CN102965332A - 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 the pig testis cloned cell line to produce live vaccines of hog cholera.
Background technology
Swine fever is a kind of height contagious disease that is 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 the category-A disease by world OIE, and China is divided into a class disease with it.It is safety and the effective vaccine of generally acknowledging in the world at present that the 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, even have eliminated swine fever.In China, the typical swine fever of the popular appearance of swine fever and the coexistence of atypia swine fever, persistent infection and inapparent infection coexistence, immunological tolerance and the coexistence of band poison syndrome etc., 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 that China prevents and treats swine fever at present, and China produces the cell that the 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 confirm the vaccine with above-mentioned cells produce 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 the vaccine batch.
In addition, because bovine viral diarrhea virus and Pestivirus suis belong to the 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, live vaccines of hog cholera immune swine with such zooblast is produced 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 high infectious pig testis clone cell of Pestivirus suis among 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 namely refer to produce the pig testis clone cell of the live vaccines of hog cholera of high-titer.
The present invention also provides a kind of preparation method of aforesaid pig testis clone cell, may further comprise the steps:
1) the pig testis cell is carried out limiting dilution and carry out cell clone and ubcellular clone, obtain the ubcellular clone strain;
2) selection is the high infectious pig testis clone cell of Pestivirus suis to the subclone strain of the highest pig testis cell of swine fever virus infection rate.
Preferably, among the preparation method of the aforesaid use pig testis of the present invention 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 ℃ of 5%CO
2Condition under be incubated at be cultured to naked eyes visible cell clone in the 96 porocyte culture plates after, carry out the ubcellular cultivation, obtain the subclone cell strain;
Preferably, among the preparation method of the aforesaid use pig testis of the present invention clone cell, described step 2) for the strain of pig testis cell subclone is inoculated in the 96 porocyte culture plates, the Pigs Inoculated pestivirus is in 37 ℃ of 5%CO
2Condition under cultivate 2h, use Immunoperoxidase monolayer cell test method(s) to detect and infect the maximum cell of virus, select the subclone strain of the highest pig testis cell of swine fever virus infection rate is the high infectious pig testis clone cell of Pestivirus suis.
The preparation method that the present invention also provides a kind of aforesaid pig testis clone cell to be used for the hog cholera venom of high titre, with above-mentioned pig testis clone cell with trysinization after, synchronously inoculation contains the cell maintenance medium of fresh spleen poison, 37 ℃ contain 5%CO
2Cultivate 2-3d in the incubator, after cell covers with individual layer, go down to posterity according to 1: 3 ratio, the viral supernatant liquid of gathering in the crops simultaneously passage cell can obtain the hog cholera venom of high titre.
Preferably, among the preparation method of the hog cholera venom of the aforesaid high titre of the present invention, described passage number is can be greater than 15 generations.
Preferably, among the preparation method of the hog cholera venom of the aforesaid high titre of the present invention, described virus titer is greater than 10
6.0TCID
50/ ml.
The present invention also provides the as above method of 1 described pig testis clone cell production live vaccines of hog cholera of a kind of use, may further comprise the steps:
1) produce preparation with the seed lot virus liquid: with pig testis clone cell claimed in claim 1 with trysinization after, synchronously inoculation contains the cell maintenance medium of fresh spleen poison, 37 ℃ contain 5%CO
2Cultivate 2-3d in the incubator, after cell covers with individual layer, go down to posterity 2 times according to 1: 3 ratio, the viral supernatant liquid of gathering in the crops simultaneously the passage cell of the s-generation, the third generation can obtain the hog cholera venom of high titre;
2) seedling is with the preparation of virus liquid: after pig testis clone cell claimed in claim 1 covers with individual layer, digest with the pancreatin that contains 0.5%EDTA, adopt simultaneously inoculated method inoculation to contain the cell maintenance medium that 5% seeding is criticized poison, put in 37 ℃ of incubators that contain 5% carbonic acid gas and cultivated 3-4 days, after cell covers with individual layer, ratio according to 1: 3 goes down to posterity with trysinization, gather in the crops simultaneously viral supernatant, use the same method and went down to posterity at least 15 generations, in per generation, all collected viral supernatant liquor as the seedling venom;
3) seedling of virus liquid: the virus liquid of above-mentioned results is added lyophilized vaccine, and freeze-drying can obtain the live vaccines of hog cholera that the pig testis clone cell is produced.
