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Serum-free full-suspension cell culture method for Newcastle disease VII type attenuated strain

A technology for cell culture and Newcastle disease virus, which is applied in the field of attenuated Newcastle disease strain serum-free full suspension cell culture, can solve the problems that poultry viruses cannot grow and reproduce and have not been discovered, the adaptability of suspension cells is weak, and the method is not right to be developed. Achieve the effect of stable virus reproduction, large amount of preparation, and high poison price

Active Publication Date: 2021-06-15
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The whole suspension culture of avian virus heterologous cells has been studied at home and abroad, but few of them are actually used for production, and most of them are in the research stage. This is related to the adaptability of viruses to heterologous cells. Different viruses have different effects on heterologous cells. Many avian viruses cannot grow and reproduce on heterologous cells or have not been discovered or the method is not right to be developed
In recent years, there have been many literature and patent reports on the full-suspension cell culture of chicken Newcastle disease virus, mainly on the Lasota strain of Newcastle disease virus. The Lasota strain is a more classic and commonly used virus strain, but its adaptability to suspension cells is weak

Method used

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  • Serum-free full-suspension cell culture method for Newcastle disease VII type attenuated strain
  • Serum-free full-suspension cell culture method for Newcastle disease VII type attenuated strain
  • Serum-free full-suspension cell culture method for Newcastle disease VII type attenuated strain

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Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Virus Best Harvesting Time

[0037] In this embodiment, the optimal harvesting time of the virus liquid is explored in the method, and the steps are as follows: observe and count the cell density under a microscope to be 7.9×10 6 , adjust the cell seeding density to 1.5×10 6 The amount of TPCK trypsin added is 1-6ug / ml, the amount of virus inoculum is 0.001-1%, shake culture at 37°C, and the rotation speed is 100-150r / min, respectively at 36h, 41h, 48h, 54h, and 60h after inoculation , 72h to harvest the virus fluid, and detect the HA titer at each time point, the results are shown in Table 1.

[0038] In this embodiment, the lesion pictures and normal control cell pictures of Newcastle disease gene type VII attenuated virus (NF02 strain) suspension cultured to 48 hours are shown in figure 1 .

[0039] Table 1 HA detection results of virus liquid at different harvest time

[0040] Virus fluid harvest time 36h 41h 48h 54h 60h 72h HA 7 ...

Embodiment 2

[0042] Example 2 Optimum Seeding Density of Cells

[0043] In this embodiment, the optimal cell seeding density is explored in the method, and the steps are as follows: observe and count the cell density to be 8.0×10 6 , adjust the cell density to 1.0×10 6 , 2.0×10 6 , 3.0×10 6 indivual. The concentration of TPCK trypsin is 1-6ug / ml, the concentration of virus inoculation is 0.001-1%, shake culture at 37°C, the rotation speed is 100-150r / min, harvest after 48-54 hours of cultivation, and detect the titer of virus HA, the results are shown in the table 2.

[0044] Table 2 HA detection of virus liquid obtained from inoculation and culture at different cell densities

[0045] Cell density 1.0x10 6

[0046] The results in Table 2 show that the cell density was 1.0×10 6 When the inoculated virus reproduces best.

Embodiment 3

[0047] Embodiment 3 TPCK trypsin optimal use concentration

[0048] This embodiment explores the optimal concentration of TPCK trypsin in the method described, and the steps are as follows: adjust the cell density to 1.0×10 according to the needs of the experiment. 6 Cells, divided into five 125ml shake flasks, add TPCK trypsin concentration respectively 1ug / ml, 2ug / ml, 3ug / ml, 5ug / ml, 6ug / ml, virus inoculation concentration is 0.001-1%, 100 Cultivate at -150r / min for 48-54 hours and harvest, and detect the titer of virus HA. The results are shown in Table 3.

[0049] Table 3 Optimum TPCK Trypsin Use Concentration HA Detection

[0050] trypsin concentration 1ug / ml 2ug / ml 3ug / ml 5ug / ml 6ug / ml HA 6 9 9 8 8

[0051] The results in Table 3 show that the virus reproduces best when the concentration of TPCK trypsin is 2-3ug / ml.

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Abstract

The invention relates to the field of cells and biological products, in particular to a serum-free full-suspension cell culture method for Newcastle disease VII type attenuated strain. In the method provided by the invention, the Newcastle disease virus strain is a Newcastle disease gene VII type virulent strain attenuated by F gene locus mutation. According to the method disclosed by the invention, a serum-free full-suspension cell culture is adopted, so that the method is not influenced by insufficient hatching eggs supply or uncontrollable factors; the method disclosed by the invention has the advantages of easy operation, large virus preparation amount and high virus content, and overcomes the defect that the supply of vaccines on the market is influenced by vaccine production stagnation caused by influence on virus reproduction due to insufficient supply of hatching eggs in virus chick embryo culture.

Description

technical field [0001] The invention relates to the field of cells and biological products, in particular to a method for culturing serum-free full suspension cells of attenuated Newcastle disease strain. Background technique [0002] Newcastle disease is a highly contagious disease caused by a virus. It mainly affects chickens, other poultry and wild birds. Humans can also be infected. The main manifestation is conjunctivitis. The disease is the most serious disease among chicken diseases, with acute onset and high mortality, causing great economic losses. Compulsory immunization is now generally carried out in various parts of our country to improve the immunity of chickens and reduce the occurrence of diseases. [0003] At present, chicken embryos are still mostly used for the cultivation of Newcastle disease virus, and the operation process is complicated, including the introduction of breeding eggs, coding eggs, disinfection, hatching in an incubator before entering, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/04
CPCC12N7/00C12N2760/18121C12N2760/18162C12N2760/18151C12N2760/18134Y02A50/30
Inventor 周明光王鑫程泰烺徐高原张锦军张宏斌金建云陈章表
Owner WUHAN KEQIAN BIOLOGY CO LTD
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