CN1117081A - Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method - Google Patents
Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method Download PDFInfo
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- CN1117081A CN1117081A CN 95102239 CN95102239A CN1117081A CN 1117081 A CN1117081 A CN 1117081A CN 95102239 CN95102239 CN 95102239 CN 95102239 A CN95102239 A CN 95102239A CN 1117081 A CN1117081 A CN 1117081A
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Abstract
The retention number of three toxin vaccine is CGMCC NO 0222. The triple weak-toxicity live vaccine contains distemper toxin greater than 10 to the power 3.5 TCID50/dosage, rabies toxin greater than 10 to the power 5.0 PFU/dosage, and pavovirus toxin greater than 10 to the power 3.5 TCID50/dosage. Its prepn process includes three toxin vaccine standard, toxin vaccine propagation and retention, vaccine prodn compounding, freeze-drying and inspection. The said vaccine has a stable toxin vaccine inheritance, lasting immunity, use safely, long storage period and other merits.
Description
The present invention relates to canine distemper, rabies, canine parvovirus disease trigeminal live vaccine, seed culture of viruses and manufacture method, belong to the veterinary medicament technical field.
Vaccine about dog is studied more both at home and abroad over nearly 20 years, but the research of bigeminy and multi-joint seedling is few, the document that nineteen eighty-three U.S. CABABSTRACTS and AGRICOLA collect has 56 pieces, have 5 pieces comprising dog with the document of the sick triple vaccine of parvovirus, rabies virus, canine distemper virus, used parvovirus vaccine majority is the weak poison of feline panleucopenia virus, the feline panleucopenia virus of deactivation or the canine parvovirus of deactivation in these reports, can not protect fully the canine parvovirus strong virus attack; The hydrophobia of connection in the seedling mostly is deactivation or low virulent strain (ERA SAD FLURY strain) is existing in pup and the 12-20 gram small white mouse brain pathogenic, so its range of application and security are restricted; In U.S.'s multiple vaccines, the strain of canine distemper virus utilization is pathogenic for A-CDV has the snow racoon dog, so topic between its security existence necessarily.Also there are above same problem in the pentavaccine of domestic development, parvovirus strain with the strain of racoon dog parvovirus, rabies virus ERA strain.
From 1970 till now, the relevant patent of the U.S. and Europe Japan has four, and US P5000951 is a kind of canine distemper inactivated vaccine patent.E P0023731A2 is the process patent that single clone prepares polyvalent vaccine, is a kind of method invention.E P0169958A2 is the patent of canine parvovirus vaccine.E P0044920A2 is for the rabies inactivated vaccine patent with the pig testis cell preparation, so the patent report of no rabies, canine distemper, canine parvovirus trigeminal live vaccine.
The manufacture method that the purpose of this invention is to provide a kind of canine distemper, rabies, canine parvovirus disease trigeminal live vaccine, seed culture of viruses and match therewith
A kind of canine distemper of the present invention, rabies, canine parvovirus disease three attenuated live vaccines seeds culture of viruses are meant that canine distemper attenuated seed culture of viruses belongs to Paramyxoviridae (Paramyxoviridae) Morbillivirus (Morbilliviridae) canine distemper virus (Caninedistemper virus), is numbered the CDV-FAC strain; The weak seed culture of viruses of rabies belongs to Rhabdoviridae (Rhabdoviridae) lyssavirus (Lyssa virus, or Rahies virus group) rabies virus (Rabies virus), is numbered CTNB
12The weak seed culture of viruses of canine parvovirus belongs to Parvoviridae, has another name called Parvoviridae (Pavoviridae), and parvovirus belongs to, and has another name called microvirus and belongs to (Parvovirus), canine parvovirus (CanineParvovirus), is numbered A
2-CPV; These three kinds of seeds culture of viruses all March 6 nineteen ninety-five, are deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO 0222.
A kind of canine distemper of the present invention, rabies, canine parvovirus disease three attenuated live vaccines are with canine distemper virus, rabies virus, and the viral liquid that three kinds of attenuated vaccine strains of canine parvovirus prepare respectively is mixed in proportion and prepares trigeminal live vaccine.This living vaccine can prevent canine distemper, rabies, three kinds of transmissible diseases of canine parvovirus disease; This three attenuated live vaccines contains canine distemper attenuated greater than 10
3.5TCID
50/ dosage, the weak poison of rabies is greater than 10
5.0PFU/ dosage, the weak poison of canine parvovirus are greater than 10
3.5TCID
50/ dosage.
The manufacture method of a kind of canine distemper of the present invention, rabies, canine parvovirus disease three attenuated live vaccines comprises that three kinds of seed culture of viruses standards, seeds culture of viruses go down to posterity and preservation, vaccine are made proportioning and frozen in check.To produce canine distemper, rabies, canine parvovirus disease three attenuated live vaccines respectively, carry out titration of virus then, according to contained immunizing dose, by canine distemper attenuated greater than 10
3.5TCID
50, the weak poison of rabies is greater than 10
5.0PFU, the weak poison of canine parvovirus are greater than 10
3.5TCID
50Mix, filter, triple vaccine is made in freeze-drying.
