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Method for preparation of swine pseudorabies virus vaccine and vaccine product

A pseudorabies virus and porcine pseudorabies technology, which can be applied to medical preparations containing active ingredients, antiviral agents, pharmaceutical formulas, etc., can solve problems such as loss of diseased pig farms, inability to protect virus strains, and low cell utilization. Achieve the effect of reducing economic losses, reducing manpower and high virus content

Inactive Publication Date: 2016-07-06
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In 2013, pig farms in Guangdong, Jiangxi, Henan, Shanxi, Hunan and other places still had some epidemic cases of porcine pseudorabies after being immunized with pseudorabies vaccine. In 2014, some pig farms in Hebei, Henan, Hubei and other places broke out. The affected pig farms have brought huge losses, which shows that the existing commercialized pseudorabies vaccines can no longer protect the virus strains that are prevalent in the market.
However, the above methods require the use of expensive microcarriers to carry out cell suspension culture, and the production cost is relatively high; when cultured with the help of microcarriers, trypsin digestion is required, the cell utilization rate after digestion is low, and the production process is complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 A kind of method for preparing pseudorabies virus vaccine

[0036] 1. According to Cellvento TM BHK-200 serum-free cell culture medium (Merck, FC00408) instructions to prepare medium for BHK-21 suspension cells, add water for injection to 90% of the total volume in the container, and pour a bag of medium (2.16g) into it In the container, stir slightly to dissolve, add sodium bicarbonate according to the weight-to-volume ratio of 2g / L, add water for injection to 1L after slight stirring, adjust the pH value to 7.2 with 1mol / L sodium hydroxide or 1mol / L hydrochloric acid, and use 0.1μm filter membrane positive pressure filter sterilization.

[0037] 2. Recover the BHK-21 passage cell line frozen in liquid nitrogen with a bottom area of ​​75cm 2 Static culture in the square flask, the amount of medium added for BHK-21 suspension cells is 20ml.

[0038] 3. After 3 days of culture, collect the living cells floating in the liquid and culture them in a 100ml sh...

Embodiment 2

[0048] Example 2 Suspension of BHK-21 cells in a bioreactor is continuously amplified in a bioreactor

[0049] 1. In the NBSCellgen1153L bioreactor, according to the suspension BHK-21 cells prepared in the steps 1 to 3 of Example 1, take a shake flask with a capacity of 250ml cells, count and take 1.2×10 9 Transfer each to a bioreactor, and add culture medium to 1.2L, so that the final concentration of cells is 1×10 6 pieces / ml. Wherein said bioreactor cultivation parameter is pH7.2, and temperature is 37 ℃, and dissolved oxygen (Do) is 60%, and stirring speed is 80rpm, and described culture medium is Cellvento TM BHK-200 serum-free cell culture medium (Merck, FC00408).

[0050] 2. Take out the cell suspension from the bioreactor every day, count after staining with trypan blue, and culture for 3 to 4 days when the cell concentration is about 4×10 6 cells / ml for further amplification.

[0051] 3. Without trypsin, directly connect the cell collection bottle and the bioreact...

Embodiment 3

[0057] Embodiment 3 A kind of method utilizing bioreactor to cultivate highly pathogenic pseudorabies virus on a large scale

[0058] 1. Take the BHK-21 suspension cells cultured in shake flasks or bioreactors for 2 to 3 days, count the cells, and after counting, add an appropriate amount of fresh medium to dilute the cells to a concentration of 0.6×10 6 pcs / ml~0.8×10 6 Individual / ml, insert in new bioreactor, bioreactor culture parameter is pH7.2, and temperature is 37 ℃, and dissolved oxygen (Do) is 60%, and stirring speed is 80rpm, and described medium is Cellvento TM BHK-200 serum-free cell culture medium (Merck, FC00408).

[0059] 2. Take samples daily to monitor the cell concentration in the bioreactor. After 48 hours of culture, the cell density reaches 3.4×10 6 pieces / ml.

[0060] 3. Insert the highly pathogenic pseudorabies virus strain according to the inoculation dose of M.O.I=0.5 according to the total number of cells in the bioreactor.

[0061] 4. When receivi...

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PUM

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Abstract

The present invention relates to a method for preparation of a swine pseudorabies virus vaccine, the method comprises the follwoing steps: (1) preparation of suspension passage cells; (2) multiplication culture of the suspension passage cells prepared by the step (1); and (3) inoculation of pseudorabies virus onto the suspension passage cells after the multiplication culture of the step (2), and multiplication culture of the pseudorabies virus. Compared with production methods in the prior art, production process is simplified, and cost is reduced. The swine pseudorabies virus vaccine can well avoid attacks from market popular highly pathogenic pseudorabies virus strains, and economic losses caused by pseudorabies can be reduced.

Description

technical field [0001] The invention relates to a method for preparing porcine pseudorabies virus vaccine and a vaccine product, belonging to the field of veterinary medicine biotechnology. Background technique [0002] Pseudorabies (Pseudoraties, Pr) is an acute infectious disease caused by porcine herpesvirus type I pseudorabies virus of the herpesviridae family and a variety of domestic and wild animals. Pigs are the natural host and reservoir of the virus. Piglets and other susceptible animals mostly have acute lethal symptoms, with obvious neurological symptoms, and the mortality rate is as high as 100%. Adult pigs are mostly recessively infected, showing reproductive disorders and respiratory symptoms. symptom. Vaccine immunization is the main measure to prevent pseudorabies. [0003] In 2013, pig farms in Guangdong, Jiangxi, Henan, Shanxi, Hunan and other places still had some epidemic cases of porcine pseudorabies after being immunized with pseudorabies vaccine. In...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/22
Inventor 田克恭孙进忠王进产张许科
Owner PU LIKE BIO ENG
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