CN108421037A - A kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture preparation method that suspends - Google Patents

A kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture preparation method that suspends Download PDF

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CN108421037A
CN108421037A CN201810312293.5A CN201810312293A CN108421037A CN 108421037 A CN108421037 A CN 108421037A CN 201810312293 A CN201810312293 A CN 201810312293A CN 108421037 A CN108421037 A CN 108421037A
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culture
liquid
antigen
porcine pseudorabies
porcine
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尹争艳
徐高原
周明光
钟恩
陈斌
方玉林
苏顺攀
陈章表
苏秀婵
黄慧君
洪灯
陈关平
曹毅
张洁
金建云
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WUHAN KEQIAN BIOLOGICAL Co Ltd
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Abstract

The invention belongs to veterinary biologics technical fields, and in particular to a kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture preparation method that suspends.The preparation method includes：Porcine pseudorabies virus, which suspends, cultivates the preparation of antigen and pig parvoviral suspension culture antigen；By after inactivation porcine pseudorabies virus and PPV Antigen Using liquid mix in proportion, add adjuvant it is fully emulsified after to get porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine.The present invention establishes the suspension culture process of porcine pseudorabies virus antigen liquid and PPV Antigen Using liquid, preparing gained viral antigen liquid using the suspension culture process has many advantages, such as that antigenic content is high, antigen is of large quantities, batch is stablized, the preparation method greatly reduces artificial use, reduce culture systems floor space and space, reduces the production cost of enterprise；Meanwhile using porcine pseudorabies of the present invention/porcine parvovirus bivalent inactivated vaccine, a needle exempts from two poison, reduces the immune time of animal, reduces Animal stress number, greatly reduces the production cost of vaccine and the aquaculture cost of raiser.

Description

A kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture that suspends Preparation method
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to a kind of porcine pseudorabies/porcine parvovirus two Join inactivated vaccine and its culture preparation method that suspends.
Background technology
Porcine pseudorabies (Pseudorabies, PR) are drawn by pseudorabies virus (Pseudorabies virus, PRV) One kind of a variety of domestic animals and wild animal that rise is by the urgency generated heat, very itched (except pig), characterized by breathing and the nervous system disease Sexually transmitted disease mainly causes farrowing sow miscarriage, production stillborn foetus, the mummification of fetus, returns feelings and match repeatly not wherein endangering maximum to pig It is pregnant；Newborn piglet mortality；Growing and fattening pigs weightening slows down and herd boar loses kind ability.The World Health Organization (OIE) by its B class animal epidemics are classified as, China is classified as two class animal epidemics.The popular of this disease is in that lasting rise becomes in the world Gesture.Pig breeding industry is had become a few days ago endangers one of maximum disease.
Porcine parvovirus (porcine parvovirus infection, PPI) is also known as pig breeding dysfunction disease.It is by pig The breeding difficulty disease of a boar caused by parvovirus.Characterized by miscarriage, stillbirth, production mummy occur for farrowing sow.Pig is thin Minor illness viral disease is a kind of pig breeding dysfunction disease caused by pig parvoviral (PPV), which is mainly shown as embryo and fetus Stillborn foetus, monster and the mummification of fetus occur for infection and death, especially first farrowing sow, but sow itself is without apparent symptom.
The effective means of the porcine pseudorabies and porcine parvovirus that prevent boar at present is immune porcine pseudorabies inactivation Vaccine and porcine parvovirus inactivated vaccines.Such product of market sale at present is single seedling, needs to be immunized respectively, increases people Work and animal stress number, and be rolling bottle technique culture, technique falls behind, and labor intensive, spinner culture area occupied is larger, Antigenic content is low, influences the using effect of vaccine.
Invention content
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of porcine pseudorabies/porcine parvovirus bigeminy Inactivated vaccine and its culture preparation method that suspends.
For achieving the above object, the technical solution adopted by the present invention is：
A kind of preparation method for the culture porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine that suspends, including walk as follows Suddenly：
(1) porcine pseudorabies virus, which suspends, cultivates the preparation of antigen：A) culture of production BHK-21 cells, including seed training It supports, level-one bioreactor expands culture and two stage biological reactor expands culture；B) continue after being inoculated with porcine pseudorabies virus strain Culture；C) porcine pseudorabies virus antigen is harvested；
(2) pig parvoviral, which suspends, cultivates the preparation of antigen：A) culture of production ST cells, including the training of ST cell spinner bottles It supports, level-one bioreactor expands culture and two level Perfusion bioreactor expands culture；B) after being inoculated with pig parvoviral strain Continue to cultivate；C) PPV Antigen Using is harvested；
(3) porcine pseudorabies virus antigen and PPV Antigen Using are mixed in proportion, add adjuvant it is fully emulsified after, Up to porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine.