The cultivation of the weak poison of rabies is: will be through aseptic, and the negative BHK normal in blocks of mycoplasma check
21Cell dissociation adds 3-5 times cultivation liquid measure of original fluid, transfers pH to 7.6-7.8, and add the virus of nutrient solution 2%, mixing be sub-packed in the cell bottle or fermentor tank in microcarrier cultivate, cultivate for 33 ℃-37 ℃ and received supernatant in 3-10 days, change again and keep liquid and receive poison more once after 3 days.Make steriling test and carry out titration of virus with the plaque method when receiving poison, results liquid is stored in-20 ℃, and the shelf time is no more than 3 months.
The going down to posterity and saving as of the weak seed culture of viruses of rabies: seed culture of viruses goes down to posterity and uses BHK
21Cell, BHK
21Cell for through 150 generations that mycoplasma, exogenous factor are up to the standards with inner cell; after growing up to individual layer; change and keep liquid; keep liquid and add 2% virus stock solution used, put 33 ℃-37 ℃ and cultivate and to receive poison in 3-10 days, the viral liquid of results is added the protective material (tread flaking milk, calf serum, 6% gelatin, 6% maltose all can) of equivalent; freeze-drying; and test, reach the seed culture of viruses standard and can be stored in-30 ℃, went down to posterity once in per 3 years.
The cultivation of the weak poison of canine parvovirus is: the healthy new cub cat of choosing, and the aseptic kidney of getting is made monolayer cell in culturing bottle or in the fermentor tank, and the pH value is 7.4-7.6, cultivates the weak poison of inoculation canine parvovirus, A 2-3 days
2-CPV breeding condition is pH7.2-7.6, under the temperature 35-38 ℃ of condition, cultivates 3-6 days, and freeze thawing is received poison or received supernatant when treating 40% above cell generation pathology; Top condition is pH7.4, and 37 ℃ of temperature were cultivated 4-5 days, and freeze thawing is received poison 3 times when treating 75% above cell generation pathology; Be stored in below-20 ℃, the shelf time can not surpass 3 months, and as surpassing titration virus again before the then freeze-drying, liquid nitrogen is preserved and can be deposited 10 years, goes down to posterity once in per 10 years.
The going down to posterity and saving as of the weak seed culture of viruses of canine parvovirus: seed culture of viruses goes down to posterity and uses A
72Cell.Conventional with A
72Cell cultures becomes individual layer, the canine parvovirus that liquid measure 2% is kept in inoculation adsorbed 1 hour for 36 ℃, add and keep 37 ℃ of cultivations of liquid 4-5 days, poison is received in freeze thawing when treating 70% above cell generation pathology, aseptic through examining, add equivalent 5% sucrose skimmed milk freeze-drying, and check, reach the seed culture of viruses standard and promptly be stored in-30 ℃, went down to posterity once in per 3 years.
Canine distemper attenuated cultivation, go down to posterity and save as: select the 6-15 age in days SPF chicken embryo that physically well develops, the preparation chick fibroblast in Tissue Culture Flask or fermentor tank, was cultivated 2-10 days for 36-39 ℃, the absorption virus inoculation, cultivated 2-10 days for 30-35 ℃, when treating 40% above cell generation pathology, cell is moved to 2-8 ℃ spend the night, receive supernatant, freeze-dried mixed with the equivalent protective material, and check reaches the seed culture of viruses standard and puts-30 ℃ of preservations, goes down to posterity once in per 3 years; Top condition is 33 ℃, cultivates 2-3 days, when treating 70% above cell generation pathology, cell is moved to 3-5 ℃ spend the night, and shakes the back repeatedly and receives supernatant, and liquid nitrogen is preserved and can be deposited 10 years, goes down to posterity once in per 10 years.
The protective material of making freeze dried vaccine is 5% sucrose skimming milk, sterilized 30-40 minutes through 110-116 ℃; through check aseptic after; and by every milliliter of adding penicillin 100-500 unit, Streptomycin sulphate 100-500 micrograms or gentamicin 100-500 units; be used to join seedling; at last vaccine is sub-packed in the blue or green bottle of trident plug, carries out vacuum-freeze-dry then.
Advantage of the present invention and positively effect are as follows:
1, the advantage of low virulent strain:
The CDV-FAC strain: to ferret small white mouse nontoxicity, can be at the chicken embryo, the chick embryo fibroblast breeding, titre is up to 10
7.3TCID
50/ ml can be in Madin-Darby canine kidney(cell line), breeds on the FL cell Vero cell, and dog is had good immunogenicity, is 10 to dog 80% immunoprotection dosage
1.4TCID
50, 100% immunoprotection dosage is 10
2.1TCID
50
CTNB
12Strain: to reaching the unlikely thermally-stabilised strain of pup in small white mouse (body weight 10-20g) brain, at BHK
21Titre can reach 10 on the cell
7.25PFU/ml, to dog, cavy, the equal no pathogenicity of rabbit, immunogenicity is close with the Flury strain, is 10 to the minimum immune dosage of dog
4.5PFU.
