CN104740627B - A kind of large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals - Google Patents

Abstract

It is that the BHK21 C13 cell densities for making suspension culture in reactor using the strain of cell suspension process culture pseudo-rabies viurs attenuated strain are more than or equal to 2 × 10 the invention discloses a kind of large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals⁶Cell/mL, then with containing pseudoabies suspend kind poison a viral maintaining liquid cultivated, then by the pseudo- mad weak viral disease venom of dog of harvest it is clarified, with seedling, packing, freeze after obtain pseudo- mad dog attenuated live vaccines for animals.The present invention can realize the weak a large amount of propagation of viral disease poison of pseudo- mad dog, greatly improve virus titer and Yield of Antigen, realize automation technolo, the quality of vaccine is improved, BHK21 C13 cell densities can not only be improved, the most suitable biochemical condition of automatic monitoring cell growth or virus breeding, improve virus titer, and process scale amplification is relatively easy, vaccine quality is stable, difference between batch is small, produces and has a extensive future.

Description

A kind of large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals

Technical field

The invention belongs to the preparation method of vaccine in biomedicine field, more particularly to one kind is given birth to suspended culture cell The method for producing pseudo- mad dog attenuated live vaccines for animals.

Background technology

Pseudoabies is a variety of domestic animals caused by Pseudorabies virus (Pseudorabies virus, PRV) and wild dynamic The acute infectious disease of thing, especially endangers one of serious infectious diseases of global pig industry.

At present, pseudoabies worldwide generally occurs, and it is pseudo- mad that oneself has more than 40 countries and regions report there occurs Dog disease.With the appearance continued to develop with velogen strain of large scale of pig farm, the generation of porcine pseudorabies and prevalence are on the rise. The own You20Duo Ge provinces and cities report of China there occurs porcine pseudorabies, and oneself is successively separated to many plants of PRV strains, and oneself is separated to report Strain have Fujian A, Shan A, capital A, AKW, SR, YN, DQ-8401, S, Y plants etc..Effectively controlling for porcine pseudorabies is there is no at present Treat medicine, rely primarily on vaccine inoculation, isolation infection swinery, eliminate subclinical infection animal controlled.

At present, the method for the pseudo- rabies vaccine of domestic production still uses primary or continuous cell line adhere-wall culture method, its Traditional rolling bottle production technology is used, with antigen batch is small, difference between batch is big, inefficiency, labor intensity are big, occupied ground Many the shortcomings of.And the present invention can not only overcome the shortcoming of above-mentioned rolling bottle production technology using suspension culture process, but also it can carry High Yield of Antigen, cost-effective, raising product quality, but at present temporarily without the suspension culture work for producing pseudo- mad dog attenuated live vaccines Skill and its relevant report of application.

The content of the invention

It is an object of the invention to provide a kind of method that pseudo- mad dog attenuated live vaccines for animals are produced with suspended culture cell, with Reach the purpose of large-scale production high-quality pseudo- mad dog attenuated live vaccines for animals.

The large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals provided by the present invention, is to utilize cell suspension process Pseudo-rabies viurs attenuated strain strain is cultivated, the BHK21-C13 cell densities of suspension culture in reactor is more than or equal to 2 × 10⁶Carefully Born of the same parents/mL, are then cultivated with kind of the viral maintaining liquid for poison that suspends containing pseudoabies, then by the pseudo- mad weak viral disease venom of dog of harvest It is clarified, with seedling, packing, it is lyophilized after obtain pseudo- mad dog attenuated live vaccines for animals.

Wherein, suspension culture BHK21-C13 cell processes are in reactor：Utilize the BHK21-C13 suspension cells of recovery Strain passage in nutrient solution, which suspends, cultivates to cell density >=2.0 × 10 of BHK21-C13 suspension cells⁶Cell/mL, will suspend Cell is transferred in reactor, adds suspension special culture media to cell density 0.2-0.5 × 10⁶Cell/mL, 36.5 ± 0.5 DEG C, pH value 7.20 ± 0.1, DO values 30%-60% (solubility percentage) is cultivated 48-72 hours under rotating speed 60-110rpm BHK21-C13 cell densities in reactor are more than or equal to 2 × 10⁶Cell/mL.