In said program, the porcine pseudorabies strain is HNX-12 plants of porcine pseudorabies virus gE gene-deleted strains, institute It is WH-1 plants of pig parvoviral to state pig parvoviral strain.
In said program, in the incubation of step (1) the production BHK-21 cells, when BHK-21 cell seeds are trained Cell viability in foster bottle reaches 95% or more, cell density and reaches 4~6 × 106When a/ml, it is seeded to level-one biological respinse Device expands culture, when the density of BHK-21 cells in level-one bioreactor reaches 2~3 × 106Cells/ml is forwarded to two level Bioreactor continues to expand culture, when the density of BHK-21 cells in two stage biological reactor reaches 2~3 × 106cells/ Ml is inoculated with porcine pseudorabies virus strain.
In said program, the condition of level-one bioreactor culture described in step (1)：Culture solution Installed liquid in reactor Amount is 60%~70%, and the inoculum density of BHK-21 cells is 5~7 × 10 in reactor5Cells/mL, cultivation temperature 37 DEG C, 80~100r/min of rotating speed, pH value 7.2~7.4, dissolved oxygen (DO) value 40%.
In said program, the condition of two stage biological bioreactor culture described in step (1) is：Culture solution Installed in reactor Liquid measure is 60%~70%, and the inoculum density of BHK-21 cells is 5~7 × 10 in reactor5Cells/mL, cultivation temperature 37 DEG C, 50~60r/min of rotating speed；PH value 7.2~7.4；Dissolved oxygen (DO) value 40%.
In said program, the inoculum concentration of porcine pseudorabies strain described in step (1) is 1%~2% (volume ratio), inoculation Afterwards, continue culture condition be：50~60r/min of rotating speed, pH value 7.2~7.4, dissolved oxygen (DO) value 40%, 37 DEG C of temperature.
In said program, the condition that porcine pseudorabies virus antigen is harvested described in step (1) is：Continue to cultivate after inoculation For 24 hours~36h harvests virus liquid as Cell viability ＜ 20%.
In said program, the condition of ST cell spinner bottle cultures described in step (2) is：Rotating speed is 9~10 turns/hour, temperature 37 DEG C of degree, incubation time 36~48 hours stop culture, with 0.25% trypsin solution vitellophag when cell covers with single layer.
In said program, the condition that level-one bioreactor described in step (2) expands culture is：ST cells in reactor Inoculum density be 4 × 105~5 × 105A/ml, microcarrier 5~9g/L of content, the liquid amount 60%~70% of culture solution stir 60~75r/min of speed is mixed, dissolved oxygen (DO) is 60%, pH value 7.2~7.4,37 DEG C of temperature, incubation time 96h~120h.
In said program, the condition that two level Perfusion bioreactor described in step (2) expands culture is：In reactor The inoculum density of ST cells is 4 × 105~5 × 105A/ml, microcarrier 5~9g/L of content, the liquid amount 60% of culture solution~ 70%, 50~60r/min of mixing speed, dissolved oxygen (DO) they are 60%, pH value 7.2~7.4,37 DEG C of temperature, incubation time 72h~ 96h。
In said program, the microcarrier is 60~250 μm of diameter, suitable for the microballon of adherent cell growth, composition Ingredient is glucan.
In said program, the inoculum concentration of pig parvoviral strain described in step (2) is 2%~5%, continues to cultivate after inoculation Condition：Mixing speed is 50~60r/min, and dissolved oxygen (DO) is 60%, pH value 7.2~7.4,37 DEG C of temperature.
In said program, the condition that parvovirus strain is harvested described in step (2) is：Continue after cultivating 48h~72h, when When lesion occur in 80% or more ST cells, parvovirus antigen is harvested.
In said program, porcine pseudorabies virus content >=10 in step (3) the porcine pseudorabies virus antigen8.0TCID50/ Ml, content >=10 of parvovirus in the parvovirus antigen7.0TCID50/ ml or valence >=2 viral hemoagglutination (HA)10。
In said program, the volume ratio of porcine pseudorabies virus antigen and PPV Antigen Using described in step (3) is 1:1 ~1:2.