A
2--the CPV strain: adapt to newborn cat nephrocyte of former generation, titre is up to 10
5.5TCID
50/ ml, in the small white mouse brain, the equal no pathogenicity of abdominal injection to the equal no pathogenicity of cat dog, does not have anti-ancestral's phenomenon, is 10 to the minimum immune dosage of dog
1.5TCID
50
2, the effectiveness of vaccine and immune persistence are good
For this confirms effectiveness of the present invention and immune persistence, the regional test of five batches of vaccine random sampling (same proof test) of centering trial production trial production vaccine in ground such as Shanghai, Beijing, Guangdong, Inner Mongol, Henan are done, 39540 of dogs of inoculation, test location participate in cooperation unit with testing data collect simultaneously to the dog of every batch of vaccine immunity randomly draw 20-35 exempt from preceding, exempt from back one month, 12 months respectively blood sampling survey RV and the neutralizing antibody of CEV, the HI antibody of CPV.Emphasis selects a test site to observe, to before and after the immunity and the epidemiology situation of check plot compare.
The testing data that collect in above area shows that immune dog did not have rabies, canine distemper and parvovirus disease in 1 year takes place, and its serum is carried out antibody test.In 5 batches of vaccines of trial production before antibody detection is exempted from through exempting from after antibody horizontal lower, or do not have antibody, exempt to produce higher antibody in back 1 month, prove non-natural infection institute extremely, be the generation of vaccine immunity stimulation body.Exempt from back 12 months blood rabies, canine distemper virus neutralizing antibody and canine parvovirus serum and suppress antibody horizontal and all be higher than the protection antibody horizontal, so think that exempting from back 12 months dogs should have provide protection, the immune persistence of vaccine is better.
Before the contrast vaccination, dog is detected every group of average out to RV＜2 of antibody, CDV＜1, CPV＜4.1. immune group: the vaccination dog, reduce 348 because of reason such as selling, 12 months follow-up study groups show that canine distemper, rabies, minute viral enteritis clinical onset, epidemic prevention effect all not occurring reaches 100%, test confirms that vaccine effect is well safe and reliable.2. control group: reduce by 19, animal doctor business personnel and be diagnosed as the rabies clinical onset and slaughter 1 (sickness rate 0.94%), 17 of canine distempers (death rate of the onset 16%) because of reason such as selling.Canine parvovirus causes dead 9 of infectious enteritis (death rate of the onset 8.4%).
3, safe reliability is good
In Shanghai, ground such as Guangdong, Guangxi, Inner Mongol, Henan, Beijing carried out regional test to the pilot scale vaccine, inoculate 39540 of dogs altogether, the data of the relevant vaccine safety of collecting show: the local dog that redness takes place accounts for 0.02% behind the dog injection triple vaccine, what systemic anaphylaxis took place accounts for 0.0025%, in Beijing one example taking place in the test, systemic anaphylaxis takes place when injecting triple vaccine for the second time, through rescuing rehabilitation, further investigation reveals that this dog in 6 months immunity cross twice home-made univalent vaccine.So analyze reason may with immune time too much and contain bovine serum in the univalent vaccine and the cell composition is too much relevant.Using none example of 39540 dosage through this triple vaccine of proof test observation proof in a word causes morbidity dead because of vaccinate.Vaccine safety good reliability of the present invention.
Fig. 1 is a process flow sheet of the present invention
Narrate embodiments of the invention below:
One, seed culture of viruses embodiment:
The classification of canine distemper of the present invention, rabies, canine parvovirus disease three attenuated live vaccines seeds culture of viruses, numbering and preserving number are that canine distemper attenuated seed culture of viruses belongs to the Paramyxoviridae Morbillivirus, is numbered the CDV-FAC strain, and the weak seed culture of viruses of rabies belongs to the Rhabdoviridae lyssavirus, is numbered CTNB
12, the weak seed culture of viruses of canine parvovirus belongs to the Parvoviridae parvovirus and belongs to, is numbered A
2-CPV, these three kinds of seeds culture of viruses all March 6 nineteen ninety-five, are deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and the preserving number of three kinds of seeds culture of viruses is CGMCC NO 0222.
Two, product embodiments:
Canine distemper of the present invention, rabies, canine parvovirus disease three attenuated live vaccines products, this three attenuated live vaccines product contains canine distemper attenuated greater than 10
3.5TCID
50/ dosage, the weak poison of rabies is greater than 10
5.0PFU/ dosage, the weak poison of canine parvovirus are greater than 10
3.5TCID
50/ dosage.
Three, manufacture method embodiment:
The manufacture method of a kind of canine distemper of the present invention, rabies, canine parvovirus disease three attenuated live vaccines comprises that three kinds of seed culture of viruses standards, seeds culture of viruses go down to posterity and preservation, vaccine manufacturing proportioning and freeze-drying check.The present invention produces canine distemper, rabies, three kinds of attenuated live vaccines of canine parvovirus disease respectively earlier, carries out titration of virus then, according to contained immunizing dose, by canine distemper attenuated greater than 10
3.5TCID
50, the weak poison of rabies is greater than 10
5.0PFU, the weak poison of canine parvovirus are greater than 10
3.5TCID
50Mix, filter, triple vaccine is made in freeze-drying.
