药业有限公司 | A kind of preparation method of live vaccines of hog cholera |
| CN107058211A (en) * | 2016-12-22 | 2017-08-18 | 武汉市工程科学技术研究院 | Carry ST cell lines and its application of rabbitization swine fever low virulent strain virus |
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| CN104099292B (en) * | 2014-07-11 | 2017-01-11 | 陕西溯源农业发展有限公司 | Selective breeding method for EST sensitive cell line containing classical swine fever virus (CSFV) C strain |
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