Construction Of MARC-145 Cells With Cathepsin H Knockdown

Cathepsin H is an important member of the lysosomal cysteine protease family and is the only known monoaminopeptidase in the papain-like family.Its expression level is increased in activated immune cells,and other tissues.Like proteases,it plays a central role in many important cells,such as degrading intracellular proteins,activating other proteases in lysosomes,remodeling extracellular matrices,and participating in apoptosis.Whether monkey Cathepsin H gene affects the replication of Pseudorabies virus(PRV)has not been reported yet.In order to establish MARC-145 cells that stably and efficiently inhibit the expression of monkey Cathepsin H gene,we used the small interfering RNA technology to target Cathepsin H gene.We also examined the effect of knockdown of Cathepsin H on PRV replication.Fluorescence microscopy,flow cytometry,viral titer determination,real-time PCR,western blot and other methods were used to detect the proliferation of PRV in shCathepsin H MARC-145 cells.The results showed that the fluorescence intensity of PRV-GFP in shCathepsin H cells was significantly lower than that in shControl cells with the prolongation of PRV-GFP infection,indicating that knockdown of Cathepsin H inhibited PRV repliCTS ion.Furthermore,shControl and shCathepsin H cells were infected with PRV-QXX.and the virus titer assay showed that the virus titer in shCathepsin H cells was significantly reduced compared to shControl cells.In addition,knockdown of Cathepsin H significantly inhibited the transcription of the PRV gB and TK mRNA,and the translation of PRV gE protein.In conclusion,our results show that knockdown of the Cathepsin H gene in MARC-145 cells has a significant inhibitory effect on PRV replication.Therefore,inhibition of the expression of Cathepsin H gene may be an effective strategy of preventing PRV infection.The results of this study will provide a new way for the prevention and treatment of pseudorabies.