The Mutation Analysis On Furin Cleavage Site Of PrM Protein And Establishment Of Cell Lines Stably Expressing Virus-like Particles Of Japanese Encephalitis Virus

Japanese encephalitis (JE), is the most important cause of epidemic encephalitis in most Asiaregions. It is serious arthropod-borne viral disease which can bring damage to animals and humans. Sofar, there is no specific therapeutic treatment, the only effective way to prevent virus infection inhumans and domestic animals is vaccination.Although traditional inactivated vaccine and attenuated live vaccine have played important roles inprevention and control of JE. However, both of them are also limited by some disadvantages such ashigh cost, lack of long-term immunity and side effects. Thus there is an urgent need to develop moreeffective vaccines and to alter the strategies against this viral disease.It has been shown M and E protein of JEV expressed in mammalian cells can assemble intovirus-like particles (VLP). Antigenic analysis and mouse experiments indicated that VLPs are similarwith the virions in inducing neutralizing antibody and protective immunity and more safety due to theabsence of viral nucleic acids. Establishment of continuously expressing eukaryotic cell lines would bean ideal way to produce VLPs in terms of safety and yield. But the fusogenic activity of VLPsassociated with cleavaging of prM by furin protease in the particles maturation process, may becytotoxic, which makes it difficult to establish cell lines expressing VLP. We manage to change thecleavage site to inhibit the toxic effect.The furin recognition site of prM is R89SRR92. In order to define the crucial amino acids forcleavaging, a series of point mutations (ΔR89, ΔR91, R89A, R91A, R92A) were introducedinto the cleavage site of prM and the mutated prM gene were cloned into the eukaryotic expressionvector pCAG. BHK-21cells were transfected with the recombinant plasmids pCAG-JEV-prM(ΔR89)E,pCAG-JEV-prM(ΔR91)E、pCAG-JEV-prM(R89A)E、pCAG-JEV-prM(R91A)E、pCAG-JEV-prM(R92A)E. Cells grew in medium supplemented with G418and identified by indirectimmunofluorescence assay (IFA) and western blot. In addition, the cells displaying high-level proteinexpression were purified through limiting dilution cloning. The results showed that cell lines ofprM(ΔR91)E, prM(R89A)E, prM(R92A)E can release uncleavage protein prM and cell lines ofprM(R91A)E can cleavage protein prM. Electron microscopy observation of secretion expressionproducts of the cell, the prM (R89A) E cells lines with uncleavage prM could be assembled intovirus-like particles (prME–VLP) with the E protein observed under the electron microscopy. Virusneutralizing antibody level in mouse immunized with prME-VLP were much lower (1:10to1:20)than those achieved by ME-VLP (1:160), and the prME-VLP provided merely0-10%protection against virus lethal challenge while protection rate of ME-VLP could arrive to90-100%.In this study, the effect of amino acid in prM cleavage site to the maturation process of VLP wasdefined by establishing cell lines stably expressing prM with mutant furin site. The protective immunitylevel inducing by prME-VLP was demonstrated, too. These established cell lines would provide a basisfor further study on effect of the cleavage event on maturation mechanism of JEV and other flavivirus.