In said program, adjuvant described in step (3) is W/O/W adjuvant, the quality and pseudorabies of the adjuvant Viral antigen is identical with PPV Antigen Using gross mass.
Microcarrier used in the present invention refers to Cytodex 1, is with cross-link dextran pedestal (cross-linked Dextran matrix) based on, the pedestal is by positively charged N, N diethylin ethyl groups (N, N- Diethylaminoethyl groups) replace, which is dispersed throughout in entire microcarrier pedestal；Used perfusion type Bioreactor refers to one is the device with stirring type bioreactor suspended culture cell, and this bioreactor has thin Born of the same parents' cut-off equipment can make being in preferable nutrient environment for cytotostatic, and detrimental metabolic waste concentration buildup is relatively low, is conducive to Higher cell density is maintained, to the larger yield for improving product.
Beneficial effects of the present invention are as follows：The present invention establishes porcine pseudorabies virus antigen liquid and PPV Antigen Using liquid Suspension culture process, preparing gained viral antigen liquid using the suspension culture process has that antigenic content is high, antigen batch Greatly, the advantages that batch is stablized, the preparation method greatly reduces artificial use, reduces culture systems floor space and sky Between, reduce the production cost of enterprise；Meanwhile epidemic disease is inactivated using porcine pseudorabies of the present invention/porcine parvovirus bigeminy Seedling, a needle exempt from two poison, reduce the immune time of animal, reduce Animal stress number, greatly reduce the cost of raiser.
Specific implementation mode
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention Content is not limited solely to the following examples.
1 porcine pseudorabies virus of embodiment, which suspends, cultivates the preparation of antigen
1.1 porcine pseudorabies virus gE gene-deleted strains (HNX-12 plants)
1.1.1 production is prepared with seed culture of viruses
1.1.1.1 seed culture of viruses is bred
By floating type BHK-21 cell inoculation cell shaking flasks, 37 DEG C, 120r/min shaking table cultures are set, Cell viability 95% is taken More than, cell density 4~6 × 106Inoculation bottle is added in the seed cell of a/ml, samples and counts after mixing, is forwarded to biological respinse Device.Dissolved oxygen (DO) electrode, pH electrodes, temperature electrode calibration, tank body high pressure sterilization are carried out before bioreactor inoculating cell.According to Volume of culture is pumped into the culture solution of 70% volume of culture in advance, and optimal culture condition is arranged：37 DEG C of temperature, inoculum density 5~7 × 105Cells/ml, 50~60r/min of rotating speed, pH value 7.2~7.4, dissolved oxygen (DO) value 40% cultivate floating type BHK-21 cells, When cell density is grown to 2~3 × 106PRV is inoculated in floating type for HNX-12 plants by cells/ml with the ratio of 1% (v/v) BHK-21 cells, 50~60r/min of rotating speed, pH value 7.2~7.4, dissolved oxygen (DO) value 40%, 37 DEG C are continued to cultivate.Inoculation is left for 24 hours Cell viability is worked as on the right side<Virus liquid is harvested when 20%, quantitative separating sets -70 DEG C or less preservations, indicates title, date, seed culture of viruses generation It is inferior.
1.1.1.2 seed culture of viruses examines viral level and pure property, should meet regulation.
1.1.1.3-15 DEG C of virus seed conservation wet poison or less preserves, and the term of validity is 6 months.
1.1.1.4 virus seed subculture should be no more than for 5 generations.
1.1.2 the preparation and inspection of seedling virus liquid
1.1.2.1 the preparation of Cells for production
1.1.2.1.1 BHK-21 cell culture in level-one bioreactor
Floating type BHK-21 cell culture expands training method using seed cascade, and the kind cell of cell shaking flask culture carries out Sampling counts and calculates Cell viability, takes 95% or more Cell viability, cell density 4 × 106A/ml~6 × 106The kind of a/ml Inoculation bottle is added in daughter cell, samples and counts after mixing, is transferred in level-one bioreactor with pipeline, tank inner cell density is made to reach To 5~7 × 105cells/ml。
Dissolved oxygen (DO) electrode, pH electrodes, temperature electrode calibration, tank body high pressure are carried out before level-one bioreactor inoculating cell Sterilizing.It is pumped into the culture solution of 70% volume of culture in advance according to volume of culture, optimal culture condition is set：37 DEG C of temperature, inoculation Density 5~7 × 105Cells/ml, 80~100r/min of rotating speed, pH value 7.2~7.4, dissolved oxygen (DO) value 40% cultivate floating type BHK-21 cells.
