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Method for replicating influenza virus in culture

A technology of canine influenza virus and virus, which is applied in the field of new immunogenic composition, vaccine for canine respiratory diseases, and selection of influenza virus grown in human embryonic kidney cells, can solve the problems that are difficult to achieve and affect the antigenicity of vaccines

Inactive Publication Date: 2013-07-03
SCHERING PLOUGH ANIMAL HEALTH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, this approach can be difficult to do and may affect the antigenicity of the resulting vaccine

Method used

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  • Method for replicating influenza virus in culture
  • Method for replicating influenza virus in culture
  • Method for replicating influenza virus in culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1: chemical reagent and biological reagent

[0115] The infection medium used contained 1 L of DMEM (Cambrex, catalog number 04-096) or equivalent, stored at 2-7°C; 20 mL of L-glutamine (Cellgro, catalog number 25-005-CV) or equivalent, stored in -10°C or colder, once thawed it can be stored at 2-7°C for up to 4 weeks; and trypsin type IX (Sigma Product No. T0303, CAS No. 9002-07-7) or equivalent, aliquoted and stored frozen at -5 to -30°C. Infection medium was freshly prepared prior to infecting cells.

[0116] Cell culture medium was prepared as follows: 1 L DMEM, 20 mL L-glutamine, 50 mL fetal bovine serum (Gibco cat# 04-4000DK) or equivalent (Note: from BSE-free countries). Store complete media at 2-7°C for no more than 30 days after preparation.

[0117] EDTA-Trypsin (Cellgro Cat. No. 98-102-CV or equivalent) used for passaging cells was stored at -5 to -30°C with expiration date indicated by the manufacturer.

Embodiment 2

[0118] Example 2: Cell Culture Preparation

[0119] Because the protease dilution used for the virus infection step can vary from batch to batch, titrate a new batch of trypsin to establish optimal levels before use. An example of a titration is a serial dilution of trypsin type IX in DMEM containing L-glutamine with a half-log dilution (10 -1 、10 -1.5 、10 -2 、10 -2.5 Wait). Using a 96-well plate containing a fresh confluent monolayer of Vero cells, wash each well of the plate twice with 280 μL of PBS. Immediately after washing, 200 μL of each dilution of trypsin type IX was added to the plate in rows. Then place the plate at 37 °C plus or minus 2 °C in 5% CO 2 and observe the cells after 4 days. The lowest dilution of trypsin that showed no or little effect on cell health was chosen as an appropriate concentration of trypsin for isolation and optimization of influenza infection. It is desirable and common to have little or no variation between wells inoculated withi...

Embodiment 3

[0126] Example 3: Limiting dilution cloning

[0127] Dilution tube preparation and sample dilution for limiting dilution clones are as follows. Set up test tubes (12 x 75 mm) on a test tube rack and label them. One sample was assayed per plate, and the dilution series for each sample was 10 -1 to 10 -10 . The diluted medium was dispensed in 1.8 mL volume to each test tube with a serological pipette. The first tube is labeled Virus Identification. Several other tubes were prepared to serve as dilution controls and to be replaced in case errors occurred during dilution. Mix the sample by vortexing for approximately 5 seconds, then pipette 200 μL of the sample to 10 -1 Dilution Tubes Prepare starting dilutions. Continue serial dilution to 10 -10 . For each dilution, samples were mixed by vortexing and pipette tips were changed between dilutions.

[0128] Dilutions were transferred to cell plates as follows. Aseptically decant the medium from the cell plate immediatel...

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Abstract

The invention is related to a method for selecting an influenza virus for growth on tissue culture cells to produce a tissue-culture adapted viral isolate. The invention also includes vaccines produced from the isolate.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Application 60 / 875287, filed December 15, 2006, and US Application 60 / 882412, filed December 28, 2006, both of which are incorporated herein by reference. technical background [0003] Influenza epidemics and pandemics have been known for centuries and have resulted in considerable loss of life. Influenza virus is a segmented RNA-containing virus belonging to the Orthomyxoviridae family. Epidemics and pandemics are caused by viruses with a new envelope component to which there is little immunity in the human population. These new components are often the result of mutations and / or mixing of human and animal influenza viruses. [0004] However, the capsid of influenza virus is slightly polycrystalline and its outer surface, as in all viruses, consists of a lipid envelope from which protrudes two protruding glycoprotein spines: hemagglutinin (HA or H) and neuraminidase (NA or N). ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/145C12N7/02
CPCC12N2760/16051C12N7/00
Inventor T·L·沃斯梅恩P·高B·A·埃迪O·Y·阿布德尔梅吉
Owner SCHERING PLOUGH ANIMAL HEALTH
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