环指蛋白31基因敲除细胞系的构建及其对口蹄疫病毒复制的影响

Construction of cell lines with ring finger protein 31 gene knockout and evaluation of its impact on the replication of foot-and-mouth disease virus

摘要:

目的 探究环指蛋白31 (ring finger protein 31, RNF31)在口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制过程中的具体功能，旨在为宿主蛋白RNF31调控FMDV复制的分子机制提供理论支撑。方法 运用CRISPR/Cas9基因编辑技术，在RNF31基因外显子区段设计2条sgRNA序列，并与pX459-puro载体连接构建重组质粒。将pX459-RNF31-sgRNA转染至PK-15细胞，经嘌呤霉素筛选，获得RNF31基因敲除的细胞系。通过Western blotting、RT-qPCR及TCID₅₀方法检测敲除RNF31基因对FMDV复制的影响。结果 与野生型细胞相比，RNF31基因敲除细胞系中FMDV的蛋白水平、mRNA水平及病毒滴度均显著升高。结论 本研究成功构建了RNF31基因敲除细胞系，证实RNF31在FMDV复制过程中扮演关键角色，研究结果为进一步探究RNF31抑制FMDV复制的机制提供了数据支持。

Abstract:

Objective To investigate the function of ring finger protein 31 (RNF31) in the replication of foot-and-mouth disease virus (FMDV) and to provide a theoretical basis for the research on the molecular mechanism by which the host protein RNF31 regulates FMDV replication.Methods CRISPR/Cas9 gene editing was employed to design two sgRNA sequences in the exon segment of RNF31, and recombinant plasmids were constructed by ligation with the pX459-puro vector. The recombinant plasmids pX459-RNF31-sgRNA were transfected into PK-15 cells, followed by screening under the action of puromycin to obtain the cell lines with RNF31 gene knockout. The effect of RNF31 gene knockout on FMDV replication was detected by Western blotting, RT-qPCR, and TCID₅₀ methods.Results Compared with wild-type cells, the knockout of RNF31 significantly increased the protein level, mRNA level, and virus titer of FMDV.Conclusion We successfully construct the cell lines with RNF31 gene knockout and prove that RNF31 plays a key role in the replication of FMDV. This result provides data support for further research on the mechanism by which RNF31 inhibits FMDV replication.