Method for suspension culture of Newcastle disease virus (NDV) by using full-suspension passage cell line

A technology of Newcastle disease virus and subculture cells, which is applied in the field of veterinary biological products, can solve the problem of inability to guarantee the source of chicken embryos, the consistency of maternal antibody levels of breeds, incomplete autoclaving of embryo body waste, and inability to ensure the stable effect of vaccines To achieve the effect of improving virus titer and vaccine titer, overcoming the shortage of chicken embryo supply, good economic benefits and application prospects

Inactive Publication Date: 2018-06-29
ZHAOQING INST OF BIOTECHNOLOGY CO LTD +2
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  • Abstract
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  • Claims
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Problems solved by technology

[0003] At present, the production of chicken Newcastle disease vaccine in my country is mainly through inoculation of 9-12 day-old chicken embryos to cultivate and prepare vaccines. In the case of vaccines, the direct use of embryoid bodies for virus propagation is easily limited by the source of non-immune embryoid bodies, resulting in the inability to guarantee the consistency of chicken embryo source, variety, and maternal antibody levels, and then affect the hatching rate of chicken embryos and the potency of the propagated virus It has a great impact, which makes it impossible to ensure that the vaccine has a stable effect between different batches
In addition, the virus suspension cultured in chicken embryos contains a large amount of miscellaneous proteins, which poses certain safety hazards in the vaccine preparation process. At the same time, the embryo body waste is often not completely autoclaved, and there is a danger of spreading the virus. Therefore, it is urgent to choose new vaccines. Newcastle disease virus vaccine production medium for production of Newcastle disease virus
[0004] At present, it has been reported that the passaged cell lines include MDCK cell line, vero cell line, marc-145 cell line, ST cell line, BHK cell line, MDBK cell line and chicken embryo passage cell DF-1 cell line to cultivate Newcastle disease virus preparation However, due to the low abundance of Newcastle disease virus receptors on the surface of the above-mentioned cells, the virus titer is low and the ability to form plaques is weak, which causes great difficulties for further in-depth research on the preparation of vaccines

Method used

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  • Method for suspension culture of Newcastle disease virus (NDV) by using full-suspension passage cell line
  • Method for suspension culture of Newcastle disease virus (NDV) by using full-suspension passage cell line
  • Method for suspension culture of Newcastle disease virus (NDV) by using full-suspension passage cell line

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Experimental program
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Embodiment 1

[0040] The cultivation method of embodiment 1 Newcastle disease virus is as follows:

[0041] Step 1: Use the second-stage culture method to press 10 -2 Newcastle disease virus was inoculated at MOI in the whole suspension cell line EB66 cells, and the cells were placed at 37°C and 5% CO after inoculation. 2 , Adsorption culture in an incubator with orbital shaker speed of 120rpm for 1h, after completion of adsorption culture, add fresh production medium twice the volume of the original growth medium, and place at 33°C, 5% CO 2 1. Continue culturing in an incubator with an orbital shaker rotating at 120rpm. On the 0th, 1st, 2nd, and 3rd day after inoculation, 1 mg / L TPCK trypsin was added. Sampling every 12h for EID 50 detection. Harvest the virus at the time when the virus titer is the highest as the seed virus for the next generation of domestication;

[0042] Step 2: Freeze-thaw and centrifuge the seed poison harvested in step 1 repeatedly for 3 times, and repeat the m...

Embodiment 2-6

[0048] The culture method of embodiment 2-6 Newcastle disease virus is as follows:

[0049] Using the second-stage culture method, the Newcastle disease virus was inoculated into the whole suspension cell line EB66 cells, and the infected cells were placed at 37°C and 5% CO 2 , Adsorption culture in an incubator with orbital shaker speed of 120rpm for 1h, after completion of adsorption culture, add fresh production medium twice the volume of the original growth medium, and place at 33°C, 5% CO 2 1. Continue culturing in an incubator with an orbital shaker rotating at 120rpm. On the 0th, 1st, 2nd, and 3rd day after inoculation, 1 mg / L TPCK trypsin was added. Sampling every 12h for EID 50 detection.

[0050] Wherein, the Newcastle disease virus exposure dose (MOI) of embodiment 2 is 10; The Newcastle disease virus exposure dose (MOI) of embodiment 3 is 5; The Newcastle disease virus exposure dose (MOI) of embodiment 4 is 1; The dose (MOI) of Newcastle disease virus in Exampl...

Embodiment 7-8

[0056] The culture method of embodiment 7-8 Newcastle disease virus is as follows:

[0057] Using the second-stage culture method, the Newcastle disease virus was inoculated into the whole suspension cell line EB66 cells, and the infected cells were placed at 37°C and 5% CO 2 , Adsorption culture in an incubator with orbital shaker speed of 120rpm for 1h, after completion of adsorption culture, add fresh production medium twice the volume of the original growth medium, and place at 33°C, 5% CO 2 1. Continue culturing in an incubator with an orbital shaker rotating at 120rpm. After inoculation, TPCK trypsin was added to the culture medium. Samples were taken every 12 hours for the detection of EID50.

[0058] Among them, the supplementary method of TPCK trypsin in embodiment 7 is: add 1 mg / L of TPCK trypsin on the 0th, 1st, 2nd and 3rd days after inoculation; Add 4 mg / L of TPCK trypsin once a day.

[0059] The virus of embodiment 7,8 harvest is carried out EID50 detection r...

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Abstract

The invention relates to a method for suspension culture of Newcastle disease virus (NDV) by using a full-suspension passage cell line. The method comprises the following steps: inoculating the full-suspension EB66 cell line having a growth density of 8*10<6> cells/mL to 16*10<6> cells/mL with the NDV by using a two-stage culture method, continuously culturing after the virus is inoculated, and sampling every 12 hours so as to determine the EID50 of the virus; harvesting the virus when the titer of the virus is maximum, and storing the harvested virus so as to complete the culture of the NDV.The method adopts the duck embryonic stem cell EB66 passage cell line to culture the chicken NDV, thus effectively improving the titer and purity of the obtained virus, further enabling produced vaccines to have higher quality, and lowering the production cost.

Description

technical field [0001] The invention relates to a full-suspension culture method of Newcastle disease virus, in particular to a method for suspension culture of Newcastle disease virus by a full-suspension subculture cell line, and belongs to the technical field of veterinary biological products. Background technique [0002] Newcastle disease (ND) is an acute septic and highly contagious infectious disease caused by Newcastle disease virus (NDV) of the family Paramyxoviridae and the genus Paramyxovirus , is one of the most harmful poultry infectious diseases in the world today, and the morbidity and mortality of chickens after infection are all above 90%. Therefore, the disease is listed as a class A infectious disease by the World Organization for Animal Health (OIE). Such diseases occur frequently in our country, seriously affecting the healthy development of my country's poultry industry. At present, the incidence of Newcastle disease in our country is still relatively hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N7/00C12M3/02C12M1/02A61K39/17A61P31/14
CPCA61K39/12A61K2039/552C12M27/02C12N5/0606C12N7/00C12N2760/18134C12N2760/18151
Inventor 温良海陈瑞爱罗顺李延鹏蔡仕君张晓楠
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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