(1), seed culture of viruses
The seed culture of viruses of making canine distemper, rabies, canine parvovirus disease trigeminal live vaccine is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center March 6 nineteen ninety-five, is responsible for the preservation and the supply of seed culture of viruses of the present invention by this center.
1, make rabies attenuated vaccine seed culture of viruses and must meet following standard:
(1), seed culture of viruses must be pure, must do bacterium, mould, mycoplasma and external source cause of disease check (method is seen veterinary biological product manufacturing and inspection procedure in January, 85 version) eligible at every turn after going down to posterity behind the freeze-drying seed culture of viruses Kaifeng and can be used for production of vaccine.
(2), effect standard: by " veterinary biological product manufacturing and inspection procedure " in January, 85 version method carry out potency test and should be not less than 10000LD small white mouse protection index
50
(3), virulence standard: to 1-3 age in days suckling mouse brain inner infection LD
50Be no less than 10
5.0/ 0.03ml.To 12-14 gram small white mouse brain inner infection LD
50Be 0.
(4), safety standards: soak into the sciatic nerves of 5 susceptible dogs with the virus of 10 vaccine doses, should not find rabies or death in 90 days.
(5), immunogenicity standard: it is 10 that the minimum immune dosage in 1 year of immunoprotection of susceptible dog is not higher than
4.5PFU.
2, make canine parvovirus attenuated vaccine seed culture of viruses and must meet following standard:
(1), pure check, must do bacterium, mould seedling, mycoplasma and the check of external source cause of disease after going down to posterity behind freeze-drying seed culture of viruses Kaifeng at every turn, eligible can be used for production of vaccine.
(2), seed culture of viruses is to the oral and collunarium inoculation 10 of 3-12 monthly age dogs
5.5TCID
50, constant, promptly not pathogenic to dog through pup interior generation 5 generation virulence.
(3), seed culture of viruses is not less than 10 in the titre of A72 cell
5.0TCID
50/ ml.The titre that goes down to posterity on newborn cat kidney primary cell is not less than 10
5.0TCID
50/ ml.
(4), seed culture of viruses is not higher than 10 to the minimum immune dosage in 1 year of pup immunoprotection
3.5TCID
50
(5), seed culture of viruses is with 20 of minimum immune dosage immunity susceptible dogs, exempts to attack poison in back 21 days, attack the anti-infective protection ratio in poison back and be not less than 95%.
3, make canine distemper virus attenuated vaccine seed culture of viruses and must meet following standard:
(1), pure check: must do bacterium, mould, mycoplasma and the check of external source cause of disease after going down to posterity behind the freeze-drying seed culture of viruses Kaifeng, eligible can be used for production of vaccine at every turn.
(2), virulence standard: 1 dog using dosage is inoculated 2 susceptible ferrets, inoculates back 21 days and has no adverse reaction.To dog and the equal no pathogenicity of small white mouse.
(3), seed culture of viruses is not less than 10 to the titre of chick embryo fibroblast
5.0TCID
50/ 0.05ml.
(4), seed culture of viruses is not higher than 10 to the dog minimum immune dosage
2.1TCID
50
(5), seed culture of viruses is with minimum immune dosage immunity susceptible dog, exempts to use in back 21 days 500ID
50CDV is strong, and poison is attacked poison, attacks malicious protection ratio and is not less than 95%.
4, seed culture of viruses going down to posterity and preserving
(1), the going down to posterity and preserving of the weak seed culture of viruses of rabies: seed culture of viruses goes down to posterity and uses BHK
21Cell, BHK
21Cell be 150 generations through mycoplasma, exogenous factor assay approval with inner cell, grow up to individual layer after, change and keep liquid, keep liquid and add 2% virus stock solution used, put 33 ℃ and cultivate and received poison in 6 days.The viral liquid of results is added the calf serum of equivalent, freeze-drying, and test, reach the seed culture of viruses standard and can be stored in-30 ℃, went down to posterity once in per 3 years.
(2), the going down to posterity and preserving of the weak seed culture of viruses of canine parvovirus: seed culture of viruses goes down to posterity and uses A
72Cell.Conventional with A
72Cell cultures becomes individual layer, the canine parvovirus that liquid measure 2% is kept in inoculation adsorbed 1 hour for 36 ℃, add and keep 37 ℃ of cultivations of liquid 4-5 days, poison is received in freeze thawing when treating 70% above cell generation pathology, aseptic through examining, add equivalent 5% sucrose skimmed milk freeze-drying, and check, reach the seed culture of viruses standard and promptly be stored in-30 ℃, went down to posterity once in per 3 years.
