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Method for culturing avian reovirus

An avian reovirus and a culture method technology, applied in the field of veterinary biological products, can solve the problems of unsatisfactory host cell performance and low efficiency of culture conditions, and achieve the effects of increasing toxin production, saving labor, and ensuring stability

Inactive Publication Date: 2015-03-04
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a method for cultivating avian reovirus, so as to solve the technical problems of unsatisfactory host cell performance and low efficiency of culturing conditions in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 (reactor microcarrier suspension culture Vero cell)

[0029] Medium: MEM serum medium (GIBCO company) 8-10% newborn bovine serum

[0030] Trypsin digestion solution: 0.25% trypsin-0.53mM EDTA

[0031] PBS solution: pH 7.6, Ca-free with 0.02% (W / v) EDTA 2+ , Mg 2+ ion

[0032] Vero cells (from the China Center for Type Culture Collection, Wuhan University) were cultured in spinner bottles. When the cells grew into a dense monolayer, the cell density reached about 2×10 6 When cells / ml is above, select healthy and well-growth monolayer cells as working cells, and the density is to detect the cell density by extracting a bottle of cells produced in the same batch of spinner bottles to make a cell suspension, and use this as the working cell. The density of the batch.

[0033] Connect the reactor gas pipe, liquid inlet pipe, liquid outlet pipe and other pipes to hydrate the cytodex TM I Microcarriers (GE HealthCare), sterilized by high pressure and damp h...

Embodiment 2

[0035] Embodiment 2 (reactor microcarrier suspension culture Vero cell---100L tank culture)

[0036] 14L small-scale BIOSTAT reactor was used to culture Vero cells (from the China Center for Type Culture Collection of Wuhan University). When the cells grew into a dense monolayer, samples were taken and counted, and the cell density reached about 5×10 6 When cells / ml is above, digest with trypsin / EDTA digestion solution to make cell suspension for later use.

[0037] 14L reactor digestion process: stop the agitation of the small reactor, allow the microcarriers to settle naturally, pump out the supernatant culture solution, and use a Ca-free solution containing 0.02% (W / v) EDTA with a pH value of 7.6 2+ , Mg 2+ Ionic PBS was washed 2 to 3 times, and the amount of EDTA-PBS solution used was 1 working volume of the reactor, and 1 working volume refers to 40% of the total volume of the reactor, and then digested with digestive solution (0.25% trypsin-0.53mMEDTA) effect. The act...

Embodiment 3

[0039] Embodiment 3 (reactor microcarrier amplifies viral arthritis virus)

[0040]Chicken viral arthritis virus: ARV S1133 strain (purchased from China Veterinary Drug Control Institute)

[0041] Maintenance solution: MEM low serum medium (GIBCO company) 1-2% fetal bovine serum

[0042] Virus seed inoculum volume: 0.1-0.5% volume

[0043] Propagation process: Use cell maintenance medium to make fresh virus seeds into virus suspension, inoculate on a 100L reactor microcarrier monolayer cell, replace with maintenance culture medium after the cells settle, maintain pH 7.4, and temperature 37°C. 48-72 hours after inoculation, samples were taken every 3 hours to detect the state of cell shedding and lesions, and the lesions were observed to reach more than 80%. They were harvested together with microcarriers, stored at -20°C, and retained samples were tested to determine the virus content.

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Abstract

The invention provides a method for culturing an avian reovirus. According to the method, because a vero cell which has excellent performance and is conducive to the proliferation of the avian reovirus is used as a host cell to be used for proliferating the avian reovirus, an optimized process for culturing the avian reovirus can be determined. A bioreactor is adopted to culture and replicate the avian reovirus, and due to the adhesion of the cell to a carrier, the suspension culture of the cell can be realized, the number of vero cells in unit volume can be increased, and further, the yield of the avian reovirus can be improved. Meanwhile, because the avian reovirus culturing process can be monitored timely and controlled quantitatively, the stability of the production process can be guaranteed. Moreover, because the content of avian viral arthritis viruses in an avian viral arthritis virus liquor cultured by adopting the method is high, the content of antigens in the prepared inactivated vaccine is high, and the effect of the inactivated vaccine is good. Furthermore, due to the way of proliferating the avian reovirus by using the bioreactor through the microcarrier, the cost can be lowered, the labor can be saved, and the stability of the product can be improved.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a method for cultivating avian reovirus. Background technique [0002] Avian reovirus (ARV) can infect chickens or turkeys and cause chicken viral arthritis, and its specific symptoms include arthritis, tenosynovitis, malabsorption, etc. The virus is ubiquitous in commercial chicken flocks, mainly infecting broiler chickens, and also infecting commercial laying hens and turkeys. The infection rate can reach more than 90%, and the mortality rate is 5% to 10%. The existence of the disease in my country was confirmed in the mid-1980s. In recent years, although chicken viral arthritis has been controlled, it still occurs. It has been reported that some 20-day-old chicks in a certain chicken farm will experience joint swelling. With the development of the disease, they will gradually appear lame, paralyzed, and inconvenient to move. It is diagnosed as chicken v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 康亚男李亚杰郁宏伟梁武杨保收
Owner TIANJIN RINGPU BIO TECH
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