1.1.2.1.2 BHK-21 cell culture in two stage biological reactor
When cell density reaches 2~3 × 10 in level-one bioreactor6After cells/ml or so, it is transferred to by pipeline It samples and counts in two stage biological reactor, after mixing, tank inner cell density is made to reach 5~7 × 105cells/ml。
Dissolved oxygen (DO) electrode, pH electrodes, temperature electrode calibration, tank body high pressure are carried out before two stage biological reactor inoculating cell Sterilizing.It is pumped into the culture solution of 70% volume of culture in advance according to volume of culture, optimal culture condition is set：37 DEG C of temperature, inoculation Density 5~7 × 105cells/ml；50~60r/min of rotating speed；PH value 7.2~7.4；Dissolved oxygen (DO) value 40%, is enlarged training It supports.
1.1.2.2 virus inoculation
When floating type BHK-21 cell densities reach 2~3 × 10 in two stage biological reactor6When cells/ml, with 1% (v/v) HNX-12 plants of PRV is inoculated in floating type BHK-21 cells by ratio, 50~60r/min of rotating speed, pH value 7.2~7.4, Dissolved oxygen (DO) value 40%, 37 DEG C are continued to cultivate.
1.1.2.3 virus harvest
Inoculation 24 hours or so, virus liquid is harvested as Cell viability ＜ 20%, indicates title, harvest date, generation etc., Sampling is inactivated for use after the assay was approved by following requirements.
1.1.2.3.1 steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
1.1.2.3.2 viral level measurement answers >=108.0TCID50/ml。
1.1.2.4 inactivation and the inspection of semifinished product
1.1.2.4.1 addition inactivation fills after virus liquid inactivation will examine qualified virus stock solution used or dilution, and concentration is added It is stirring while adding for the BEI to final concentration of 0.005mol/l of 0.2mol/l, after mixing, kept for 36~37 DEG C, inactivation 48 Hour, 2~4 hours periods stirred 1 time.
When 1.1.2.4.2 inactivation being blocked to terminate, the 50%Na of filtration sterilization is added in inactivation of viruses liquid immediately2S2O3 (sodium thiosulfate) solution, make its final concentration of 2.0%, stir.2~8 DEG C of preservations are set, sampling is examined by following requirements It tests after qualification for use.
1.1.2.4.4 steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
1.1.2.4.5 inactivation is examined the virus liquid after inactivation, is inoculated with well-grown BHK-21 cell monolayers (25cm2Cell bottle), every bottle of 2ml, 37 DEG C of absorption discards virus liquid after 1 hour, and cell maintenance medium 5ml is added, sets and is trained at 37 DEG C It is harvested after supporting 3, multigelation 2 times, centrifuging and taking supernatant 2ml is inoculated with BHK-21 cells again, so 2 generation of blind passage again, should observe Less than cytopathy.
1.2 WH-1 plants of pig parvovirals
1.2.1 production is prepared with seed culture of viruses
1.2.1.1 seed culture of viruses is bred
Seed culture of viruses is inoculated in by the amount of virus liquid and 1 ﹕ 10 of growth-promoting media by ST cells using synchronous inocalation method, sets 37 DEG C of rotation trainings It supports, when cytopathy (CPE) reaches 80% or more, collects virus liquid.Qualified virus liquid will be examined to mix, quantitative separating ,- 15 DEG C or less preservations, indicate title, harvest date, Virus passages etc..
1.2.1.2 seed culture of viruses examines viral level and pure property, should meet regulation.
1.2.1.3-15 DEG C of virus seed conservation wet poison or less preserves, and the term of validity is 6 months.
1.2.1.4 virus seed subculture should be no more than for 5 generations.
1.2.2 the preparation and inspection of seedling virus liquid
1.2.2.1 the preparation of Cells for production
1.2.2.1.1 cell Proliferation：The ST cells for having grown up to single layer with the digestion of 0.25% trypsin solution, by 1:3 ratios pass For Multiplying culture.The ST cells that will be enlarged by culture are transferred in rolling bottle, and adjustment rotating speed is 9~10 turns/hour, is cultivated at 37 DEG C, When cell covers with single layer (36~48 hours), with 0.25% trypsin solution vitellophag, by 1:3 ratios pass on Multiplying culture.