The nutrient solution or suspension special culture media are：Sodium acid carbonate is added by 1.15g/L in BBSM culture mediums, then is added Plus 3%-5% (percent by volume) NBCS, regulation pH value to 7.2 ± 0.5.

Cultivate pseudo- mad dog production is with suspension virus liquid process：Qualified BHK21-C13 will be cultivated in reactor to suspend carefully Born of the same parents discard suspension special culture media, add viral maintaining liquid and recover to former volume of culture, then add pseudo- mad dog suspension by 1% volume Kind of poison, at 36.5 ± 0.5 DEG C, pH value 7.40-7.60, DO value 30%-60% (solubility percentage), under rotating speed 60-110rpm Culture, virus liquid is harvested when cytopathy >=75%.

The viral maintaining liquid is：Sodium acid carbonate is added by 1.15g/L in BBSM culture mediums, then adds 1%-3% (volumes Than) NBCS, pH value 7.4 ± 0.5.

In above-described production method, depending on production scale from small to large reactor volume be followed successively by 5L, 50L, 500 or 5000L；And, the reactor of the large-scale big volume of production and application is used in previous small-scale production container in the reactor and trained Foster BHK21-C13 suspension cells.Cultural method and condition of culture in 5L, 50L, 500L, 5000L reactor is identical.

In the production method, it is with the pseudo- mad dog base of BHK21-C13 suspension cell cultures that the pseudoabies, which suspends and plants a poison, Pseudo- mad dog production kind of the poison that suspends of plinth kind poison culture, specific acquisition process is as follows：

1) poison culture is planted on pseudo- mad dog basis

Take that adhere-wall culture obtains grows fine and BHK21-C13 attached cells of confluent monolayers, discards original fluid, The viral maintaining liquid (maintaining liquid is with claim 5) of the weak seed culture of viruses poison containing 1% (percent by volume) pseudoabies is added, in 37 DEG C of trainings Support, virus liquid, -20 DEG C of freezen protectives, sampling detection TCID are harvested when cytopathy >=75% (number percent)₅₀, often 0.1mL viral levels answer >=10^6.0TCID₅₀；

2) pseudo- mad dog production kind of the poison culture that suspends

Qualified suspension BHK21-C13 cells will be cultivated in reactor, discard cell nutrient solution, add viral maintaining liquid extensive It is multiple to plant poison, pH value 7.40-7.60, DO20%- to former volume of culture, then by the pseudo- mad dog basis of 1% (percent by volume) addition Add up the culture 72-96h that suspends under the conditions of 60% (solubility percentage), rotating speed 60-110rpm, 36.0 ± 0.5 DEG C of temperature, treat thin Virus liquid, -20 DEG C of freezen protectives are harvested during born of the same parents' lesion >=75% (number percent)；

To adapt to large-scale production, which pseudo- mad dog production also pass on expansion culture including in the reactor with kind of the poison that suspends The part of volume to needed for planting poison batch, i.e., by step 2) the reacted device of virus liquid of harvest expands culture 1-2 for obtained production Suspended with pseudoabies and plant poison batch.

In the production method, the clarification of the pseudo- mad weak viral disease venom of dog for animals is using 0.45 μm of filter core positive press filtration；

It is lyophilized to include：The pre-freeze stage：Product is down to -42 DEG C and maintains 2h by normal temperature inlet, 1-2h；The lyophilization stage： Product is risen to -8 DEG C from -42 DEG C and maintains 10h by 1-2h；Product is risen to 0 DEG C from -8 DEG C and maintains 0.5-1h by 0.5-1h；Desorption Drying stage：Product is risen to 28 DEG C from 0 DEG C and maintains 7h by 1-2h, is freezed and is terminated.