Preferably, the aforesaid pig testis clone cell of the present invention is produced in the method for live vaccines of hog cholera, and tiring of described living vaccine is every part viral level 〉=9000 rabbit infective dose, and every part contains cell venom 〉=0.01ml.
Therefore, the invention provides and use aforesaid pig testis clone cell, 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 the 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 the swine Fever Vaccine that malicious method is produced with poison, virus titer is high, be not vulnerable to the pollution of BVDV, the pure property of vaccine is good; The method that the band poison passes poison reached at least 15 generations so that the cell of each recovery can continue to go down to posterity, and had reduced recovery cell and the number of times that connects poison, had simplified production technique, and had 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 used growth media prescription of the present invention is: 91%v/vDMEM liquid, 9%v/v calf serum PH are adjusted into 7.1; The prescription 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, available 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) CSFV infects the foundation of ST cell detection method 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 at 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 trysinization after, inoculate toxic cell maintenance medium; (2) after cell grows up to individual layer, went down to posterity by 1: 3, harvested cell supernatant simultaneously, second, third supernatant of results is criticized poison as seeding in generation; (3) the harvested cell supernatant that uses the same method, as the vaccine venom, the band poison passes poison and can go down to posterity more than at least 15 generations
For making the present invention easier to understand, the below will further set forth specific embodiments of the invention.Such as special declaration not, general experimental technique of the present invention can use this area test method commonly used to carry out.
Cell growth medium in the embodiment of the 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 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 of cell check: without mycoplasma contamination
2. exogenous virus detects and describes the exogenous virus detection method as an example of bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) example.
Utilize round pcr, the ST cell is carried out bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) detect.The ST cell should not carry BVDV as a result.Detection method: with the individual layer ST cell of trysinization in the growth of T25 cell bottle, harvested cell, the centrifugal 10min of 1000rpm, abandoning supernatant is washed one time with PBS, the centrifugal collecting cell precipitation.With reference to the TRIzol specification sheets of Invitrogen, extract cell total rna.The cDNA that the RNA that extracts carries out obtaining after the reverse transcription is as pcr template, adopt the BVDV Auele Specific Primer, carry out pcr amplification, amplified production carries out the agarose gel electrophoresis analysis, amplify purpose band person and carry BVDV, do not amplify purpose band person and then 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 behind the extraction 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
The individual layer ST cell that is in logarithmic phase is become 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 the 96 porocyte culture plates, mark is carried out in the hole of containing individual cells after, 96 orifice plates are placed contain 5% CO2, cultivate in 37 ℃ of cell culture incubators until produce till the naked eyes visible cell clone.In this process, can according to the Growth of Cells situation, change liquid to clone cell.When the Growth of Cells in the hole reaches individual layer, it is digested, place 24 porocyte culture plates, in containing 5%CO
2, continue in 37 ℃ of cell culture incubators to cultivate.After cell covers with 24 holes, after its digestion, place 6 orifice plates to continue to cultivate, as possible, the cell of cloning is carried out frozen conservation, with for subsequent use.10000rpm after conditioned medium is collected, behind the centrifugal 10min, 0.45 μ m membrane filtration is removed cell debris.