1.2.2.1.2 level-one bioreactor culture cell, which is digested with 0.25% trypsin solution in rolling bottle, has grown up to single layer ST cells, after counting press 4 × 105~5 × 105The density of a/ml is inoculated with level-one bioreactor, (described micro- equipped with microcarrier 60~250 μm of Carrier diameters are suitable for adherent cell growth, and group is divided into glucan composition) 5~9g/L, the dress liquid of culture medium Amount 60%~70%, adjustment mixing speed are 60~75r/min, and dissolved oxygen (DO) is 60%, pH value 7.2~7.4,37 DEG C of temperature, Cultivate 96h~120h；
1.2.2.1.3 two level Perfusion bioreactor culture cell：When the cell in level-one bioreactor is in micro- load After covering with single layer on body, growth-promoting media in drain tank washs cell 2 times with PBS (0.01mol/L, pH value 7.2).Injection 0.25% Pancreatin is digested, and after cell dissociation gets off on carrier, is injected suitable cell growth medium, is terminated digestion.Take cell Suspension, is inoculated with two level Perfusion bioreactor after counting, cell-seeding-density is 4 × 105~5 × 105A/ml, addition it is micro- Carrier (60~250 μm of the microcarrier diameter is suitable for adherent cell growth, and group is divided into glucan composition) 5~9g/L, training The liquid amount 60%~70% of base is supported, mixing speed is adjusted to 50~60r/min, and dissolved oxygen (DO) is 60%, pH value 7.2~7.4, 37 DEG C of temperature cultivates 72h~96h；
1.2.2.2 the cell that virus inoculation is worked as in bioreactor on carrier covers with before single layer (72h~96h), is discharged The DMEM culture solutions containing 2% serum are added in growth-promoting media in tank, and by 2%~5% amount inoculation kind poison, adjustment bioreactor stirs It is 50~60r/min to mix speed, and dissolved oxygen (DO) is 60%, and 7.2~7.4,37 DEG C of pH value continues to cultivate.
1.2.2.3 virus harvest connects after poison since 18 hours the cytopathy from sampling in 6 hours, when 80% with Upper cell occurs when lesion (48~72 hours), harvest virus, indicates title, harvest date, generation etc., and following requirements are pressed in sampling Inactivation is for use after the assay was approved.
1.2.2.3.1 steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
1.2.2.3.2 viral level measures WH-1 plants of every milliliter of viral levels of pig parvoviral and answers >=107.0TCID50, or Viral hemoagglutination (HA) valence answers >=210。
1.2.2.4 inactivation and the inspection of semifinished product
1.2.2.4.1 addition inactivation fills after virus liquid inactivation will examine qualified virus stock solution used or dilution, and concentration is added It is stirring while adding for the BEI to final concentration of 0.005mol/l of 0.2mol/l, after mixing, kept for 36~37 DEG C, inactivation 48 Hour, 2~4 hours periods stirred 1 time.
When 1.2.2.4.2 inactivation being blocked to terminate, the 50%Na of filtration sterilization is added in inactivation of viruses liquid immediately2S2O3 (sodium thiosulfate) solution, make its final concentration of 2.0%, stir.2~8 DEG C of preservations are set, sampling is examined by following requirements It tests after qualification for use.
1.2.2.4.3 steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
1.2.2.4.4 inactivation is examined
The virus liquid after inactivation is taken, 2 bottles of ST cells are inoculated in 5% ratio in virus liquid and growth-promoting media using Synchronos method, 37 DEG C of cultures harvest after observing 2~3 days, and multigelation 2 times, the cell culture for harvesting freeze thawing as above inoculates ST cells, such as This 2 generation of blind passage, cell answer disease-free change to generate.
2 porcine pseudorabies of embodiment/porcine parvovirus bivalent inactivated vaccine
2 vaccine preparations
2.1 water phases prepare the two kinds of virus liquid (porcine pseudorabies virus that will have been inactivated：Pig parvoviral) press 1: 1 (volume Than) be uniformly mixed.Wherein porcine pseudorabies virus inactivation provirus content answers >=108.0TCID50Disease before/ml, pig parvoviral inactivation Malicious content answers >=107.0TCID50/ ml or valence >=2 viral hemoagglutination (HA)10。
2.2 emulsification：By water phase and adjuvant according to 1:It is emulsified after 1 ratio (mass ratio) mixing.Vaccine is drawn in sampling 10.0ml is added in centrifuge tube, is centrifuged 15 minutes with 3000r/min, and the water phase that tube bottom is precipitated should be no more than 0.5ml.
