Prokaryotic Expression And Identification Of Swine Ribonuclease L(sRNase L)and Its Interaction With PRRSV Proteins

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease which is caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRS caused significant economic loss to the swine industry worldwide.The disease is one of the most economically significant viral disease of swine,frustrating challenge to the global swine industry.In 2006,the outbreak of highly pathogenic porcine reproductive and respiratory syndrome in China was characterized by high fever,high morbidity and mortality.Ribonuclease L is one of the most important antiviral protein(antiviral protein,AVP),with is induced by interferon,participate in the specific immune and nonspecific immunity.It plays a very important role in the antiviral process.Activated RNase L cleaves single stranded RNAs,including viral RNAs and cellular RNAs.The activated RNase L is inhibited by hydrolysis of viral RNA to reduce virus replication.However,the interaction between RNase L and PRRSV proteins affecting the replication and propagation of PRRSV has never been reported.Our research is divided into the following three parts:sRNase L gene was amplified purpose and cloned into prokaryotic expression vectorpGEX-4T,The recombinant plasmid p GEX-4T-sRNase L was transformed into the hostexpression bacteria BL21.The soluble protein was obtained by IPTG induction,and theGST label was removed by rTEV protease was showed that the expressed sRNase L has thefunction of cutting RNA.In this study,luciferase interaction system was constructed to screen PRRSV proteins that interact with sRNase L.All the coding protein genes of PRRSV and sRNase L gene were cloned into two plasmids of luciferase interaction system.It was found that PRRSV proteins interacting with sRNase L are Nsp4,Nsp12 and N.The results of Co-IP showed that the Nsp4,Nsp12 and N proteins of PRRSV interact with sRNase L.It was further showed that N sp4,Nsp12 and N proteins co-localize with sRNase L in cells by IFA.RNase L plays an important role in the antiviral process of the organism,but the antiviral effect and mechanism of sRNase L on PRRSV have not been elucidated.Nsp4 is the most important proteolytic enzyme in virus replication and has the function of cutting protein.The function of Nsp12 protein is not yet studied and may play a role in the transcription of PRRSV.N protein is the nucleocapsid protein of PRRSV and plays an important role in the assembly process of the virus.We found that sRNase L interact with PRRSV Nsp4,Nsp12 and N protein which may affect the role in PRRSV replication,transcription,assembly,and then play important role in anti-PRRSV activity.This study lays an important foundation for further study of anti-PRRSV mechanism of sRNase L,and provides new ideas and methods for the infection and prevention and PRRSV.