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Virus and vaccine of porcine reproductive and respiratory syndrome and preparation method of same

A porcine PRRS virus and production method technology, applied in the field of veterinary biological products, can solve problems such as limiting product output, improving quality, reducing production costs, monitoring and controlling environmental conditions, and small surface area for cell growth, etc., to achieve The effect of uniform and stable product quality, large production scale and high product quality

Active Publication Date: 2011-02-23
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing large-scale production of PRRS virus adopts the traditional culture mode of rotary bottle culture production, has following shortcoming: (1) workshop type production, labor intensity is big, and floor space is big, and the surface area that unit volume provides cell growth is little; 2) The cell growth density is low, and the yield is low; (3) After the virus liquid is harvested, subsequent mixing has differences between batches, and the uniformity is not good; (4) Various monitoring and control environmental conditions are limited during cultivation, etc.
Above-mentioned shortcoming has caused PRRS virus titer not high, has limited the output of product, the improvement of quality and the reduction of production cost

Method used

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  • Virus and vaccine of porcine reproductive and respiratory syndrome and preparation method of same
  • Virus and vaccine of porcine reproductive and respiratory syndrome and preparation method of same
  • Virus and vaccine of porcine reproductive and respiratory syndrome and preparation method of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Large-scale production of porcine PRRS virus and live vaccine by tidal microcarrier suspension bioreactor

[0052] The microcarrier used in the present embodiment is polyester fiber, and the virus strain that is used to prepare PRRS virus antigen is NVDC-JXA1-R strain (preservation number No.2467 is deposited in China's common microbial culture preservation management center), The cell line African green monkey kidney cells (Marc-145 cells) with good sensitivity to PRRS virus was used as the cell line for seedling production, and a 20L carrier bottle tidal bioreactor was selected.

[0053] 1).Inoculation and culture of cells

[0054] Firstly, the cell growth medium (MEM medium containing 5% bovine serum, pH 7.25) was used to amplify and culture Marc-145 cells with a spinner bottle. In a 20L bioreactor, add a sterile carrier with a concentration of 1300g, digest and disperse the spinner bottle cells, and make the cell density 2.0×10 7 The cells / g microcarrier...

Embodiment 2

[0064] Example 2 Tidal Microcarrier Suspension Bioreactor Large-scale Production of Porcine PRRS Virus and Inactivated Vaccine

[0065] The preparation method of the PRRS virus solution is the same as in Example 1, and the virus strain is selected from the virulent strain NVDC-JXA1 of porcine PRRS virus. The prepared virus liquid was carried out according to the inspection procedure of highly pathogenic porcine reproductive and respiratory syndrome inactivated vaccine (JXA1 strain). Add formaldehyde to the qualified virus liquid according to the ratio of 0.1% V / V, mix well, place it at 4°C for inactivation for 48 hours, take 94 parts (parts by volume) of the inactivated virus liquid, add 6 parts (parts by volume) of Tween Mix -80 to make a water phase; take 94 parts (parts by volume) of white oil for injection, add 6 parts (parts by volume) of Siben-80 to mix, add and stir until transparent, heat to 121°C, and sterilize under a pressure of 103kPa For later use, it is the oil ...

Embodiment 3

[0066] Embodiment 3 Suspension culture technology compares with traditional spinning bottle culture technology

[0067] 1) Producing porcine PRRS in a 10L spinner bottle with a cell density of 1×10 5cells / ml cell suspension 2L, cultivate at 37°C until the second day to inoculate with M.O.I. of 0.001, add maintenance solution to 3L after virus adsorption for 1 hour, culture at 37°C for 96h, harvest liquid, and freeze at -20°C.

[0068] 2) Producing porcine PRRS virus in a 10L bioreactor, the method steps are the same as in Example 1.

[0069] 3) Experimental results: After Marc-145 cells were cultured in a 10L spinner bottle for 2 days, the number of cells could increase by 3 times, reaching 6×10 8 (3×10 5 cells / ml); when cultured at a density of 65g / L microcarrier in a 10L reactor, the density can reach 3.0×10 after 6 days of cultivation 10 (3.0×10 6 cells / ml), the large-scale tidal cell microcarrier suspension culture system and the commonly used spinner bottle culture sy...

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Abstract

The invention discloses a method for preparing virus of porcine reproductive and respiratory syndrome on a large scale. In the method, the virus of the porcine reproductive and respiratory syndrome is prepared in a cell microcarrier suspension culture system by a bioreactor. The method comprises the following steps of: inoculating host cells for preparing the virus to a carrier tank containing culture solution and a microcarrier, and mixing the cells and the microcarrier uniformly to ensure that the cells are attached to the microcarrier; providing sufficient nutrients and appropriate gas environment for the cells under the appropriate culture environment to ensure that the cells are grown until the cells are in an amount which are 10 to 20 times of the inoculation concentration on the microcarrier; preparing virus suspension from the virus of the porcine reproductive and respiratory syndrome by using cell maintenance culture solution to ensure that the suspension is adsorbed to the cells; culturing the virus under the appropriate culture environment; culturing continuously for 2 to 3 days to obtain virus solution; and after the virus solution passes inspection, performing freeze thawing on the virus solution twice at the temperature of -20 DEG C, and inactivating and purifying to prepare an inactivated vaccine of the porcine reproductive and respiratory syndrome or adding a freeze-drying protective agent for freeze drying to prepare a live vaccine of the porcine reproductive and respiratory syndrome. The method has large production scale, high yield of single batch and low production cost.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and relates to a porcine blue ear disease virus, a vaccine and a production method thereof. Background technique [0002] Pig blue-ear disease, also known as porcine reproductive and respiratory syndrome, is a viral infectious disease that endangers the pig industry. In the summer and autumn of 2006, pig "high fever" epidemics occurred in some provinces in southern my country. The Ministry of Agriculture actively organized scientific research personnel to jointly tackle the problem, and identified the pig PRRS variant virus as the main pathogen, which was named "highly pathogenic pig PRRS". Until today, the epidemic situation is still severe. Like other viral diseases, there is currently no effective therapeutic drug for the treatment of PRRS. Immunization is the most effective way to prevent and control the disease. Currently commercialized PRRS Vaccines are mainly attenu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P31/14C12N7/00A61K39/12
Inventor 张许科孙进忠乔荣岑
Owner PU LIKE BIO ENG
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