The present invention produces pseudo- mad dog attenuated live vaccines for animals with suspended culture cell, realizes large-scale production high-quality for animals Pseudo- mad dog attenuated live vaccines.The core of this method is to suspend to cultivate BHK21-C13 cells and connect on a large scale using bioreactor Pseudo- mad kind of dog poison is planted, regulation culture parameters make Pseudorabies virus amount reproduction, so as to obtain the pseudo- mad dog of high concentration and high titre Virus, virus liquid is clarified, dilution, lyophilized prepare pseudo- mad dog attenuated live vaccines for animals.BHK21- is used using what the present invention was provided C13 suspended culture cells prepare pseudo- mad dog attenuated live vaccines technique for animals, are used as the BHK21-C13 of Pseudorabies virus host cell Cell suspension cultures are extremely successful, and volume of culture reaches 2-4 × 10 up to 5000L, cell density⁶Cell/mL.The present invention can The weak a large amount of propagation of viral disease poison of pseudo- mad dog are realized, virus titer and Yield of Antigen is greatly improved, realizes automation technolo, improve epidemic disease The quality of seedling, solving traditional spinner culture BHK21-C13 cells production, Pseudorabies virus yields poorly, vaccine potency is low, immune Dosage is big to wait technical barrier.Present invention overcomes current technique is cumbersome, antigenic content is low, effect is low, and dosage is big, difference between batch is big Deng technical barrier, BHK21-C13 cell densities can not only be improved, the most suitable biochemical bar of automatic monitoring cell growth or virus breeding Part, improves virus titer, and process scale amplification is relatively easy, and vaccine quality is stable, difference between batch is small, production and application prospect It is wide.

The present invention is described in further details with reference to specific embodiment.

Brief description of the drawings

Fig. 1 is the process chart that present invention suspended culture cell prepares pseudo- mad dog attenuated live vaccines for animals

Embodiment

The acquirement approach of various biomaterials described in embodiment be only to provide it is a kind of test obtain approach with up to To specifically disclosed purpose, the limitation to biological material source of the present invention should not be turned into.In fact, used biomaterial Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to carrying in embodiment Show and be replaced.

Embodiment is implemented lower premised on technical solution of the present invention, gives detailed embodiment and specific Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.

Embodiment 1, with suspended culture cell prepare pseudo- mad dog attenuated live vaccines for animals

As shown in figure 1, the present invention prepares the technical process of pseudo- mad dog attenuated live vaccines for animals with suspended culture cell Including following five steps：1st, suspension BHK21-C13 kinds cell culture；2nd, suspension BHK21-C13 cells 5L, 50L, 500L, 5000L bioreactor cultures；3rd, the weak poison of pseudo- mad dog, which suspends, plants poison culture；4th, pseudo- mad dog attenuated live vaccines production is trained with suspension venom Support；5th, clarification, with seedling, packing, freeze.

First, suspend culture BHK21-C13 kind cells

1st, cell recovery is planted

The BHK21-C13 frozen a suspension cell strain is taken out from liquid nitrogen, 37 DEG C of fast melts, 1000rpm centrifuges 5 points Clock, abandons supernatant, adds suspension special culture media and (prepares：Sodium acid carbonate is added by 1.15g/L in BBSM culture mediums, then is added 3%-5% (percent by volume) NBCS, regulation pH value to 7.2 ± 0.5.BBSM culture mediums are purchased from the clear big section of day one in Beijing Skill Co., Ltd, product code：MD910), it is resuspended, regulation inoculum density is 0.2-0.5 × 10⁶Cell/mL, goes to 125mL thin Cultivated in born of the same parents' blake bottle, volume of culture：30-50mL, 37 DEG C, 100rpm cultures.

2nd, passage

Sample and count after cell culture 48h, if cell density >=2.0 × 10⁶Passage is carried out during cell/mL, otherwise Continue to cultivate, take the cell for needing to pass on, add suspension special culture media (preparing ibid), cell liquid is diluted to 0.2-0.5 ×10⁶Cell/mL, will the cell liquid that diluted dispense blake bottle in suspend culture 1-3 generations, volume needed for being expanded to reactor and Density (cell density >=2.0 × 10⁶Cell/mL).

