Improve The Yield Of CA16 Virus Like Particles And Determine The Immunogenicity Based On The Modification Of Sequence And Promoter

Both CoxsackievirusA16(CA16)and Enterovirus71(EV71)are two major pathogenic agents of hand,foot and mouth disease(HFMD),which frequently infects among young children around the world because of possessing no obvious differences in various regions.Although majority of HFMD patients accompany with mild clinical symptoms,some cases of them may develop severe nervous system diseases which may ultimately lead to death.The large scale of epidemic of HFMD recently indicates that it is posing a serious threat to the public health of children in Asia-Pacific regions,especially in China.Due to lacking specific therapeutic interventions and effective prevention and control strategies,there is a common sense for researchers that development of preventive vaccines should be the most approach for preventing the disease epidemic.A inactivated whole-virus vaccine has been approved in China mainland for well protection in severe HFMD causing by EV71.However,the monovalent EV71 vaccine has no ability to crossly protect against CA16 infection,so this may indicate that it cannot effectively prevent the overall HFMD epidemics.Therefore,speeding up the study of various kinds of CA16 vaccines are highly desirable.Virus Like Particles(VLPs)have developed into a new vaccine type because of similar natural viral particles,lack of nucleic acids,complete epitopes,high safety and immunogenicity.Compared with the development of EV71 VLP vaccine,the study of CA16 VLP vaccine was hysteretic because of poor yield.Therefore,the overriding objective of this study intended to improve the yield under the insect baculovirus expression system combining with the modifications of sequences and promoters.Hence,a series of recombinant plasmids co-expressing P1 and 3CD were obtained in this paper.To overcome the bottleneck of CA16 VLPs vaccine development,this study sought to construct a highly productive plasmid to improve the production yield relying on baculovirus expression systerm,the coden optimization of the P1 ori target gene sequence was designed to obtain P1 opt,the exchange of 3CD target gene sequence into BJ3 CD was also designed,the replacement of P10 promoters into ECMV promoter was designed,and the optimized process parameters were also screened.What is more,as a candidate vaccine against HFMD,the antigenicity and immunogenicity of purified CA16 VLPs were also evaluated.In summary,five different recombinant plasmids were successfully constructed marking Y(P1ori-3CD-PFD),BY(P1ori-BJ3CD-PFD),BU(P1opt-BJ3CD-PFD),BEY(P1ori-ECMV-BJ3CD-PFD)and BEU(P1opt-ECMV-BJ3CD-PFD)based on the Bac-to-Bac expression system respectively.And the results of optimization expression parameters were T=21?,TOH=120h and MOI=0.1.Under the optimization expression conditions,our present study provided a recombinant plasmid BU(?14.7mg/L)that gave the highest total VLPs yield than the original plasmid Y(?2.60mg/L)in sf9 cells,which corresponding to?5.7-fold.More essentially,the purified VLPs possessed the similar structure to inactivated CA16 virus particles and induced high neutralization antibody titers and IgG antibody titers in mice.The high productive recombinant plasmid and optimized prameters may drive the large scale production of CA16 VLPs in the future.Meanwhile,the VLPs releasd into the supernatant because of ECMV promoter regulation slow down the cell lysis and intracellular contaminants.Take these results altogether suggested that our work is a meaningful attempt to increase the yield of CA16 VLPs and provides a approach to overcome the bottleneck in vaccine production.