(3), canine distemper attenuatedly going down to posterity and preserving: selecting the 9 ages in days SPF chicken embryo that physically well develops for use, asepticly get fetus, decaptitate and internal organ, shred, with 37 ℃ of digestion of 0.25% pancreatin solution 30 minutes, sucking-off enzyme liquid is washed 3 times with Hank ' s liquid then, after adding the piping and druming of adequate nutrition liquid, left standstill 10 minutes, sucking-off top does not have the piping and druming once more in aseptic bottle of visible tissue piece liquid, leaves standstill 10 minutes, and sucking-off top liquid mixes with sucking-off for the first time, make cell suspension, nutritive medium is for containing 199 substratum of 5-10% bovine serum.Be diluted to every milliliter and contain cell count 80-100 ten thousand, be sub-packed in the culturing bottle, grew up to good monolayer cell in 2-3 days in 37 ℃ of cultivations, abandon nutrient solution, press 2-2.5% of growth media and add seed culture of viruses, adsorbed 1 hour, shake 1 time the centre, changes to keep 35 ℃ of liquid and cultivated 4-5 days, when waiting 70% above cell generation pathology, cell is moved to 4 ℃ spend the night, receive supernatant.Qualified through steriling test, promptly mix with the calf serum of equivalent, packing freeze-drying, and check reach seed culture of viruses standard-30 ℃ preservation, go down to posterity once in per 3 years.
(2), the manufacturing of vaccine
1, the cultivation of the weak poison of rabies: will be through aseptic, the negative BHK normal in blocks of mycoplasma check
21Cell is with 0.25% trysinization, 3-5 times cultivation liquid measure (50% the MEM nutrient solution that adds original fluid, 50% 0.5% lactoalbumin hydrolysate EarleShi damping fluid, 10% calf serum, green grass or young crops, each 100 units/ml of strepto-poison transfer pH to 7.6-7.8 with 7.5% sodium bicarbonate), and add the virus of nutrient solution 2%, mixing is sub-packed in the cell bottle, cultivates for 37 ℃ and receives supernatant in 6 days, changes to keep liquid and receive poison more once after 3 days again.Per three bottles of mixing are made sterility test respectively and are carried out titration of virus with drink spot method (seeing appendix) when receiving poison.Results liquid is stored in-20, and the shelf time is no more than 3 months.
2, the cultivation of the weak poison of canine parvovirus: the healthy new cub cat of choosing, etherization, water logging is dead, and suitably thimerosal is washed 3 times, last bubble 20 minutes, the aseptic kidney of getting goes coating to shred, and washes 3 times with EagleShi liquid, add 37 ℃ of digestion of 0.25% pancreatin 40 minutes, remove trypsin solution, wash 3 times with EagleShi liquid, piping and druming adds adequate nutrition liquid (50%199 substratum, 40%0.5% lactoalbumin hydrolysate, 10% calf serum, celebrating big mould malicious 100 units/ml, 1.1/ ten thousand glutaminase 7.5%NaHCO
3Transfer pH to 7.4-7.6), left standstill 10 minutes, upper liquid is poured in the feeding solution, add nutritive medium piping and druming once more, left standstill 10 minutes, pour out upper liquid again, so repeat 3 times; Upper liquid is all collected in the same bottle, every about 500-1000ml of feline kidney cells Ensure Liquid liquid, be sub-packed in the culturing bottle again, cultivate for 37 ℃ and grew up to individual layer in 2-3 days, the weak poison of the canine parvovirus of inoculation culture liquid measure 2%, 36 ℃ adsorbed 1 hour, add and keep liquid (3% calf is equipped with 199 clear substratum, includes 100 units and celebrates big toxin/ml, and pH is 7.4), cultivated 4-5 days for 37 ℃, poison is received in freeze thawing when treating 70% above cell generation pathology, and sterility test and titration of virus are made in per three bottles of mixing respectively when receiving poison, are stored in-20 ℃, shelf time can not surpass 3 months, then freezes Yu Qianying titration virus again as surpassing.
3, the poison of canine distemper is weak cultivates: cultural method is with the seed culture of viruses manufacturing.
(3), the proportioning of vaccine and freeze-drying
1, above method preparation and the virus stock solution used through being up to the standards can be used for freeze-drying.
2, the protective material of making freeze dried vaccine be 5% sucrose skimming milk, through 110-116 ℃ of sterilizations 30-40 minutes, be used to join seedling through checking after aseptic.
3, join seedling-growing method: the seedling ratio is joined in the titre decision according to every kind of virus, and behind the seedling thorough mixing, filtering can the packing freeze-drying.In order to suppress assorted bacterium, every milliliter adds penicillin 100-500 units, Streptomycin sulphate 100-500 micrograms or gentamicin 100-500 units.
4, the freeze-drying of vaccine: vaccine is sub-packed in the blue or green bottle of trident plug, carries out vacuum-freeze-dry then.
5, freeze-drying finishes promptly to be in Freeze Drying Equipment under the vacuum dress attitude and presses the trident plug, takes out the back and presses upper aluminum cap.
6, every bottle of vaccine should indicate the vaccine title, in batches, loading amount.Some bottles of vaccines are loaded in the packing box, and with specification sheets.Every box vaccine label indicates goods title, seedling composition, authentication code, lot number, loading amount, check number tree, usage, and store method, effect phase, date of built and factory's name etc.
7, all manufacturings and freeze-drying process, must detail record in special-purpose statistical form, and be marked with experiment people and collator.
(4), the check of vaccine
1, steriling test: undertaken by " inspection after construction relevant regulations " in " veterinary biological product manufacturing and inspection procedure ".Should make assorted bacterium counting and pathogenicity bo when freeze dried vaccine has bacteria growing identifies.Assorted bacterium pathogenicity bo is identified by " inspection after construction relevant regulations ".Assorted bacterium counting: the nonpathogenic bacteria number of every milliliter of vaccine is no more than 1000.