2nd, reactor, which suspends, cultivates BHK21-C13 cells

1st, BHK21-C13 cells 5L reactors, which suspend, cultivates

Step one is cultivated to obtained satisfactory suspension cell (cell density >=2.0 × 10⁶Cell/mL, survival rate It is blended in more than 90%) 250-1000mL in special inoculation bottle.Cell in inoculation bottle is transferred in 5L reactors, added outstanding Floating special culture media (preparing ibid) is to 5000mL, and cell density is diluted to 0.2-0.5 × 10⁶Cell/mL, sets cell culture Temperature：36.5 ± 0.5 DEG C, pH value：7.20 ± 0.1, DO value：20%-60% (saturation degree percentage), rotating speed is close with cell Degree, volume of culture, agitating mode it is different and different, typically in 60-110rpm.Sample and count after cell culture 48h, work as cell Density >=2.0 × 10⁶Cell liquid is gone in 50L reactors during cell/mL and cultivated, or (be can be used for as culture virus use Pseudo- mad dog production kind of the poison culture that suspends, it is different according to the malicious demand of production, thereby using the bioreactor culture of different size), Otherwise continue to cultivate to required cell density.Through surveying, cell density reaches 2-4 × 10⁶Cell/mL.

2nd, BHK21-C13 cells 50L reactors, which suspend, cultivates

(cell density >=2.0 × 10 that meet the requirements for taking step 1 to cultivate⁶Cell/mL) suspension cell, add 50L it is anti- Answer in device, add suspension special culture media (composition is ibid) to 50L, cell density is diluted to 0.2-0.5 × 10⁶Cell/mL, Set cell culture temperature：36.5 ± 0.5 DEG C, pH value：7.20 ± 0.1, DO value：20%-60% (saturation degree percentage), rotating speed With cell density, volume of culture, agitating mode it is different and different, typically in 60-110rpm.Sampled after cell culture 48h Count, when cell density >=2.0 × 10⁶During cell/mL, cell can be gone in 500L reactors and continue to cultivate or culture virus Use, otherwise continue to cultivate to required cell density.

3rd, BHK21-C13 cells 500L reactors, which suspend, cultivates

(cell density >=2.0 × 10 that meet the requirements for taking step 2 to cultivate⁶Cell/mL) suspension cell, add 500L it is anti- Answer in device, add suspension special culture media (composition is ibid) to 500L, cell density is diluted to 0.2-0.5 × 10⁶Cell/ ML, sets cell culture temperature：36.5 ± 0.5 DEG C, pH value：7.20 ± 0.1, DO value：20%-60% (saturation degree percentage), Rotating speed with cell density, volume of culture, agitating mode it is different and different, typically in 60-110rpm.After cell culture 48h Sampling is counted, when cell density >=2.0 × 10⁶During cell/mL, cell can be gone to 5000L reactors and continue to cultivate or virus Culture is used, and otherwise continuing culture, (cell density reaches 2-4 × 10 to required cell density⁶Cell/mL).

4th, BHK21-C13 cells 5000L reactors, which suspend, cultivates

(cell density >=2.0 × 10 that meet the requirements for taking step 3 to cultivate⁶Cell/mL) suspension cell, add 5000L In reactor, suspension special culture media (composition is ibid) about 4500L is added, cell density is diluted to 0.2-0.5 × 10⁶Carefully Born of the same parents/mL, set cell culture temperature：36.5 ± 0.5 DEG C, pH value：7.20 ± 0.1, DO value：20%-60% (saturation degree percentages Than), rotating speed with cell density, volume of culture, agitating mode it is different and different, typically in 60-110rpm.Cell culture Sample and count after 48h, when cell density >=2.0 × 10⁶It is used for Virus culture during cell/mL, otherwise continues to cultivate to required thin (cell density reaches 2-4 × 10 to born of the same parents' density⁶Cell/mL).

3rd, pseudo- mad dog, which suspends, plants poison culture

1st, poison culture is planted on pseudo- mad dog basis

Adhere-wall culture：From liquid nitrogen take out one freeze BHK21 attached cells strain, 37 DEG C of fast melts, 1000rpm from The heart 5 minutes, abandons supernatant, adds a small amount of MEM nutrient solutions (MEM culture mediums, with 7.5% (quality percent by volume W/V) bicarbonate Sodium solution adjusts pH value to 7.2 ± 0.5, addition 8%-10% (percent by volume) NBCS；MEM culture mediums are purchased from suitable Emerging city Sai Er bio tech ltd, product code：75cm is added after 1605-030) being resuspended²In Tissue Culture Flask, add MEM nutrient solutions are to 30mL, to 37 DEG C of incubator quiescent cultures, taken after cell covers with individual layer or continue passage.