Three, the screening of high infective ST cell clone
With the ST cell subsets of enlarged culturing on 6 orifice plates, with carrying out cell counting after the trysinization, according to 2 * 10
4Per 96 holes of individual cell/, inoculate 96 porocyte culture plates, repeat 1 hole, connect malicious CSFV virus liquid behind the inoculation 24h, the poison amount that connects virus inoculation with MOI=0.1, every hole adds 200 μ L virus stock solution useds, 37 ℃ hatch 1h after, discard virus liquid, add cell maintenance medium, in 5%CO2, after cultivating 2d in 37 ℃ of incubators, detect cell infection virus situation with the test of Immunoperoxidase monolayer cell, the hole that positive cell number is many is the high ST cell subsets of CSFV infection rate, is required high infective ST clone cell.Then with its enlarged culturing on 6 orifice plates, use 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 purpose 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 high infective ST clonal cell line ST-B2 (preserving number CCTCC C2011101) that obtains is with after the trysinization, the inoculation seeding is criticized poison synchronously, cultivate 3-4d in 37 ℃ of incubators, ratio according to 1: 3 (volume ratio) goes down to posterity, gather in the crops simultaneously supernatant and carry out freezing preservation as seedling with venom, went down to posterity at least 15 generations according to same method, gather in the crops the viral supernatant in per generation as the seedling venom.
1, the mensuration of CSFV virus titer on positive ST clonal cell line ST-B2
The band poison is passed CSFV virus continuous multiplication culture on high infections clone cell ST-B2 that malicious method goes down to posterity and obtains, adopt indirect immunofluorescence method to measure the titre of the CSFV virus liquid of each generation of gathering in the crops, specific as follows: as will to be in the ST-B2 cell trysinization of 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 icing the methyl alcohol fixed cell 10 minutes, then add 1%BSA and sealed 30 minutes, after PBS washes twice, add the good primary antibodie of dilution, 37 ℃ hatch 2h after, PBS washes 5 times, add good two anti-of dilution, hatch 1h for 37 ℃, PBS washes 5 times, then under inverted microscope, observe the fluorescence in each hole, utilize Reed-Muench Liang Shi method to calculate the titre of virus.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 30 generations of continuous passage, and cell growth state is good, and every 5 generations cell is inoculated CSFV virus, measures the CSFV titre, and virus titer is stabilized in 10
6.5TCID
50About/ml, chose for 10 generations and use the seed lot cell with interior clone cell as producing.
3, produce the preparation of using the seed lot poison
After the ST-B2 cell covers with individual layer, after digesting with the pancreatin that contains 0.5%EDTA, take simultaneously inoculated method to contain the cell maintenance medium of fresh spleen poison with the MOI=0.1 inoculum size, put in 37 ℃ of incubators that contain 5% carbonic acid gas and cultivated 2-3 days, after cell covers with individual layer, according to going down to posterity with the ratio according to 1: 3 after the trysinization, gather in the crops simultaneously viral supernatant, in second, third generation of results,, viral supernatant was criticized poison as seeding.
4, the seedling preparation of cell venom
After the ST-B2 cell covers with individual layer, digest with the pancreatin that contains 0.5%EDTA, adopt simultaneously inoculated method inoculation to contain the cell maintenance medium that the 5%v/v seeding is criticized poison, put in 37 ℃ of incubators that contain 5% carbonic acid gas and cultivated 3-4 days, after cell covers with individual layer, go down to posterity with trysinization according to 1: 3 ratio, gather in the crops simultaneously viral supernatant, use the same method and went down to posterity at least 15 generations, in per generation, all collected viral supernatant as the seedling venom, is stored in-70 ℃ 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, the result is without bacterium, mould, mycoplasma growth.The 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.Each is received inferior virus liquid and adds lyophilized vaccine; live vaccines of hog cholera is made in freeze-drying, and vaccine batch is 20101101,20101102,20101103, measures with rabbit respectively; every part viral level 〉=9000 rabbit infective dose, every part contains cell venom 〉=0.01ml.