2.3 packing：By the qualified vaccine quantitative separating of emulsification, lid, labeling are rolled.
The poison valency measures of 3 antigen of embodiment
3 porcine pseudorabies virus TCID50Measurement
3.1.1 virus is made 10 times with DMEM maintaining liquids (containing 2% newborn bovine serum) to be serially diluted, takes 10-5、10-6、10-7、10-8、10-95 dilutions, each dilution is inoculated with 96 hole Microtitration plates, one tandem totally 8 hole, while setting cell pair According to 8 holes, per 100 μ l of hole.
3.1.2 100 μ l BHK-21 cell suspensions are added in every hole again.
3.1.3 contain 5%CO at 37 DEG C2Incubator in culture observation 4~6 days, when 80% cytopathy (cell aggregation Protuberance, cell outline are fuzzy, circle contracts, and finally fall off disintegration) it is judged to infect, cytopathy hole count is recorded, by Reed-Muench methods Calculate TCID50.As a result 1 is see the table below, illustrates that, with the BHK-21 cells of batch, use is of the present invention by the experimental data of table 1 The suspension antigen of suspension process culture improves nearly 10 times than the antigen valence of the rolling bottle antigen using traditional rolling bottle technique culture, Antigenic content is substantially increased, production cost is reduced.
Table 1 is compared with the malicious valence of batch cell suspension antigen and rolling bottle antigen
Cell batch Cell generation Antigen lot number Suspension antigen Antigen lot number Rolling bottle antigen
0301 F6 0403 109.0TCID50/ml 0402 107.5TCID50/ml
0301 F7 0405 108.7TCID50/ml 0404 107.7TCID50/ml
0301 F8 0407 108.8TCID50/ml 0406 107.8TCID50/ml
The TCID of 3.2 pig parvovirals50Assay method
3.2.1 virus is made 10 times with DMEM maintaining liquids (containing 2% newborn bovine serum) to be serially diluted, takes 10-5、10-6、10-7、10-8、10-95 dilutions, each dilution is inoculated with 96 hole Microtitration plates, one tandem totally 8 hole, while setting cell pair According to 8 holes, per 100 μ l of hole.
3.2.2 100 μ l ST cell suspensions are added in every hole again.
3.2.3 contain 5%CO at 37 DEG C2Incubator in culture observation 4~6 days, when 80% cytopathy (cell aggregation Protuberance, cell outline are fuzzy, circle contracts, and finally fall off disintegration) it is judged to infect, cytopathy hole count is recorded, by Reed-Muench methods Calculate TCID50.2 are the results are shown in Table, is illustrated by the experimental data of table 2, with the ST cells of batch, using suspension work of the present invention The suspension antigen of skill culture improves nearly 10 times than the antigen valence of the rolling bottle antigen using traditional rolling bottle technique culture, improves Antigenic content reduces production cost.
Malicious valence comparison of the table 2 with the suspension antigen and rolling bottle antigen of batch cell culture
Cell batch Cell generation Antigen lot number Suspension antigen Antigen lot number Rolling bottle antigen
0401 F10 0517 108.0TCID50/ml 0516 106.5TCID50/ml
0401 F11 0520 107.6TCID50/ml 0519 106.7TCID50/ml
0401 F12 0523 107.7TCID50/ml 0522 106.6TCID50/ml
4 security study of embodiment is used
The susceptible piglet 5 of 4 safety verification, 28~35 age in days health, per incidence 2 times of immunizing doses (4ml) of intramuscular injection The present invention prepare gained porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine, be observed continuously 14, immune swine answers equal nothing Adverse reaction, and all strong work.Illustrated by the experimental data of table 3, the porcine pseudorabies/pig prepared with the antigen for the culture that suspends After 2 multiple dose of parvovirus bivalent inactivated vaccine is immune, have no adverse reaction safely.
The safety of 3 vaccine of table
5 effect research of embodiment
5.1 porcine pseudorabies virus part, the following optional one of method
(1) mouse immune attacks malicious method：With 4~5 week old Balb/c mouse 10, each hind leg muscle injection present invention prepares institute Obtain porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine 0.2ml, commodity list seedling Reearch in Pseudorabies virus gE Gene missing inactivation epidemic disease Seedling (HNX-12 plants), after being immunized 28, together with control mice 10, each foot pad injection PRV HNX strain virus liquid 0.1ml (5 × 104.0TCID50/ ml), it observes 14.Control mice should be 9 at least dead, and immune mouse should at least survive 9.