Passage：Take the attached cell for covering with individual layer, discard original fluid, add digestive juice (0.25% trypsase- 0.02%EDTA) 5-10ml, by required ratio addition MEM nutrient solutions after cell dissociation is scattered, cell liquid is dispensed in proportion to In neoblast bottle, to 37 DEG C of incubator quiescent cultures, taken after cell covers with individual layer or continue passage.

Plant poison culture in basis：Take and grow fine and BHK21-C13 attached cells of confluent monolayers, discard original fluid, Add viral maintaining liquid and (maintain formula of liquid：MEM culture mediums, are adjusted with 7.5% (quality percent by volume W/V) sodium bicarbonate solution PH value is saved to 7.4 ± 0.5, addition 1%-3% (percent by volume) NBCS；MEM culture mediums are purchased from Yixing City Sai Er Bio tech ltd, product code：Original volume 1605-030) is supplemented to, then it is pseudo- mad in the ratio addition of 1% (volume ratio) Kind of dog poison (such as Bartha-K61 strains, originate Harbin Veterinary Medicine Inst., China Academy of Agriculture), in 37 DEG C of cultures, when thin Virus liquid is harvested during born of the same parents' lesion >=75% (number percent) and obtains pseudo- mad dog basis kind poison, -20 DEG C of freezen protectives, sampling detection TCID₅₀, >=10 are answered per 0.1mL viral levels^6.0TCID₅₀。

2nd, pseudo- mad dog production kind of the poison culture that suspends

Qualified suspension BHK21-C13 cells (step 2 is obtained, and the amount of taking is adjusted according to scale) will be cultivated in reactor, Original fluid is discarded, viral maintaining liquid is added and (maintains formula of liquid：BBSM culture mediums press the sodium acid carbonate that 1.15g/L is added, addition 1%-3% (percent by volume) NBCS, regulation pH value to 7.4 ± 0.5) recover to former volume of culture, adds 1% Malicious (above-mentioned steps 1 are obtained) is planted on (percent by volume) pseudo- mad dog basis, sets the pH value of Virus culture as 7.40-7.60, DO： 20%-60% (saturation degree percentage), rotating speed：60-110rpm, temperature：36.0 ± 0.5 DEG C, add up the culture 72-96h that suspends, treat Virus liquid is harvested during cytopathy >=75% (number percent) as the production kind poison, -20 DEG C of freezen protectives of suspending.

When more mass producing, production kind of the venom that suspends of harvest can be used, reacted device continuously cultivates 2-3 for conduct again Suspend production kind poison, -20 DEG C of preservations.

4th, pseudo- mad dog production suspension venom culture

1st, suspension virus liquid is used in the pseudo- mad dog production of 50L bioreactor cultures

Qualified suspension BHK21-C13 cells (step 2 acquisition) will be cultivated in reactor, discard suspension special culture media, Add viral maintaining liquid and (maintain formula of liquid：BBSM culture mediums add sodium acid carbonate by 1.15g/L, add 1%-3% (volumes hundred Point ratio) NBCS, regulation pH value to 7.4 ± 0.5) recover to former volume of culture, add 1% (percent by volume) pseudo- mad Dog, which suspends, produces kind of malicious (step 3 is obtained), sets the pH value of Virus culture as 7.40-7.60, DO values：20%-60% (saturations Spend percentage), rotating speed：60-110rpm, temperature：36.0±0.5℃.Suspend culture, treats cytopathy >=75% (quantity percentage Than) when harvest virus liquid, -20 DEG C of freezen protectives.Sampling detection TCID₅₀, often viral level answers >=10 in 0.1mL^6.0TCID₅₀。 Satisfactory virus liquid is used to prepare vaccine.