The method of rendeing a service with rabbit effect quarantine seedling is as follows:
(1) the inferior virus liquid of every receipts dilutes 3 * 10 with sterile saline
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.Behind family's rabbit inoculation, respectively survey body temperature morning and afternoon 1 time, after 48 hours, surveyed body temperature 1 time every 6 hours, according to the body temperature reaction with attack malicious result and carry out synthetic determination.
1. after the rabbit vaccination, the body temperature reaction normal is as follows:
Typing 48～96 hours latent period of thermal response (++), 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 typing heat that is judged to of poison.
Slight fever is reacted 48～96 hours (+) latent period, and body temperature rise is obvious curve, has the inferior normal temperature that surpasses of 2 temperature at least more than 0.5 ℃, 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 surpass 96 hours to 120 hours and thermal response occurs.
The body temperature reaction is the secondary peak, and once the peak meets typing thermal response (++) or slight fever reaction (+) standard person, must attack poison.Attack behind the poison when reactionless, this rabbit thermal response can be judged to typing heat or slight fever reaction.
Reactionless (-) body temperature is normal.
2. the result judges:
After annotating seedling, when 2 rabbit all are typing thermal response (++), or 1 rabbit is typing thermal response (++), when 1 rabbit is slight fever reaction (+) in addition, vaccine is judged to qualified.
After annotating seedling, when 1 rabbit is typing thermal response (++) or slight fever reaction (+), 1 rabbit is suspicious reaction (±) in addition; Or 2 rabbits are when all being slight fever reaction (+), can be after annotating seedling attack poison (inoculate fresh spleen and drench poison or freeze-drying poison) on the 7th～10.When attacking poison, add 2 of contrast rabbits, attacking the toxic agent amount is 50～100 times of emulsions.Every rabbit ear vein injection 1ml.
The body temperature reaction normal of attacking behind the poison is as follows:
In 24～72 hours latent period of thermal response (+), body temperature rise is obvious curve, surpasses normal temperature more than 1 ℃, 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 surpass 36 hours and do not descend.
Reactionless (-) body temperature is normal.
After attacking poison, when 2 contrast rabbits all are typing thermal response (++), or 1 rabbit is typing thermal response (++), and 1 rabbit is slight fever reaction (+) in addition, and 2 annotated seedling rabbit all reactionless (-), and vaccine is declared qualified.
After annotating seedling, be typing heat (++) or slight fever reaction (+) if any 1 rabbit, in addition 1 rabbit is suspicious reaction (±) or without thermal response (-), can adopt suspicious reaction rabbit or reactionless rabbit and cut open the method for killing or adopting the painstaking effort isolated viral, distinguishes whether inapparent infection; Or behind the notes seedling, 2 rabbits all are the slight fever reaction, also can be to 1 rabbit isolated viral wherein.Method is: between 96～120 hours, rabbit is cutd open extremely after the vaccination, taked spleen, make 50 times of dilution emulsions (spleen emulsion should be aseptic) with physiological saline, or take painstaking effort (whole blood), inoculate 2 rabbit, every rabbit ear vein injection 1ml.All have 1 rabbit typing thermal response (++) to occur 24～72 hours latent period, and it is qualified that vaccine can be judged to.
