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BHK21 cell group with reconstructed innate immune system and cell clone potentiation application

An immune system and innate technology, applied in the field of biotechnology and veterinary biological product engineering, can solve the problems of undiscovered BHK21 cell population and its cell cloning application, and achieve the effect of increasing the proliferation titer

Active Publication Date: 2018-11-27
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Looking at the existing technology, it can be found that those skilled in the art often use gene knockout methods to inhibit the proliferation of viruses, and the inventors have never discovered the application of the BHK21 cell population and its cell clones remodeled by the innate immune system in virus proliferation

Method used

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  • BHK21 cell group with reconstructed innate immune system and cell clone potentiation application
  • BHK21 cell group with reconstructed innate immune system and cell clone potentiation application
  • BHK21 cell group with reconstructed innate immune system and cell clone potentiation application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Acquisition of BHK21-KO-IM cell population and formation of single cell clones

[0050] 1. Construction of pUC-gRNA-library

[0051] The genome sequence of the golden hamster was analyzed, and the respective gene sequences of receptor proteins, effector proteins, stress proteins, protein kinases, interferons and their downstream regulatory products of the innate immune system were identified, according to the CRISPR / Cas9 system. Sequence identification for gene editing requires selection of target sequences. Typically at least two target sequences are selected per gene sequence. For each target sequence, a pair of DNA oligo primers were designed. The primers used were primers with SEQ ID NOs: 1-446.

[0052] Each pair of target sequence primers is individually annealed to generate DNA duplexes with cohesive ends. The gRNA expression vector backbone (containing U6 promoter, RNA scaffold, and polyA signal sequence) was treated with restriction endonuclease B...

Embodiment 2

[0059] Example 2 Screening of BHK21 cell clones with high PRV production and application of virus proliferation

[0060] 1. The BHK21-KO-IM cell population was sorted by flow cytometry to form single cell clones in a 96-well plate. After recovery, 177 actual cell clones were obtained. The cell density in each well was adjusted to approximately the same, and the cells expanded. Duplicate plates were made for comparison of virus proliferation efficiency.

[0061] 2. Remove the culture supernatant and inoculate 200ul / well of virus proliferation maintenance solution containing the same amount of porcine pseudorabies virus (PRV) respectively. The cytopathic changes were observed every day, and the virus proliferation was completed in 72 hours. After three freeze-thaw cycles, the titer of the virus proliferation samples in each well was determined. Specifically, for each virus sample, 10 -6 , 10 -7 , 10 -8 After dilution, it was inoculated on ordinary monolayer BHK21 cells, and ...

Embodiment 3

[0063] Example 3 Screening of JEV high-yielding BHK21 cell clones and their application in virus proliferation

[0064] 1. The BHK21-KO-IM cell population was sorted by flow cytometry to form single cell clones in a 96-well plate. After recovery, 177 actual cell clones were obtained. The cell density in each well was adjusted to approximately the same, and the cells expanded. Duplicate plates were made for comparison of virus proliferation efficiency.

[0065] 2. Remove the culture supernatant and inoculate 200ul / well of virus proliferation maintenance solution containing the same amount of porcine Japanese encephalitis virus (JEV) respectively. The cytopathic changes were observed every day, and the virus proliferation was completed in 96 hours. After three freeze-thaw cycles, the titer of the virus proliferation samples in each well was determined. Virus titer detection method According to the quality standards of veterinary biological products of the Ministry of Agricultur...

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Abstract

The invention discloses a method of enhancing viral multiplication and constructs a BHK21 cell group with a reconstructed innate immune system for viral proliferation. The method disclosed by the invention opens a new concept for viral multiplication and expands the application range of the BHK21 cells, thereby laying a solid foundation for researching vaccines capable of preventing and treating viruses.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and veterinary biological product engineering, and more particularly relates to a BHK21 cell group that randomly knocks out innate immune system effector protein molecules and the application of the cell clone and proliferation virus. Background technique [0002] BHK21 cells are currently the commonly used host cells for the proliferation of livestock and poultry vaccine viruses. Major research institutions and veterinary vaccine manufacturers are conducting researches such as seed cell screening, cell suspension culture, and virus proliferation techniques for the proliferation of various viruses. research hotspot. The innate immune system of host cells is the body's defense mechanism that is produced in the early stage of phylogeny and appears in the initial stage of the host's anti-infection response, and performs immune functions. Function. Host cells sense the invasion of pathogens th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/85C12N15/90C12N5/10C12R1/93
CPCC12N5/0686C12N7/00C12N9/22C12N15/85C12N15/907C12N2710/16752C12N2510/00C12N2760/18152C12N2770/24152C12N2810/10C12N2800/107
Inventor 冯磊陈丽侯继波
Owner JIANGSU ACAD OF AGRI SCI
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