4 porcine pseudorabies virus part of table-mouse attacks malicious method
Illustrated by the experimental data of table 4, the porcine pseudorabies/porcine parvovirus two prepared with the antigen for the culture that suspends After connection inactivated vaccine is immune, reference《Reearch in Pseudorabies virus gE Gene lacks inactivated vaccine (HNX-12 plants)》Efficacy test method, In mouse challenge viral dosage, in the case that control group experiment is set up, three batches of experiment seedling immune groups reach 90% protective rate, commodity list The immune group of seedling reaches 90% protective rate, and compared with commodity list seedling, bivalent inactivated vaccine can also reach preferable immune effect.
(2) piglet immunological attacks malicious the method susceptible piglet 5 of 28~35 ages in days health, every incidence intramuscular injection present invention Prepare gained porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine 2ml, commodity list seedling Reearch in Pseudorabies virus gE Gene missing is gone out Live vaccine (HNX-12 plants), after being immunized 28, together with control pig 5, each collunarium inoculation PRV HNX strain virus liquid 1ml (107.0TCID50/ ml), it observes 14.Compare pig should at least 4 hairs disease, and at least 2 death, immune swine should at least 4 guarantors Shield.
Body temperature reacts after 5 porcine pseudorabies virus part of table-piglet attacks malicious method Immunization
Body temperature reacts after 6 porcine pseudorabies virus part of table-piglet attacks malicious method Immunization
Illustrated by the experimental data of table 5 and table 6, the tiny disease of porcine pseudorabies/pig prepared with the antigen for the culture that suspends After viral disease bivalent inactivated vaccine is immune, reference《Reearch in Pseudorabies virus gE Gene lacks inactivated vaccine (HNX-12 plants)》Efficacy test Method, in piglet challenge viral dosage, in the case that control group experiment is set up, immune group reaches 90% or more protective rate, commodity list The immune group of seedling reaches 90% protective rate, and compared with commodity list seedling, bivalent inactivated vaccine can also reach preferable immune effect.
5.2 pig parvoviral parts
With the above HI negative antibodies cavys of weight 350g 4, each intramuscular injection present invention prepares gained porcine pseudorabies/pig Parvovirus bivalent inactivated vaccine 0.5ml, commodity list seedling porcine parvovirus inactivated vaccines (WH-1 plants).After 28 days, together with The identical control cavy of condition 2, blood sampling measure antibody.1: 8 should all be no more than by compareing guinea pig serum HI antibody titers, should be extremely Rare 3 immune guinea pigs Serum HI antibody potency is not less than 1: 64.Above-mentioned requirements are such as not achieved, can recheck 1 time.
The antibody level of 7 pig parvoviral part of table
Illustrated by the experimental data of table 7, reference《Porcine parvovirus inactivated vaccines (WH-1 plants)》Efficacy test mark Standard, the porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine prepared with the antigen for the culture that suspends is immune, and immune group 4/4 is anti- Body level is not less than 1:64, control group is feminine gender, and experiment is set up, while 4/4 antibody level of immune group of commodity list seedling is not less than 1:64, compared with commodity list seedling, bivalent inactivated vaccine can also reach preferable immune effect.
Obviously, above-described embodiment be only intended to clearly illustrate made by example, and not limitation to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified It moves within still in the protection domain of the invention.

Claims (10)

1. a kind of preparation method for the culture porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine that suspends, which is characterized in that packet Include following steps：
（1）Porcine pseudorabies virus, which suspends, cultivates the preparation of antigen：a）The culture of production BHK-21 cells, including seed culture, Level-one bioreactor expands culture and two stage biological reactor expands culture；b）Continue to train after inoculation porcine pseudorabies virus strain It supports；c）Porcine pseudorabies virus antigen liquid is harvested, obtaining porcine pseudorabies virus through inactivation inactivates liquid；
（2）Pig parvoviral, which suspends, cultivates the preparation of antigen：a）The culture of production ST cells, including the culture of ST cell spinner bottles, Level-one bioreactor expands culture and two level Perfusion bioreactor expands culture；b）Continue after inoculation pig parvoviral strain Culture；c）PPV Antigen Using liquid is harvested, obtaining pig parvoviral through inactivation inactivates liquid；
（3）By porcine pseudorabies virus inactivate liquid and pig parvoviral inactivation liquid mix in proportion, add adjuvant it is fully emulsified after, Up to porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine.