After annotating seedling, when other response situation occurring and can't judge, can heavily examine.Do the effect inspection with rabbit, should be above 3 times.Through check, 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 all is typing thermal response (++), or 1 rabbit is typing thermal response (++), when 1 rabbit is slight fever reaction (+) in addition, 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, reaction knows that 1 rabbit is typing thermal response (++) according to body temperature, 1 rabbit is suspicious reaction (±) in addition, attacking poison on 7th～10 after annotating seedling (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).When attacking poison, add 2 of contrast rabbits, attacking the toxic agent amount is 100 times of emulsions.Every rabbit ear vein injection 1ml.After attacking poison, 2 contrast rabbits all are typing thermal response (++), or 1 rabbit is typing thermal response (++), and 1 rabbit is slight fever reaction (+) in addition, and 2 annotated seedling rabbit all reactionless (-), and vaccine is declared 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 all is typing thermal response (++), or 1 rabbit is typing thermal response (++), when 1 rabbit is slight fever reaction (+) in addition, vaccine is judged to qualified.And 1/10
62 of extent of dilution ear vein injection rabbit, every 1ml, reaction knows that 1 rabbit is typing thermal response (++) according to body temperature, 1 rabbit is suspicious reaction (±) in addition, attacking poison on 7th～10 after annotating seedling (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).When attacking poison, add 2 of contrast rabbits, attacking the toxic agent amount is 100 times of emulsions.Every rabbit ear vein injection 1ml.After attacking poison, 2 contrast rabbits all are typing thermal response (++), and 2 annotated seedling rabbit all reactionless (-), and vaccine is declared 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 all is typing thermal response (++), or 1 rabbit is typing thermal response (++), when 1 rabbit is slight fever reaction (+) in addition, 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, reaction knows that 1 rabbit is typing thermal response (++) according to body temperature, 1 rabbit is suspicious reaction (±) in addition, attacking poison on 7th～10 after annotating seedling (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).When attacking poison, add 2 of contrast rabbits, attacking the toxic agent amount is 100 times of emulsions.Every rabbit ear vein injection 1ml.After attacking poison, 2 contrast rabbits all are typing thermal response (++), or 1 rabbit is typing thermal response (++), and 1 rabbit is slight fever reaction (+) in addition, and 2 annotated seedling rabbit all reactionless (-), and vaccine is declared qualified.See Table in detail 1.
The result that table 1 rabbit effect quarantine seedling is renderd a service
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (11)
1. the high infectious pig testis clone cell of a Pestivirus suis is characterized in that, preserving number is CCTCC NO:C2011101.
2. the preparation method of a pig testis clone cell as claimed in claim 1 is characterized in that, may further comprise the steps:
1) the pig testis cell is carried out limiting dilution and carry out cell clone and ubcellular clone, obtain the ubcellular clone strain;
2) selection is the high infectious pig testis clone cell of Pestivirus suis to the subclone strain of the highest pig testis cell of swine fever virus infection rate.
3. the preparation method of use pig testis clone cell according to claim 2 is characterized in that, 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 ℃ of 5%CO
2Condition under be incubated at be cultured to naked eyes visible cell clone in the 96 porocyte culture plates after, carry out the ubcellular cultivation, obtain the subclone cell strain;
4. the preparation method of use pig testis clone cell according to claim 2 is characterized in that, described step 2) for the strain of pig testis cell subclone is inoculated in the 96 porocyte culture plates, the Pigs Inoculated pestivirus is in 37 ℃ of 5%CO
2Condition under cultivate 2h, use Immunoperoxidase monolayer cell test method(s) to detect and infect the maximum cell of virus, select the subclone strain of the highest pig testis cell of swine fever virus infection rate is the high infectious pig testis clone cell of Pestivirus suis.
5. a right to use requires 1 described pig testis clone cell to be used for the preparation method of the hog cholera venom of high titre, it is characterized in that, with pig testis clone cell claimed in claim 1 with trysinization after, synchronously inoculation contains the cell maintenance medium of fresh spleen poison, 37 ℃ contain 5%CO
2Cultivate 2-3d in the incubator, after cell covers with individual layer, go down to posterity according to 1: 3 ratio, the viral supernatant liquid of gathering in the crops simultaneously passage cell can obtain the hog cholera venom of high titre.
6. the preparation method of the hog cholera venom of high titre according to claim 5 is characterized in that, described passage number is can be greater than 15 generations.
7. the preparation method of the hog cholera venom of high titre according to claim 5 is characterized in that, described virus titer is greater than 10
6.0TCID
50/ ml.
8. a right to use requires 1 described pig testis clone cell production Pestivirus suis.