2, safety verification:
The small white mouse proof test: get 8 18-20 gram small white mouses, each intracranial inoculation 0.03ml vaccine, other gets 8 each abdominal injection 0.5ml of same weight, observes 7 days.Viewing duration is if any one group has 2 or 2 above small white mouses untoward reaction due to the vaccine itself to occur, for defective.If untoward reaction due to the non-vaccine itself occurs for a short time more than 2 or 2 from mouse, then be no result, Ying Chongjian; If heavily inspection is not judged to defective.
The dog proof test: get 3 susceptible dogs, press the approach that indicates on the label, each injects the vaccine of 10 using dosages, observes day by day to 21 days.Untoward reaction due to the appearance vaccine itself is declared defective; If untoward reaction is not due to this product, declare no result, can heavily examine, if heavily inspection then declare defective.
3, efficacy test:
Canine distemper virus: canine distemper virus content in the vaccine is carried out histocyte medium lethal dose (TCID with chick embryo fibroblast
50) mensuration TCID
50Titre is not less than 10
3.5It is qualified to be judged to, defective can heavily the inspection once.
Rabies virus: measure the weak malicious plaque unit (PFU) of mad dog in the vaccine with micro-plaque method, be provided with standard rabies serum neutralization test contrast.Under the controlled trial establishment situation, rabies virus content is not less than 10 in every batch and the per two batches of vaccines
5.0PFU/ agent.Vaccine is undertaken by the effectiveness calibrating in the version " veterinary biological product manufacturing and inspection procedure " in January, 85 " Rabies Vaccine is made and inspection procedure " mouse protection assessment of indices, must not be lower than 10000LD to the protection index of small white mouse
50For qualified, the failure can heavily examine twice.
Canine parvovirus: use A
72Cell carries out the cell medium lethal dose mensuration (TCID of microcomponent to the content of canine parvovirus in the vaccine
50), before measuring with in 37 ℃ of water-baths of Rabies Antiserum of the no canine parvovirus antibody of vaccine and equivalent with 1 hour.TCID
50Measure titre and do not hang down 10
3.5It is qualified to be judged to, defective can heavily the inspection once.
4, residue moisture content check: every batch of freeze-dried vaccine is appointed and is taken out 4 samples, measures moisture content by version " veterinary biological product manufacturing and inspection procedure " in January, 85 " residue moisture content assay method ".Each sample surplus water part should be no more than 4%.
5, vacuum tightness is measured by version " veterinary biological product manufacturing and inspection procedure " in January, 85 " relevant regulations of inspection after construction " and is undertaken.
6, physical behavior check: freeze dried vaccine is undertaken by version " veterinary biological product manufacturing and inspection procedure " in January, 85 " inspection after construction relevant regulations ".
7, all checkout procedures and assay, must detail record in the statistical form of special use.
8, finished product keeps sample: undertaken by version " veterinary biological product manufacturing and inspection procedure " in January, 85 " relevant regulations of inspection after construction ".
(5), the preservation of vaccine and use
1, effective preservation period of freeze dried vaccine: calculate Start Date from efficacy test, 2-8 ℃ of preservations were no longer than 1 year.10-30 ℃ of preservations were no longer than 2 months.
2, this vaccine is specialized in and is used for zooprophylazis canine distemper, rabies and three kinds of transmissible diseases of canine parvovirus enteritis.
3, freezing in vaccine with 1ml physiological saline or dissolved in distilled water l bottle three is an once subcutaneous or intramuscular inoculation amount of dog, pup first immunisation in 8 age in week, and every 3-4 all secondary immunity, interval immunity in 1 year later on is once.Adult dogs initial immunity injection 2 times, at interval 3-4 weeks, later annual immunity once.
4, the syringe needle and the syringe of immunity use must be sterilized, but the sterilization of unavailable chemicals, otherwise have a strong impact on vaccine potency.Period of pregnancy, bitch and unhealthy dog were not used.Anaphylaxis may be occurred to indivedual dogs, the suprarenin treatment can be used.
Claims (9)
1, a kind of canine distemper, rabies, canine parvovirus disease three attenuated live vaccines seeds culture of viruses is characterized in that: canine distemper attenuated seed culture of viruses belongs to Paramyxoviridae (Paramyxoviridae) Morbillivirus (Morbilliviridae) canine distemper virus (Caninedistemper virus), is numbered the CDV-FAC strain; The weak seed culture of viruses of rabies belongs to Rhabdoviridae (Rhabdoviridae) lyssavirus (Lyssa virus, or Rabies virus group) rabies virus (Rahies virus), is numbered CTNB
12The weak seed culture of viruses of canine parvovirus belongs to Parvoviridae, has another name called Parvoviridae (Pavoviridae) parvovirus and belongs to, and has another name called microvirus and belongs to (Parvovirus), canine parvovirus (Canine Parvovirus), is numbered A
2-CPV; The preserving number of these three kinds of seeds culture of viruses is CGMCC NO 0222.