2nd, suspension virus liquid is used in the pseudo- mad dog production of 500L bioreactor cultures

Qualified suspension BHK21-C13 cells (step 2 acquisition) will be cultivated in reactor, discard suspension special culture media, Add viral maintaining liquid and (maintain formula of liquid：BBSM culture mediums press the sodium acid carbonate that 1.15g/L is added, and add 1%-3% new born bovines Serum, regulation pH value to 7.4 ± 0.5) recover to former volume of culture adds the pseudo- mad dog suspension production of 1% (percent by volume) Malicious (step 3 is obtained) is planted, the pH value of Virus culture is set as 7.40-7.60, DO values：20%-60%, rotating speed：60-110rpm, Temperature：36.0±0.5℃.Suspended culture, and virus liquid, -20 DEG C of freezen protectives are harvested when cytopathy >=75%.Sampling detection TCID₅₀, often viral level answers >=10 in 0.1mL^6.0TCID₅₀.Satisfactory virus liquid is used to prepare vaccine.

3rd, suspension virus liquid is used in the pseudo- mad dog production of 5000L bioreactor cultures

Qualified suspension BHK21-C13 cells (step 2 acquisition) will be cultivated in reactor, discard suspension special culture media, Add viral maintaining liquid and (maintain formula of liquid：BBSM culture mediums add sodium acid carbonate by 1.15g/L, add 1%-3% (volumes hundred Point ratio) NBCS, regulation pH value to 7.4 ± 0.5) recover to former volume of culture, add 1% (percent by volume) pseudo- mad Dog, which suspends, produces kind of malicious (step 3 is obtained), sets the pH value of Virus culture as 7.40-7.60, DO：20%-60% (saturation degrees Percentage), rotating speed：60-110rpm, temperature：36.0±0.5℃.Suspend culture, treats cytopathy >=75% (percent by volume) When harvest virus liquid, -20 DEG C of freezen protectives.Sampling detection TCID₅₀, often viral level answers >=10 in 0.1mL^6.0TCID₅₀.Will symbol Closing desired virus liquid is used to prepare vaccine.

5th, clarify, with seedling, packing, lyophilized, product inspection

1st, clarify

The suspension production venom that step 4 is harvested proposes that thaw at RT is clarified with 0.45 μm of sterilizing from freezer In filter pretreatment virus liquid to the transferring storage tank with cooling system.

2nd, with seedling, packing

Qualified semi-finished product will be examined to add appropriate freeze drying protectant quantitative separating, every part of vaccine at least contains 5000TCID₅₀。 It is rapid after packing to carry out vacuum freezedrying.

3rd, freeze

The pre-freeze stage：Product is down to -42 DEG C and maintains 2h by normal temperature inlet, 1-2h；

The lyophilization stage：Product is risen to -8 DEG C from -42 DEG C and maintains 10h by 1-2h；0.5-1h rises product from -8 DEG C To 0 DEG C and maintain 0.5-1h；

The adsorption stripping and dry stage：Product is risen to 28 DEG C from 0 DEG C and maintains 7h by 1-2h, is freezed and is terminated.

4th, product inspection

The detection of following items is carried out to finished product seedling：Physical behavior, steriling test, residual moisture are determined, vacuum is determined, Safety verification, efficacy test, heat stabilization test, baterial endotoxin test, abnormal toxicity tests.Related detecting method and standard Meet《Republic of China Veterinary Pharmacopoeia 2010 edition the 3rd》Regulation.

Assay is as shown in table 1.

The pseudo- mad dog attenuated live vaccines product inspection result for animals of table 1

As can be seen from Table 1, the present invention prepares the production technology of pseudo- mad dog attenuated live vaccines for animals with suspended culture cell Unit article yield is improved, high cell densities culture and expression is realized, production cost, labor intensity is reduced, it is ensured that The quality of vaccine product is mass produced, produces and has a extensive future.