9. the method that right to use requires 1 described pig testis clone cell to produce live vaccines of hog cholera is characterized in that, may further comprise the steps:
1) produce preparation with the seed lot virus liquid: with pig testis clone cell claimed in claim 1 with trysinization after, synchronously inoculation contains the cell maintenance medium of fresh spleen poison, 37 ℃ contain 5%CO
2Cultivate 2-3d in the incubator, after cell covers with individual layer, go down to posterity 2 times according to 1: 3 ratio, the viral supernatant liquid of gathering in the crops simultaneously the passage cell of the s-generation, the third generation can obtain the hog cholera venom of high titre;
2) seedling is with the preparation of virus liquid: after pig testis clone cell claimed in claim 1 covers with individual layer, digest with the pancreatin that contains 0.5%EDTA, adopt simultaneously inoculated method inoculation to contain the cell maintenance medium that 5% seeding is criticized poison, put in 37 ℃ of incubators that contain 5% carbonic acid gas and cultivated 3-4 days, after cell covers with individual layer, ratio according to 1: 3 goes down to posterity with trysinization, gather in the crops simultaneously viral supernatant, use the same method and went down to posterity at least 15 generations, in per generation, all collected viral supernatant liquor as the seedling venom;
3) seedling of virus liquid: the virus liquid of above-mentioned results is added lyophilized vaccine, and freeze-drying can obtain the live vaccines of hog cholera that the pig testis clone cell is produced.
10. pig testis clone cell according to claim 8 is produced the method for live vaccines of hog cholera, it is characterized in that tiring of described living vaccine is every part viral level 〉=9000 rabbit infective dose, and every part contains cell venom 〉=0.01ml.
11. a right to use requires 1 described pig testis clone cell to produce live vaccines of hog cholera.
Priority Applications (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278009A (en) * | 2014-09-12 | 2015-01-14 | 江苏省农业科学院 | Serum-free culture media and application thereof as well as culture method of classical swine fever virus |
| CN104940922A (en) * | 2015-07-02 | 2015-09-30 | 瑞普（保定）生物药业有限公司 | Preparation method of swine fever live vaccine |
| 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 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002020048A1 (en) * | 2000-08-10 | 2002-03-14 | Tsinghua University | A classical swine fever virus epitope vaccine and its producing method |
| US7332170B1 (en) * | 2005-12-23 | 2008-02-19 | The United States Of America, As Represented By The Secretary Of Agriculture | Classical swine fever virus virulence determinant and a novel classical swine fever vaccine |
| 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 |
| CN101492657A (en) * | 2008-12-26 | 2009-07-29 | 天津瑞普生物技术股份有限公司 | Swine fever live vaccine ST cell culture method |
| CN101797380A (en) * | 2010-01-28 | 2010-08-11 | 洛阳普莱柯生物工程有限公司 | Method for preparing hogcholera vaccine |
-
2011
- 2011-11-30 CN CN201110391978.1A patent/CN102965332B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002020048A1 (en) * | 2000-08-10 | 2002-03-14 | Tsinghua University | A classical swine fever virus epitope vaccine and its producing method |
| US7332170B1 (en) * | 2005-12-23 | 2008-02-19 | The United States Of America, As Represented By The Secretary Of Agriculture | Classical swine fever virus virulence determinant and a novel classical swine fever vaccine |
| 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 |
| CN101492657A (en) * | 2008-12-26 | 2009-07-29 | 天津瑞普生物技术股份有限公司 | Swine fever live vaccine ST cell culture method |
| CN101797380A (en) * | 2010-01-28 | 2010-08-11 | 洛阳普莱柯生物工程有限公司 | Method for preparing hogcholera vaccine |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278009A (en) * | 2014-09-12 | 2015-01-14 | 江苏省农业科学院 | Serum-free culture media and application thereof as well as culture method of classical swine fever virus |
| 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 |
| CN104940922A (en) * | 2015-07-02 | 2015-09-30 | 瑞普（保定）生物药业有限公司 | Preparation method of swine fever live vaccine |
| 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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| CN102965332B (en) | 2014-06-04 |
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