2, a kind of canine distemper, rabies, canine parvovirus disease three attenuated live vaccines, it is characterized in that: this three attenuated live vaccines contains canine distemper attenuated greater than 10
3.5TCID
50/ dosage, the weak poison of rabies is greater than 10
5.0PFU/ dosage, the weak poison of canine parvovirus are greater than 10
3.5TCID
50/ dosage.
3, the manufacture method of a kind of canine distemper, rabies, canine parvovirus disease three attenuated live vaccines, comprise that three kinds of seed culture of viruses standards, seeds culture of viruses go down to posterity and preservation, vaccine manufacturing proportioning and freeze-drying check, it is characterized in that: will produce canine distemper, rabies, canine parvovirus attenuated live vaccines respectively, carry out titration of virus then, according to contained immunizing dose, by canine distemper attenuated greater than 10
3.5TCID
50, the weak poison of rabies is greater than 10
5.0PFU, the weak poison of canine parvovirus are greater than 10
3.5TCID
50Mix, filter, triple vaccine is made in freeze-drying.
4, manufacture method according to claim 3 is characterized in that: the cultivation of the weak poison of rabies is: will be through aseptic, and the negative BHK normal in blocks of mycoplasma check
21Cell dissociation adds 3-5 times cultivation liquid measure of original fluid, transfers pH to 7.6-7.8, and add the virus of nutrient solution 2%, mixing be sub-packed in the cell bottle or fermentor tank in microcarrier cultivate, cultivate for 33 ℃-37 ℃ and received supernatant in 3-10 days, change again and keep liquid and receive poison more once after 3 days; Make steriling test and carry out titration of virus with the plaque method when receiving poison, results liquid is stored in-20 ℃, and the shelf time is no more than 3 months.
5, manufacture method according to claim 3 is characterized in that: the going down to posterity and saving as of the weak seed culture of viruses of rabies: seed culture of viruses goes down to posterity and uses BHK
21Cell, BHK
21Cell for through 150 generations that mycoplasma, exogenous factor are up to the standards with inner cell; after growing up to individual layer; change and keep liquid; keep liquid and add 2% virus stock solution used, put 33 ℃-37 ℃ and cultivate and to receive poison in 3-10 days, the viral liquid of results is added the protective material (tread flaking milk, calf serum, 6% gelatin, 6% maltose all can) of equivalent; freeze-drying; and test, reach the seed culture of viruses standard and can be stored in-30 ℃, went down to posterity once in per 3 years.
6, manufacture method according to claim 3 is characterized in that: the cultivation of the weak poison of canine parvovirus is: the healthy new cub cat of choosing, the aseptic kidney of getting, make monolayer cell in culturing bottle or in the fermentor tank, the pH value is 7.4-7.6, cultivates 2-3 days, the weak poison of inoculation canine parvovirus, A
2The breeding condition of-CPV is pH7.2-7.6,35-38 ℃, to cultivate 3-6 days, and freeze thawing is received poison or is received supernatant when treating 40% above cell generation pathology; Top condition is pH7.4, and 37 ℃ of temperature are cultivated when treating 75% above cell generation pathology in 4-5 days freeze thawing and received poison 3 times; Be stored in below-20 ℃, the shelf time can not surpass 3 months, and as surpassing titration virus again before the then freeze-drying, liquid nitrogen is preserved and can be deposited 10 years, goes down to posterity once in per 10 years.
7, manufacture method according to claim 3 is characterized in that: the going down to posterity and saving as of the weak seed culture of viruses of canine parvovirus: seed culture of viruses goes down to posterity and uses A
72Cell, conventional with A
72Cell cultures becomes individual layer, the canine parvovirus that liquid measure 2% is kept in inoculation adsorbed 1 hour for 36 ℃, add and keep 37 ℃ of cultivations of liquid 4-5 days, poison is received in freeze thawing when treating 70% above cell generation pathology, aseptic through examining, add equivalent 5% sucrose skimmed milk freeze-drying, and check, reach the seed culture of viruses standard and promptly be stored in-30 ℃, went down to posterity once in per 3 years.
8, manufacture method according to claim 3, it is characterized in that: canine distemper attenuated cultivation, go down to posterity and save as: select the 6-15 age in days SPF chicken embryo that physically well develops, the preparation chick fibroblast, in Tissue Culture Flask or fermentor tank, cultivated 2-10 days for 36-39 ℃, the absorption virus inoculation, cultivated 2-10 days for 30-35 ℃, when treating 40% above cell generation pathology, cell is moved to 2-8 ℃ spend the night, receive supernatant, freeze-dried mixed with the equivalent protective material, and check reaches the seed culture of viruses standard and puts-30 ℃ of preservations, goes down to posterity once in per 3 years; Top condition is 33 ℃, cultivates 2-3 days, when treating 70% above cell generation pathology, cell is moved to 3-5 ℃ spend the night, and shakes the back repeatedly and receives supernatant, and liquid nitrogen is preserved and can be deposited 10 years, goes down to posterity once in per 10 years.