Claims (12)

1. a kind of large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals, is to utilize cell suspension process culture pseudoabies Malicious low virulent strain, makes the BHK21-C13 cell densities of suspension culture in reactor reach 2-4 × 10⁶Cell/mL, then with containing puppet Rabies suspend kind poison a viral maintaining liquid cultivated, then by the pseudo- mad weak viral disease venom of dog of harvest it is clarified, with seedling, divide Dress, it is lyophilized after obtain pseudo- mad dog attenuated live vaccines for animals, viral level in the pseudo- mad weak viral disease venom of dog described in per 0.1mL >= 10^6.0TCID₅₀；

The nutrient solution or suspension special culture media of suspension culture BHK21-C13 cells are in reactor：Pressed in BBSM culture mediums 1.15g/L adds sodium acid carbonate, then adds 3%-5% NBCSs, regulation pH value to 7.2 ± 0.5 by percent by volume；

Cultivate pseudo- mad dog production is with suspension virus liquid process：The BHK21-C13 suspension cells for cultivating qualified in reactor are abandoned Fall the special culture media that suspends, add viral maintaining liquid and recover to former volume of culture, then pseudo- mad dog suspension kind is added by 1% volume Poison, under 36.0 ± 0.5 DEG C, pH value 7.40-7.60, DO value 20%-60%, rotating speed 60-110rpm cultivate, treat cytopathy >= Virus liquid is harvested when 75%.

2. production method according to claim 1, it is characterised in that：Suspend culture BHK21-C13 cell mistakes in reactor Cheng Wei：Using the BHK21-C13 suspension cells strain of recovery, passage suspension is cultivated to BHK21-C13 suspension cells in nutrient solution Cell density >=2.0 × 10⁶Cell/mL, suspension cell is transferred in reactor, adds suspension special culture media close to cell Spend 0.2-0.5 × 10⁶Cell/mL, in 36.5 ± 0.5 DEG C, pH value 7.20 ± 0.1, DO value 30%-60%, rotating speed 60-110rpm BHK21-C13 cell densities in lower culture 48-72 hours to reactor are more than or equal to 2 × 10⁶Cell/mL.

3. production method according to claim 1 or 2, it is characterised in that：The viral maintaining liquid is：In BBSM culture mediums Sodium acid carbonate is added by 1.15g/L, then adds 1%-3% NBCSs, pH value 7.4 ± 0.5 by volume.

4. production method according to claim 3, it is characterised in that：Depending on production scale, reactor volume is successively from small to large For 5L, 50L, 500 or 5000L；And, the reactor of the large-scale big volume of production and application uses previous small rule in the reactor The BHK21-C13 suspension cells cultivated in mould production containers.

5. production method according to claim 4, it is characterised in that：Culture in 5L, 50L, 500L, 5000L reactor Method and condition of culture are identical.

6. production method according to claim 3, it is characterised in that：It is to use BHK21- that the pseudoabies, which suspends and plants a poison, Basic pseudo- mad dog production kind of the poison that suspends for planting poison culture of the pseudo- mad dog of C13 suspension cell cultures, specific acquisition process is as follows：

1) poison culture is planted on pseudo- mad dog basis

Take that adhere-wall culture obtains grows fine and BHK21-C13 attached cells of confluent monolayers, discards original fluid, adds The viral maintaining liquid of the weak seed culture of viruses of the pseudoabies containing percent by volume 1% poison, in 37 DEG C of cultures, when cytopathy number percent >= Virus liquid, -20 DEG C of freezen protectives, sampling detection TCID are harvested when 75%₅₀, >=10 are answered per 0.1mL viral levels^6.0TCID₅₀；

2) pseudo- mad dog production kind of the poison culture that suspends

Qualified suspension BHK21-C13 cells will be cultivated in reactor, discard cell nutrient solution, add viral maintaining liquid recover to Former volume of culture, then add pseudo- mad dog basis kind poison, pH value 7.40-7.60, DO value solubility percentage by percent by volume 1% Add up the culture 72-96h that suspends under the conditions of 20%-60%, rotating speed 60-110rpm, 36.0 ± 0.5 DEG C of temperature, treat cytopathy parameter Virus liquid, -20 DEG C of freezen protectives are harvested during amount percentage >=75%.

7. production method according to claim 6, it is characterised in that：The pseudo- mad dog production is also included with kind of the poison that suspends will Step 2) the reacted device of virus liquid of harvest expands production that culture 1-2 generations obtain and suspended kind of a poison batch with pseudoabies.

