Preparation Of Monoclonal Antibody Against Capsid Protein Of Japanese Encephalitis Virus And Its Application In The Study Of Viral Cell Entry Mechanism

Japanese encephalitis virus(JEV)is a member of the flavivirus family of flaviviridae.Its genome is a single-stranded positive-stranded RNA virus without segments.It can infect many hosts and is a zoonotic disease.The pig is the main amplifying host,which can cause sow reproductive disorders.Once the disease is occured,it will bring serious economic losses to the pig industry.Currently,there are no specific treatment methods or drugs for the disease,which can only be controlled by supportive therapy or vaccine immunization.Although progress has been made in vaccine research and development,the disease is still a major public health problem in South Asia,Eastern Europe and Southeast Asia.Therefore,it is urgent to find a new and effective strategy for the prevention and treatment of antiviral infections.As a product of new biotechnology,monoclonal antibodies can be used not only to make kits to detect antigens in pig blood,but also to monitor the health status of pigs effectively.They can also be used as an effective biological tool to study the function of viral proteins in laboratory.In this study,we purified the capsid protein of JEV by prokaryotic expression,prepared monoclonal antibodies against JEV capsid protein,and used the prepared monoclonal antibodies to explore the mechanism of JEV cell entry.The full text is as follows:1.Prokaryotic expression and purification of Japanese encephalitis virus Capsid proteinIn this study,the codon of JEV Capsid protein at position 7-246 was analyzed and optimized by Rare Codon Caltor software to reduce the rare codon contained in the gene,so that the protein encoded by the gene could be more easily expressed in E.coli.The optimized gene was cloned into prokaryotic expression vector pColdI and transformed into E.coli Rossetta 2(R2).After inducing expression,the recombinant protein was successfully expressed by SDS-PAGE identification.After a large number of inducing expression,the recombinant protein was purified,and the purified protein with high purity and concentration was obtained.2.Preparation of monoclonal antibodies against Japanese encephalitis virus Capsid proteinBalb/c mice were immunized with 100 ?g/mice emulsified recombinant Capsid protein after renaturation by dialysis and removed the imidazole and other harmful substances.Balb/c mice were immunized with 100 ?g/mice after emulsification.After repeated immunization,mice with antibody levels up to standard were euthanized.Mice spleen was taken out from sterile super-clean platform to prepare spleen cells and to fuse with prepared myeloma cells.Three hybridoma cell lines with stable secretion of antibodies were obtained after multiple screening.They are 3C6E7,3C6F11 and 3C6G8 respectively.Western blot results showed that the antibodies secreted by the three hybridoma cells could specifically recognize the recombinant Capsid protein,and the viral proteins produced by BHK-21 cells,3D4/21 cells and PK-15 cells infected with JEV.DTMUV,CSFV,PRV,PRRSV and JEV were infected respectively.The monoclonal antibody could only recognize the proteins produced by JEV infection.Confocal observation showed that JEV infected BHK-21 cells were incubated with hybridoma cells secreting antibodies,and the specific fluorescence of the antibodies secreted by the three cells was observed in the cytoplasm.3.Application of JEV Capsid protein monoclonal antibody to explore the mechanism of JEV cell entryJEV capsid protein monoclonal antibody was used to study whether JEV entry depended on cytoskeleton structure.Nocodazole and Colcemid could destroy microtubule structure.After 50 ?M Nocodazole treatment,the number of copies of JEV genome decreased by 86%compared with the control group.Colcemid treatment also produced similar results.JEV capsid protein monoclonal antibody was used to identify JEV.Similar results were found by Western blot and IFA,suggesting that JEV can be identified by Western blot and IFA.Invasion of BHK-21 cells depends on microtubule structure.Jasplakinolide can inhibit the dynamic changes of actin.After 2.5 ?M Jasplakinolide treatment,the copy number of JEV genome decreased by 75%.Similar results were found by Western blot and IFA,suggesting that microfilament structure is needed for JEV to invade BHK-21 cells.Rhosin,NSC23766 and ML141 can inhibit the signal transduction process of intracellular cytoskeleton dynamic changes.JEV internalization was significantly reduced after 250 ?M Rhosin and 100 nM NSC23766 pretreatment,while no significant changes were observed in ML 141 treatment group.The results of Western blot and IFA showed that RhoA and Racl regulation played a major role in JEV internalization into BHK-21 cells.These results suggest that JEV invasion of BHK-21 cells depends on cytoskeleton participation.