9, manufacture method according to claim 3; it is characterized in that: the protective material of making freeze dried vaccine is 5% sucrose skimming milk, sterilized 30-40 minutes through 110-116 ℃; through check aseptic after; and by every milliliter of adding penicillin 100-500 unit, Streptomycin sulphate 100-500 micrograms or gentamicin 100-500 units; be used to join seedling; at last vaccine is sub-packed in the blue or green bottle of trident plug, carries out vacuum-freeze-dry then.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95102239 CN1117081A (en) | 1995-03-16 | 1995-03-16 | Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 95102239 CN1117081A (en) | 1995-03-16 | 1995-03-16 | Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1117081A true CN1117081A (en) | 1996-02-21 |
Family
ID=5074241
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 95102239 Pending CN1117081A (en) | 1995-03-16 | 1995-03-16 | Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1117081A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997049825A1 (en) * | 1996-06-27 | 1997-12-31 | Merial | Canine herpesvirus based recombinant live vaccine, in particular against canine distemper, rabies or the parainfluenza 2 virus |
| CN101914503A (en) * | 2010-07-30 | 2010-12-15 | 中国农业科学院哈尔滨兽医研究所 | Canine distemper attenuated vaccine strain and application thereof |
| CN101942419A (en) * | 2010-07-30 | 2011-01-12 | 中国农业科学院哈尔滨兽医研究所 | Dog parvovirus attenuated vaccine strain and application thereof |
| CN101612396B (en) * | 2009-07-17 | 2011-12-07 | 齐鲁动物保健品有限公司 | Canine distemper live vaccine and preparation method thereof |
| CN102971010A (en) * | 2010-02-26 | 2013-03-13 | 梅里亚有限公司 | Recombinant CDV compositions and uses thereof |
| CN101573137B (en) * | 2006-12-27 | 2014-05-14 | 硕腾P有限责任公司 | Methods of vaccine administration |
| CN104208669A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Canine distemper and canine parvovirus disease bivalent vaccine and preparation method thereof |
| CN107537033A (en) * | 2016-06-23 | 2018-01-05 | 普莱柯生物工程股份有限公司 | Vaccine combination, kit and its application |
| CN107988174A (en) * | 2017-12-13 | 2018-05-04 | 新疆农业大学 | A kind of rabies-canine distemper-the strain of canine parvovirus gene recombined virus, construction method and its application |
| CN109735504A (en) * | 2018-12-20 | 2019-05-10 | 北京大北农科技集团股份有限公司动物医学研究中心 | Canine distemper virus attenuated vaccine strain and its application |
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1995
- 1995-03-16 CN CN 95102239 patent/CN1117081A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997049825A1 (en) * | 1996-06-27 | 1997-12-31 | Merial | Canine herpesvirus based recombinant live vaccine, in particular against canine distemper, rabies or the parainfluenza 2 virus |
| FR2750865A1 (en) * | 1996-06-27 | 1998-01-16 | Rhone Merieux | RECOMBINANT LIVE VACCINE BASED ON CANIN HERPESVIRUS, IN PARTICULAR AGAINST SQUARE DISEASE, RABIES OR PARAINFLUENZA VIRUS TYPE 2 |
| CZ297022B6 (en) * | 1996-06-27 | 2006-08-16 | Merial | Recombinant canine herpes virus expressing at least one antigen, particularly antigen of canine distemper, rabies, canine parvovirus, and parainfluenza type 2, and vaccine based on such virus |
| CN101573137B (en) * | 2006-12-27 | 2014-05-14 | 硕腾P有限责任公司 | Methods of vaccine administration |
| CN101612396B (en) * | 2009-07-17 | 2011-12-07 | 齐鲁动物保健品有限公司 | Canine distemper live vaccine and preparation method thereof |
| CN102971010A (en) * | 2010-02-26 | 2013-03-13 | 梅里亚有限公司 | Recombinant CDV compositions and uses thereof |
| CN101914503B (en) * | 2010-07-30 | 2012-04-18 | 中国农业科学院哈尔滨兽医研究所 | Canine distemper attenuated vaccine strain and application thereof |
| CN101942419A (en) * | 2010-07-30 | 2011-01-12 | 中国农业科学院哈尔滨兽医研究所 | Dog parvovirus attenuated vaccine strain and application thereof |
| CN101914503A (en) * | 2010-07-30 | 2010-12-15 | 中国农业科学院哈尔滨兽医研究所 | Canine distemper attenuated vaccine strain and application thereof |
| CN104208669A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Canine distemper and canine parvovirus disease bivalent vaccine and preparation method thereof |
| CN104208669B (en) * | 2013-09-30 | 2016-09-07 | 郑州后羿制药有限公司 | A kind of canine distemper, canine parvovirus disease bigeminy vaccine and preparation method thereof |
| CN107537033A (en) * | 2016-06-23 | 2018-01-05 | 普莱柯生物工程股份有限公司 | Vaccine combination, kit and its application |
| CN107988174A (en) * | 2017-12-13 | 2018-05-04 | 新疆农业大学 | A kind of rabies-canine distemper-the strain of canine parvovirus gene recombined virus, construction method and its application |
| CN109735504A (en) * | 2018-12-20 | 2019-05-10 | 北京大北农科技集团股份有限公司动物医学研究中心 | Canine distemper virus attenuated vaccine strain and its application |
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