8. production method according to claim 7, it is characterised in that：The clarification of the pseudo- mad weak viral disease venom of dog for animals is used 0.45 μm of filter core positive press filtration；

It is lyophilized to include：

The pre-freeze stage：Product is down to -42 DEG C and maintains 2h by normal temperature inlet, 1-2h；

The lyophilization stage：Product is risen to -8 DEG C from -42 DEG C and maintains 10h by 1-2h；Product is risen to 0 DEG C from -8 DEG C by 0.5-1h And maintain 0.5-1h；

The adsorption stripping and dry stage：Product is risen to 28 DEG C from 0 DEG C and maintains 7h by 1-2h, is freezed and is terminated.

9. production method according to claim 1 or 2, it is characterised in that：The clarification of the pseudo- mad weak viral disease venom of dog for animals is adopted With 0.45 μm of filter core positive press filtration；

It is lyophilized to include：

The pre-freeze stage：Product is down to -42 DEG C and maintains 2h by normal temperature inlet, 1-2h；

The lyophilization stage：Product is risen to -8 DEG C from -42 DEG C and maintains 10h by 1-2h；Product is risen to 0 DEG C from -8 DEG C by 0.5-1h And maintain 0.5-1h；

The adsorption stripping and dry stage：Product is risen to 28 DEG C from 0 DEG C and maintains 7h by 1-2h, is freezed and is terminated.

10. production method according to claim 3, it is characterised in that：The clarification of the pseudo- mad weak viral disease venom of dog for animals is used 0.45 μm of filter core positive press filtration；

It is lyophilized to include：

The pre-freeze stage：Product is down to -42 DEG C and maintains 2h by normal temperature inlet, 1-2h；

The lyophilization stage：Product is risen to -8 DEG C from -42 DEG C and maintains 10h by 1-2h；Product is risen to 0 DEG C from -8 DEG C by 0.5-1h And maintain 0.5-1h；

The adsorption stripping and dry stage：Product is risen to 28 DEG C from 0 DEG C and maintains 7h by 1-2h, is freezed and is terminated.

11. production method according to claim 5, it is characterised in that：It is to use BHK21- that the pseudoabies, which suspends and plants a poison, Basic pseudo- mad dog production kind of the poison that suspends for planting poison culture of the pseudo- mad dog of C13 suspension cell cultures, specific acquisition process is as follows：

1) poison culture is planted on pseudo- mad dog basis

Take that adhere-wall culture obtains grows fine and BHK21-C13 attached cells of confluent monolayers, discards original fluid, adds The viral maintaining liquid of the weak seed culture of viruses of the pseudoabies containing percent by volume 1% poison, in 37 DEG C of cultures, when cytopathy number percent >= Virus liquid, -20 DEG C of freezen protectives, sampling detection TCID are harvested when 75%₅₀, >=10 are answered per 0.1mL viral levels^6.0TCID₅₀；

2) pseudo- mad dog production kind of the poison culture that suspends

Qualified suspension BHK21-C13 cells will be cultivated in reactor, discard cell nutrient solution, add viral maintaining liquid recover to Former volume of culture, then add pseudo- mad dog basis kind poison, pH value 7.40-7.60, DO value solubility percentage by percent by volume 1% Add up the culture 72-96h that suspends under the conditions of 20%-60%, rotating speed 60-110rpm, 36.0 ± 0.5 DEG C of temperature, treat cytopathy parameter Virus liquid, -20 DEG C of freezen protectives are harvested during amount percentage >=75%.

12. production method according to claim 11, it is characterised in that：The clarification of the pseudo- mad weak viral disease venom of dog for animals is used 0.45 μm of filter core positive press filtration；

It is lyophilized to include：

The pre-freeze stage：Product is down to -42 DEG C and maintains 2h by normal temperature inlet, 1-2h；

The lyophilization stage：Product is risen to -8 DEG C from -42 DEG C and maintains 10h by 1-2h；Product is risen to 0 DEG C from -8 DEG C by 0.5-1h And maintain 0.5-1h；

The adsorption stripping and dry stage：Product is risen to 28 DEG C from 0 DEG C and maintains 7h by 1-2h, is freezed and is terminated.