2. preparation method according to claim 1, which is characterized in that the porcine pseudorabies strain is porcine pseudorabies disease HNX-12 plants of malicious gE gene-deleted strains, the pig parvoviral strain are WH-1 plants of pig parvoviral.
3. preparation method according to claim 1, which is characterized in that pseudorabies in the porcine pseudorabies virus antigen liquid Content >=10 of virus8.0TCID50/ ml, content >=10 of parvovirus in the parvovirus antigen liquid7.0TCID50/ ml or Viral hemoagglutination valence >=210。
4. preparation method according to claim 1, which is characterized in that the porcine pseudorabies virus inactivation liquid and the tiny disease of pig The volume ratio of poison inactivation liquid is 1:1~1:2.
5. preparation method according to claim 1, which is characterized in that the adjuvant is W/O/W adjuvant, the assistant The quality of agent inactivates liquid with porcine pseudorabies virus and the pig parvoviral inactivation gross mass of liquid is identical.
6. preparation method according to claim 1, which is characterized in that step（1）Described in level-one bioreactor expand Culture and two stage biological reactor expand the condition cultivated：Installed liquid measures be 60% ~ 70%, cell-seeding-density be 5 ~ 7 × 105Cells/mL, cultivation temperature are 37 DEG C, pH value 7.2 ~ 7.4, oxygen dissolving value 40%.
7. preparation method according to claim 1, which is characterized in that step（1）Described in porcine pseudorabies strains connect Kind amount volume ratio is 1% ~ 2%, after inoculation, holding 50 ~ 60r/min of rotating speed, and pH value 7.2 ~ 7.4, oxygen dissolving value 40%, 37 DEG C of temperature, after Continuous culture for 24 hours ~ 36h harvests porcine pseudorabies virus antigen liquid as Cell viability ＜ 20%.
8. preparation method according to claim 1, which is characterized in that step（2）Described in ST cell spinner bottle cultures item Part is：Rotating speed is 9 ~ 10 turns/hour, and 37 DEG C of temperature, incubation time 36 ~ 48 hours, when cell covers with single layer, stopping is cultivated, With 0.25% trypsin solution vitellophag.
9. preparation method according to claim 1, which is characterized in that step（2）Described in level-one bioreactor expand Culture and two level Perfusion bioreactor expand the condition cultivated：Be inoculated with post-reactor in ST cells density be 4 × 105~5 × 105A/ml, microcarrier 5 ~ 9g/L of content, the liquid amount 60% ~ 70% of culture solution, dissolved oxygen 60%, pH value 7.2 ~ 7.4, 37 DEG C of temperature, the time that level-one bioreactor expands culture are 96h ~ 120h, and two stage biological reactor expands the time of culture For 72h ~ 96h.
10. preparation method according to claim 1, which is characterized in that step（2）Described in pig parvoviral strain inoculation Measure volume ratio be 2% ~ 5%, continue to cultivate after inoculation, control mixing speed be 50 ~ 60r/min, dissolved oxygen 60%, pH value 7.2 ~ 7.4,37 DEG C of temperature continues after cultivating 48h ~ 72h, when lesion occur in 80% or more ST cells, harvests parvovirus antigen.
CN201810312293.5A 2018-04-09 2018-04-09 A kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture preparation method that suspends Pending CN108421037A (en)

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CN111035756A (en) * 2019-12-23 2020-04-21 武汉科前生物股份有限公司 Porcine pseudorabies virus and porcine epidemic diarrhea virus bivalent inactivated vaccine and preparation method thereof
CN112877299A (en) * 2021-01-25 2021-06-01 武汉科前生物股份有限公司 Porcine epidemic diarrhea and porcine parvovirus combined vaccine and application thereof

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CN111041002A (en) * 2019-12-23 2020-04-21 武汉科前生物股份有限公司 Bivalent inactivated vaccine of porcine epidemic diarrhea virus variant 2a and 2b and preparation method thereof
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CN111035756B (en) * 2019-12-23 2023-08-15 武汉科前生物股份有限公司 Porcine pseudorabies virus and porcine epidemic diarrhea virus combined inactivated vaccine and preparation method thereof
CN112877299A (en) * 2021-01-25 2021-06-01 武汉科前生物股份有限公司 Porcine epidemic diarrhea and porcine parvovirus combined vaccine and application thereof
CN112877299B (en) * 2021-01-25 2022-09-06 武汉科前生物股份有限公司 Porcine epidemic diarrhea and porcine parvovirus bivalent vaccine and application thereof

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