Construction Of Influenza Virus High-producing Cell Line MDCK-Tpl2^-/- And DDX21 Knockout Cell Line By Using CRISPR/Cas9 Geneediting System

Influenza vaccination is the most important way to prevent influenza at present.The traditional influenza vaccine is produced in the way of chicken embryo inoculation,but there are some limitations in this way.Cell culture influenza vaccine has attracted more and more attention.How to improve the titer of influenza virus in cell culture is one of the current research directions.This study intends to use CRISPR/Cas9 gene editing technology to transform the MDCK cell line,in order to obtain a stable passage of the dog-derived cell lines and increase the replication of influenza virus.Tpl2 is the key regulator of the regulation of I(IFN?/?)and II(IFN?)IFN,which is essential for the effective immune response during virus infection.In order to establish a new MDCK cell line supporting the efficient reproduction of influenza virus,CRISPR/Cas9 gene editing technology was used to construct Tpl2 knockout MDCK cells.Tests for characteristics,extraneous agents,endogenous agents and tumorigenicity on cells according to Chinese Pharmacopeia Book III.Influenza virus was inoculated cells(MOI=0.1),the hemagglutination titers of the cell culture supernatants at 12 h,24 h,48 h,72 h,and the TCID₅₀titer at 72 h were determined.The target genome sequencing results showed that a MDCK cell(MDCK-Tpl2^-/-)knocked out of Tpl2 stably was obtained;The Tpl2 knock out cell line is similar to wild type cells in growth characteristics,and the test of epithelial cell morphology,intracellular bacteria,fungi,mycoplasma,virus and tumorigenicity are negative.After inoculating virus for 12 h,24 h,48 h,and 72 h,the hemagglutination titers of MDCK-Tpl2^-/-cell culture supernatant increased by 1.78-2.5 times.At 72 h after virus inoculation,the TCID₅₀titer of the MDCK-Tpl2^-/-cell culture supernatants virus increased2.8-times.The results showed that the using of Tpl2-deficient MDCK cell lines can increase influenza virus production to provide the foundation for upgrading the vaccine industry.In the early stage of influenza virus infection,DDX21 can block the replication of the virus by combining the virus PB1;with the infection of the virus,the virus NS1 can compete with the PB1 to combine DDX21,block the function of DDX21 and promote the replication of the virus.In this study,CRISPR/Cas9 double plasmid system was used to establish DDX21 knockout 293T/DF-1/MDCK cell lines respectively.According to the design principle,we designed sg RNA of DDX21 gene,constructed recombinant plasmid pUC19-DDX21-sg RNA,transfected 293T/DF-1/MDCK cells with p CAG-Cas9-EGFP,screened single cell clone by flow sorting method,and screened the 293T/DF-1/MDCK cell line of DDX21 gene knockout through target genome sequencing.The sequencing results showed that 1 strains of293T/DF-1/MDCK cell lines had been knocked out of DDX21 gene,which laid the foundation for clarifying the mechanism of DDX21 on the role of virus replication and antiviral natural immunity at the cell level.