Expression Of Recombinant CSFV E2 Protein And Identification Of Its Biological Activity

Classical swine fever?CSF?,caused by Classical swine fever virus?CSFV?,is an OIE-listed,highly contagious,often fatal disease of swine.CSF is characterized by fever,high mortality,immunosuppression and reproduction failure.CSFV is a small,enveloped virus with a non-segmented positive-stranded RNA genome which belongs to the Pestivirus genus of the Flaviviridae family.The envelope proteins of CSFV are E^rns?E1 and E2.The E2protein on the surface of viral envelope is one of the virulence factors in pigs and the most important protective antigen of CSFV that participate in the infection process of virus.To date,the role of E2 protein in viral entry into host cells have been poorly identified.And the E2-based CSF subunit vaccine plays an important role in CSF control.To carry out the research on diagnose application and pathogenic mechanism we generated a suspension cell line that secretes CSFV E2 protein and then identified its biological activity.The specific primers were designed according to the JL1?06?strain published on Genbank.Then we used fusion RT-PCR amplified the ectodomain?ED?of the E2 gene with the Strep and Flag tags were introduced to its C terminus.Then the modified E2 gene was cloned into a lentiviral expression vector pLVX-IRES-ZsGreen1 to construct a recombinant plasmid,pLVX-E2?ED?-Flag/Strep.Subsequently we used auxiliary plasmids and the pLVX-E2?ED?-Flag/Strep plasmid co-transfected HEK-293T cell to generate a lentivirus and transduced it into adherent CHO cells and suspension 293 cells,and then we used limiting dilution assay,ELISA,SDS-PAGE,and western blot to select and identify the positive cell clones of CHO named as CHO/E2 cell line.By using FACS we selected the positive cells as a recombinant 293s/E2 cell line.The E2 protein concentration,detected by BCA method,in293s/E2 culture supernatant is much higher than that in CHO/E2 cell culture.Western blot and SDS-PAGE analysis revealed that the E2 protein could stably secrete to cell culture for at least 20 passages and the concentration of E2 protein in the medium is10.87?g/mL.Furthermore we immunized the New Zealand white rabbits with purified recombinant E2 protein,after immunization a quite high antibody level was detected and the titer of serum neutralizing antibody is 1:3200,which implies a good immunogenicity of the recombinant E2 protein.We used the purified E2 protein to block PK-15 cells,and the titer of progeny virus in the cell culture indicated a decrease of CSFV infectivity.Then we incubate PK-15 cells with E2 protein and CSFV,after 1h attachment at 4?a nd 2h internalization at 37?we quantitated the virus genome copies of CSFV by qRT-PCR,the results revealed that E2 protein impressed the internalization of CSFV into PK-15 cells and has no influence on virus attachment.Our study generated a 293s/E2 suspension cell line that can stably express E2 protein with a good immunogenicity in a secretory form.By using the E2 protein expressed by this cell line we identified that CSFV E2 protein plays an important role in the virus internalization into host cells.Our study laid a foundation for discovering receptors,host-factors interact with E2 protein,establishing CSFV diagnostic method.