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Mammalian cell non-susceptible H9N2 subtype cold-adapted avian influenza virus rescue

An avian influenza virus, mammalian technology, applied in the direction of antisense single-stranded RNA virus, virus, antiviral agent, etc., can solve the problems of weak replication ability and difficult rescue success, and achieve the goal of increasing infection efficiency and improving rescue success rate. Effect

Inactive Publication Date: 2019-10-08
YANGZHOU UNIV
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Problems solved by technology

[0011] The 293T and MDCK co-cultured cells can be co-transfected with the pHW2000 expression vector to rescue the 8 fragments from the strains of the H9N2 subtype, while some avian influenza virus cold-adapted strains with weak replication ability in mammalian cells can be co-cultured with 293T and MDCK Difficult rescue success in cells

Method used

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  • Mammalian cell non-susceptible H9N2 subtype cold-adapted avian influenza virus rescue
  • Mammalian cell non-susceptible H9N2 subtype cold-adapted avian influenza virus rescue
  • Mammalian cell non-susceptible H9N2 subtype cold-adapted avian influenza virus rescue

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Embodiment 1

[0030] 1. TCID of cold-adapted attenuated strains on different cells 50 Determination of

[0031] The day before inoculation, the cells to be tested, 293T, MDCK, Vero, COS-1, CEF, and DF-1, were plated in a 96-well cell culture plate. After the cells formed a monolayer, the culture supernatant was discarded and washed twice with sterile PBS. Add the Opti-MEM serum-reduced medium with a final concentration of 2 μg / mL TPCK trypsin, and inoculate the virus solution into the culture plate after 10-fold serial dilution. Each dilution is connected to 4 wells, with 0.1 mL per well. After infection, the cells are 33°C, 5% CO 2 Continue to cultivate under the conditions, count the number of positive wells after 48h, and calculate the virus TCID according to the Reed-Muench method 50 . The results showed (see Table 1) that canine kidney cell MDCK is a common influenza virus susceptible cell, and the TX strain lost the ability to replicate effectively on MDCK after the cold-adapted pa...

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Abstract

The invention relates to the technical field of animal vaccines, and especially relates to a rescue method for a H9N2 subtype cold-adapted live attenuated vaccine transfected with 293T and DF-1 co-cultured cells. A H9N2 subtype cold-adapted attenuated strain TX-25-CE30 is selected to construct 8 plasmids expressing the gene, the internal gene of TX-25-CE30 is used as a skeleton, the parental strain TX strain HA and NA genes are taken as external genes, the human embryonic kidney cell 293T and chicken fibroblast cell line DF-1 co-cultured cells are transfected, the strain that has weaker ability to infect mammalian cells is rescued and named as rTXca-HA-NA, and the immune protection rate of the recombinant virus after immunization of the chicken to a homologous strain can reach 100%.

Description

technical field [0001] The invention relates to the technical field of animal vaccines, in particular to a rescue method for H9N2 subtype cold-adapted attenuated live vaccines transfected with 293T and DF-1 co-cultured cells. Background technique [0002] Avian influenza is an important disease that seriously endangers the healthy development of my country's chicken industry and human health. There are many serotypes. While doing a good job in biosecurity, vaccine immunization is still the main means of preventing and controlling these diseases. H9N2 subtype avian influenza is endemic in many Asian countries including China and South Korea, and is endemic in my country. Poultry infected by the virus can cause clinical symptoms such as reduced egg production and growth retardation, and often die when mixed with other diseases, thus causing considerable economic losses to the poultry industry. Since 1998, China has been conducting an immunization program using inactivated oil...

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Application Information

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IPC IPC(8): C12N15/85C12N7/00A61K39/145A61P31/16C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/16C12N7/00C12N15/85C12N2760/16134C12N2760/16152C12N2760/16162
Inventor 彭大新查夕馨陈素娟秦涛
Owner YANGZHOU UNIV
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