非洲猪瘟病毒p30蛋白在293T细胞中的瞬时表达

Transient Expression of African Swine Fever Virus p30 Protein in 293T Cells

为真核表达非洲猪瘟病毒p30蛋白,探究其对炎症反应相关基因表达的影响.实验根据NCBI上的p30基因序列合成基因,将其克隆至真核表达载体pIRES2-EGFP中,再将重组质粒转染到293T细胞中,培养24 h后通过荧光显微镜观察质粒的转染情况,以western blots试验检测目的蛋白的表达情况,最后再利用RTqPCR检测过表达p30蛋白的293T细胞中NF-κB信号通路和炎症反应相关基因的mRNA转录情况.结果显示,293T细胞转染重组质粒24 h后,约80%的细胞可显示绿色荧光,并且western blots检测到一条可与Flag标签抗体特异性反应的条带,大小与预期相符.RT-qPCR试验表明,过表达ASFV p30蛋白的293T细胞中,炎性反应相关细胞因子TNF-α和IL-6的mRNA表达水平得到显著上调,上调倍数为对照组的2.988~3.605倍.试验可为ASFV抗原抗体检测技术的研发提供前期基础,为ASFV分子致病机制研究提供参考.

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-αand 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.