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Method for culture of Newcastle disease virus by suspension cells and application

A technology of Newcastle disease virus and suspension cells, applied in the field of virus suspension culture, which can solve the problems of complex process, low virus titer, and inability to use large-scale production

Pending Publication Date: 2019-08-16
成都史纪生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Cells are a good medium for proliferating viruses. It has been reported that MDCK cells, DF1 cells, Vero cells, ST cells, Marc-145 cells, BHK cells, and MDBK cells were used to prepare Newcastle disease virus liquid, but the virus titer is low and cannot be used for mass production
In addition, there are also reports of using EB66 cells to prepare Newcastle disease virus liquid. Although the titer of the prepared virus liquid can reach the standard for preparing vaccines, the production process adopts the second-stage culture method, that is, after the cells are inoculated, they need to be adsorbed and cultured for 1 hour, and then replenished. Add fresh production medium twice the volume of the original growth medium for cultivation, the process is complicated and easy to cause pollution
There is no report on the use of this cell for the culture of genotype VII Newcastle disease virus

Method used

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  • Method for culture of Newcastle disease virus by suspension cells and application
  • Method for culture of Newcastle disease virus by suspension cells and application
  • Method for culture of Newcastle disease virus by suspension cells and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The method for using suspension cells to cultivate Newcastle disease virus in embodiment 1 shake flask

[0023] Take three 250ml shake flasks to culture AGE1 cells, when the cell density reaches 8.0×10 6 genotype VII Newcastle disease virus aSG10 strain virus was inoculated according to the MOI of 0.0001, and the serum-free DMEM medium with a volume ratio (volume accounting for the total volume after addition, the same below) of 10% was added at the same time, and the final concentration was added 16U / ml trypsin solution, add the same dose of trypsin solution once a day, adjust the culture temperature to 35 ° C, 8% CO 2 Cultivate under conditions for 72 hours, collect the virus, freeze and thaw three times, and measure the titer of HA (hemagglutination test) and virus content.

Embodiment 2

[0024] The method for using suspension cells to cultivate Newcastle disease virus in the embodiment 2 bioreactor

[0025] The AGE1 cells were amplified and passaged into a 7L bioreactor, the volume of the culture medium was 4-5L, the pH value was set to 7.15, the dissolved oxygen value was set to 60%, the speed was set to 150r / min, and cultured at 37°C. When the cell density is 8.0×10 6 When it is above / ml, inoculate Newcastle disease virus aSG10 strain virus, add the serum-free DMEM medium that volume ratio is 10% at the same time, and add the trypsin solution that final concentration is 16U / ml, add the same dose of trypsin solution once a day , the culture temperature was adjusted to 35°C, cultured for 72 hours, and the virus liquid was harvested. A total of 3 batches of Newcastle disease virus were prepared. The results showed that the HA titer of the harvested virus was 11-12 log2, and the virus content was 10 9.2 ~10 9.5 EID 50 / 0.1ml. The results are detailed in T...

experiment example 1

[0029] Experimental Example 1 Optimum Inoculation Cell Density Test of aSG10 Strain Virus

[0030] Take three 250ml shake flasks to culture AGE1 cells, respectively culture to a cell density of about 4.0×10 6 / ml, 8.0×10 6 / ml, 1.2×10 6 / ml, and then adjust the cell density precisely to 4.0×10 6 / ml, 8.0×10 6 / ml, 1.2×10 6 / ml, that is, when the cell density fails to reach the required density, continue to culture, and when the cell density is greater than the required density, it is diluted to the required density in a certain proportion. Then adjust the cell volume to 80ml / bottle, inoculate Newcastle disease virus aSG10 strain virus, add serum-free DMEM medium with a volume ratio of 10%, and trypsin solution with a final concentration of 16U / ml, and put it on a shaker , 160r / min, 35℃, 8%CO 2 to cultivate. The same dose of trypsin solution was added once a day, after 72 hours, the virus was harvested, frozen and thawed three times, and the HA titer and virus content we...

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Abstract

The invention relates to the field of virus suspension culture and provides a method for culture of Newcastle disease virus by suspension cells. The invention provides application of a DMEM culture medium to increasing of cell agglomeration rate in a process of virus culture with the suspension cells. The invention further provides a method for culturing gene VII-type Newcastle disease virus by the suspension cells. The method is characterized by including: culturing AGE1 cells until density reaches (8-12)*10<6> / ml, performing inoculation of the Newcastle disease virus, adding a serum-free DMEM culture medium accounting for 10%-20% of the volume of a cell culture system, daily adding pancreatic enzymes in final concentration of 8-16U / ml, culturing at a temperature of 33-37 DEG C, and collecting the virus. The AGE1 is used for culturing the gene VII-type Newcastle disease virus for the first time, and by adoption of the serum-free DMEM culture medium and other reasonable process parameters, high-titer Newcastle disease virus can be obtained.

Description

technical field [0001] The invention relates to the field of virus suspension culture. Background technique [0002] Chicken Newcastle disease (ND) is one of the most harmful viral infectious diseases to the poultry industry. . The results of systematic molecular epidemiological surveys show that in recent years, the ND epidemic strains in my country are mainly genotype VII, which is significantly different from the widely used vaccine strains La Sota and Clone 30 in China. Many routine vaccine immunizations have been carried out, but there is still the possibility of infection with virulent strains causing epidemics. Clinically, the main manifestation is the occurrence of atypical ND, which proves that the immune protection of the currently used vaccines is insufficient, so the development of immunogenicity is more effective. Good vaccines that better match the current circulating strains have become the trend and urgent task of ND vaccine research and development. [0003...

Claims

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Application Information

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IPC IPC(8): C12N7/00
CPCC12N7/00C12N2760/18151
Inventor 邢刚李成山岳丰雄丁莉左榕琳黄杰王洁清魏胜男李晏齐廖鏖江勇
Owner 成都史纪生物制药有限公司
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