Identification Of The Interaction Between 1B Protein Of Encephalomyocarditis Virus And RACK1

Encephalomyocarditis virus(EMCV)belongs to the Picornaviridae family,which is currently divided into 12 genera:Aphtovirus,Avihepatovirus,Cardiovirus,Enterovirus,Erbovirus,Hepatovirus,Kobuvirus,Parechovirus,Tescovirus,Tremovirus,Sapelovirus and Senecavirus.The EMCV only harbors one serotype named as Mengo virus,.In 1945,EMCV was isolated from a captive male gibbon which died suddenly of pulmonary edema and myocarditis in Miami of USA.As the surface protein of Mengo virus,capsid protein VP1 and VP2 has a depressed structure which considered as the binding sites with host cell receptor.So far,the function of 1B protein is far unclear although 1B monoclonal antibody has been identified and two epitopes for B cell activity have been found.Here,a prokaryotic expression vector pET-28a-1B was constructed,and then transformed into BL21 competent cell to express 1B protein in inclusion bodies.Subsequently,1B protein was purified from the inclusion body for animals,immunization.The serum positive detection using ELISA analysis on immunized mice the spleen cells of BALB/c mice were fused with myeloma cells SP2/0.After three times of sub-cloning,the positive hybridomas cell were screened by ELISA coated with His-1B and acquired a plant of 1B monoclonal antibody.1B monoclonal antibody was prepared and detected using 1B monoclonal antibody in western blot and immunofluorescence.It was shown that successfully prepared 1B monoclonal antibody can be used for western blot and indirect immunofluorescence.The anti-1B monoclonal antibody was shown to be highly specific to 1B protein.The yeast two hybrid techniques is an effective method for identification of protein interaction in vivo.It is often used to screen the interacted protein from a constructed cDNA library with known proteins..The yeast two hybrid technology is an important means of interaction between protein and protein,because it still has some limitations,for example,cause many false positives.So in biochemistry,there are many methods used to study the interaction between protein and protein,such as co-immunoprecipitation,confocal,affinity chromatography,these methods are adopted to study the in vitro interaction between protein and protein.The Receptor for Activated C Kinase 1(RACK1)is a member of the tryptophanaspartate repeat(WD-repeat)family of proteins.RACK1 interacts many proteins,it has a significant role to play in cancer.It necessary for RACK1 study.In this study,110 positive clones were got by different auxotrophic medium screening and we obtained 6 interaction partners of EMCV 1B after sequencing these clones.Finally,chose RACK1 to investigate further considered the function of RACK1 in viral replication.The interaction of 1B and RACK1 was idendtified To map the interacting domain of RACK1,the results suggested that EMCV 1B interacted with WD6-7 of RACK1.We detected EMCV infected in BHK-21 cells in different time and concentration by western blot,the result showed EMCV infection can be reduced RACK1 expression We detected western blot after the infection of BHK-21 cells which were pretransfected with the siRNA specific for RACK1 by EMCV.In conclusion,our research showed that RACK1 was inhibited to EMCV replication.The molecular mechanism of RACK1 regulating EMCV replication will be further study in the future.