Transient Expression of African Swine Fever Virus p30 Protein in 293T Cells
To eukaryotic express the p30 protein of African swine fever virus,the author explores its impact on the expression of inflammatory response related genes.This experiment synthesized the p30 gene based on NCBI website,and cloned it into the eukaryotic expression vector pIRES2-EGFP,then transfected the recombi-nant plasmid into 293T cells,and observed the transfection status through fluorescence microscope after 24 hours of cultivation.Western blots assay was used to detect the expression of the target protein,and finally,RT-qPCR was used to detect the mRNA level of NF-κB and inflammatory response related genes in 293T cells overexpress-ing p30 protein.The results showed that after transfection of the recombinant plasmid with 293T cells for 24 hours,about 80%of the cells could display green fluorescence,and western blots could detect a band that could react specifically with Flag labeled antibodies,with a size that was consistent with expectations.RT-qPCR assay showed that in 293T cells overexpressing ASFV p30 protein,the mRNA expression levels of TNF-α and IL-6 were up regulated significantly,with an up-regulation multiple of 2.988～3.06 times that of the control group.This experiment can provide a preliminary basis for the development of ASFV antigen antibody detection technol-ogy and provide reference for the molecular pathogenesis of ASFV.
万方数据
维普
