Method for large-scale suspension culture of fowl adenoviruses

A technology of suspension culture and poultry adenovirus, applied in the direction of using microorganisms, viruses, viruses/phages, etc., can solve the problems of relying on serum, complicated process, and unsatisfactory titer

Pending Publication Date: 2021-10-29
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chicken embryo fibroblast inactivated vaccines were also eliminated due to complex process, high cost and low potency
At present, it is more feasible to culture on adherent LMH cells, harvest the virus liquid, and prepare finished vaccines after inactivation. However, this process is produced by traditional adherent production methods such as spinner bottles or cell factories. Not ideal, relying on serum, labor-intensive, limited by the culture space and cannot be scaled up on a large scale

Method used

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  • Method for large-scale suspension culture of fowl adenoviruses
  • Method for large-scale suspension culture of fowl adenoviruses
  • Method for large-scale suspension culture of fowl adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Domestication of LMH cells

[0022] The inoculation process of avian adenovirus (type 4, type 8b, type D11, type 8a) of avian adenovirus (group Ⅰ type 4, type 8b, type 8a) antigen inoculation of LMH cell proliferation in a bioreactor full suspension: that is, full suspension LMH cell recovery and 500ml shake flask culture; small reactor ( 5L~25L) cell culture, bioreactor cell scale-up culture (100~1500L), virus inoculation, virus liquid harvesting and inactivation, and semi-finished products prepared into multiple inactivated vaccines and then immunized 35-day-old SPF chickens, respectively measured for 21 days And 28 days immune antibody.

[0023] 1. Suspension acclimatization of LMH cells

[0024] Adherent LMH cells P3 were resuscitated with DMEM medium containing 10% fetal bovine serum, and then passaged to P4 after dense growth. Select the well-growing cells of the P4 generation, and gradually adapt to the culture with DMEM medium containing different s...

Embodiment 2

[0063] Embodiment 2: preparation vaccine

[0064] 1. Inoculate whole-suspension LMH cells and adherent cells with virus liquid inoculated with 4 types of poultry adenovirus group I, respectively, and inactivate the virus liquid, and emulsify the inactivated semi-finished products to make Newcastle disease, avian influenza (H9 subtype), and avian adenovirus (group I) Type 4) triple inactivated vaccine, immunize 35-day-old SPF chickens respectively, and inject 0.2ml intramuscularly into each chicken. After 21 days of immunization, blood was collected, serum was separated, and antibodies were tested by agar amplification method. After 21 days of immunization, the virus was challenged at the same time. All immunized chickens and control chickens were intramuscularly injected with avian adenovirus group I type 4 YBAV-4 strain virus solution, each 0.2ml (including 2×10 5.5 TCID 50 ). Observe 7 days after attacking the virus, and count the protection rate of attacking the virus. ...

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Abstract

The invention provides a method for large-scale suspension culture of fowl adenoviruses. The method provided by the invention can be used for obtaining high-titer fowl adenoviruses of I group 4 type, 8b type, D11 type, 8a type and the like, a domesticated LMH cell used in the method is a chicken liver cancer suspension LMH cell, and the collection number of the domesticated LMH cell is CCTCC NO: C2021202. According to the method disclosed by the invention, adherent LMH cells are domesticated into suspension cells capable of realizing stable and continuous passage by adopting a gradual serum reduction method. Then, full suspension culture is carried out on the LMH cells by using a bioreactor, then, step-by-step amplification suspension culture is carried out on the LMH cells, and then, aviadenovirus antigens are respectively inoculated and proliferated on the suspension cells. By use of the method disclosed by the invention, high-titer aviadenovirus suspension culture technology virus antigens can be obtained, and the titer of the antigens is about 5-10 times of that of existing vaccine regulation antigens.

Description

technical field [0001] The invention belongs to the technical field of virus culture, and in particular relates to a method for large-scale suspension culture of poultry adenoviruses, that is, domesticated suspension LMH cells (chicken liver cancer cells) are used to suspension culture poultry adenoviruses (type 4, type 8b, D11 of group I) type) method, comprising the steps of suspension acclimation of adherent LMH cells, culture of suspension cells and suspension culture amplification of poultry adenovirus (group I type 4, type 8b, type D11, type 8a). Background technique [0002] In the past ten years, my country's avian adenovirus inactivated vaccines have mainly experienced tissue inactivated vaccines, chicken embryo inactivated vaccines and cell inactivated vaccines. Tissue inactivated vaccine is a vaccine made from the liver homogenate of infected chickens, which is extracted with chloroform and inactivated with formaldehyde. Chicken embryo inactivated vaccine uses SP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N1/36C12N7/00
CPCC12N5/0693C12N1/36C12N7/00C12N2710/10252
Inventor 王玉红韩建文关秀春李王强刘岳郑长虹王鹏听黄理进贾建斌贠鲁祥黄新李晓林楚电峰杜元钊范根成
Owner YEBIO BIOENG OF QINGDAO
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