TSG 101基因敲低对猪伪狂犬病病毒体外增殖的影响

Effect of TSG 101 Gene Knockdown on Proliferation of Pseudorabies Virus in vitro

本研究旨在使用慢病毒包装敲低技术构建敲低肿瘤易感基因(tumor susceptibility gene 101,TSG 101)的慢病毒质粒及稳转PK15细胞系,并验证该敲低细胞系对伪狂犬病病毒(pseudorabies virus,PRV)感染的影响。针对TSG 101基因构建并筛选,成功获得重组慢病毒,用获得的重组慢病毒感染PK15细胞,并使用嘌呤霉素进行筛选,获得稳转敲低TSG 101基因的PK15细胞系。通过Real-time PCR以及Western blot方法对所构建的细胞系进行编辑效率检测;使用CCK-8法对筛选出的细胞系进行细胞活力及生长状况检测;通过流式细胞术、Real-time PCR、Western blot以及子代病毒滴度方法验证该细胞系对PRV复制的影响;最后对PRV的吸附、进入以及复制阶段的试验探究敲低TSG 101基因对于PRV感染有影响的阶段。结果表明,1)本研究所构建的细胞系中TSG101的表达明显降低,并将该细胞系命名为sh-TSG101细胞系;2)细胞活性检测结果显示,敲低TSG 101基因对细胞活性及生长情况无显著影响;3)使用PRV-GFP、PRV-QXX感染sh-TSG101,体外试验证明敲低TSG 101对PRV的增殖有显著的抑制作用。4)病毒的吸附、进入以及复制试验证明,敲低TSG 101在PRV的复制阶段有明显的抑制作用。综上,本研究成功构建TSG 101基因敲低的PK15细胞系,体外敲低TSG101能够显著抑制PRV增殖,为PRV等DNA病毒性疾病的防控提供了新思路,并为进一步探究TSG101调控PRV感染的分子机制奠定了基础。

The aim of this study is to use lentiviral packaging knockdown technology to construct lentiviral plasmids for knocking down tumor susceptibility gene 101(TSG 101)and stably transfect PK15 cell lines,and to verify the impact of this knockdown cell line on PRV infection.A recombinant lentivirus was successfully constructed and screened for the TSG 101 gene,and PK15 cells were infected with the obtained recombinant lentivirus.Puromycin was used for screening to obtain PK15 cell lines that knocked down the TSG 101 gene.Perform editing efficiency testing on the constructed cell line using Real time PCR and Western blot methods;Use CCK-8 method to detect cell viability and growth status of the selected cell lines;Verify the effect of this cell line on PRV replication through flow cytometry,Real time PCR,Western blot,and progeny virus titer methods;Finally,experiments were conducted on the adsorption,entry,and replication stages of PRV to explore the impact of knocking down the TSG 101 gene on PRV infection.The results showed that:1)the expression of TSG101 was significantly reduced in the cell line constructed in this study,and the cell line was named sh-TSG101 cell line;2)The results of cell viability testing showed that knocking down the TSG 101 gene had no significant effect on cell viability and growth;3)Using PRV-GFP and PRV-QXX to infect sh-TSG101,in vitro experiments have shown that knocking down TSG 101 has a significant inhibitory effect on PRV proliferation.4)The adsorption,entry,and replication experiments of the virus have shown that knocking down TSG 101 has a significant inhibitory effect on the replication stage of PRV.In summary,this study successfully constructed PK15 cell lines with TSG 101 gene knockdown.Knocking down TSG 101 in vitro can significantly inhibit PRV proliferation,providing new ideas for the prevention and control of DNA viral diseases such as PRV,and laying a foundation for further exploring the molecular mechanism of TSG101 regulating PRV infection.