Preparation And Evaluation Of Dengue Type 3 Virus E Protein Subunit Vaccine And E Protein Dimer Expression Test

Objective(s): Dengue fever is one of the most prevalent insect-borne diseases in the world,with different outbreaks in different provinces in China every year,bringing a huge burden to our society and economy.Although there have been a large number of studies on dengue virus,there is no specific antiviral drug for dengue fever.Vaccination to prevent dengue virus infection is still the mainstream research direction.There are currently two licensed dengue vaccines.However,the protective effects of the two dengue vaccines against severe dengue fever caused by different dengue serotypes still need to be further evaluated.Due to the widespread use of recombinant proteins and the rapid development of subunit vaccines in recent years,dengue subunit vaccines have received increasing attention due to their safety,economy and ease of scale-up,and E protein dimer epitope antibodies have shown strong potential to avoid ADE effects.Therefore,in this study,Dengue type 3 virus isolated from Xishuangbanna was used as a vaccine strain to express its E protein monomer and dimer by using mammalian cell expression system,and the monomer E protein was used as a subunit vaccine for animal immune efficacy evaluation,laying a foundation for the development of dengue subunit vaccine.Methods: The gene sequence of dengue virus type 3 strain isolated from the serum of dengue patients in Xishuangbanna was used as the template,and the full length of E protein gene was obtained by PCR with specific primers.Then the signal peptide and sequence of the vector were optimized,the transmembrane region and stem region of E protein were removed,and EDⅠ,EDⅡ and EDⅢ were used as the target sequences.Human Ig G Fc terminal sequence was added to the C terminus of E protein,and the eukaryotic expression plasmid and secretory expression plasmid of E protein were constructed by enzyme digestion and ligation.The intracellular expression plasmid was transfected into 293 F cells and its expression was verified by WB.At the same time,the transfection reagent and expression time were optimized to obtain the best transfection conditions.After that,protein G column was used to purify the target protein,and the purified target protein was mixed with Addvax adjuvant to immunize Balb/C mice aged 6-8 weeks.Serum of the mice was collected for serum antibody titer and neutralization ability assessment.In addition,based on the successful expression of E monomer protein,the sequence of monomer amino acid(aa)was modified,and the dimer sequence was formed by cysteine mutation at L107 C,A257C and A313 C.Swiss-modle online software was used for homologous recombinant modeling,and the dimer expression plasmid was constructed by enzyme cleavage and enzyme linkage.After transfection of the plasmid into 293 F cells,the protein expression was verified by reduction and non-reduction WB assay.Results: In this study,the E protein of Dengue virus type 3 isolated from Xishuangbanna was used as the target gene.By optimizing the transfection reagent,transfection time and vector signal peptide,the intracellular high expression vector Albp2A-E-Fc and the high secretion expression vector CD33p2A-E were successfully constructed,and the protein was successfully expressed by WB experiment.After the protein G column was used to purify the intracellular protein,high purity E-Fc protein was obtained.After immunizing Balb/C at 6-8 weeks of age with Addvax adjuvant,the body could stimulate the production of a large number of specific antibodies.The titer of the neutralizing antibody against dengue type 3 virus was 1:512.On the other hand,the E protein dimer expression plasmid was correctly constructed by enzyme digestion and sequencing verification,and the protein was successfully expressed by reduction and non-reduction WB experiments.Conclusion(s): In this study,mammalian cells were used as the expression system.The E protein expression vector of DENV type 3 virus was successfully constructed and the verified protein was successfully expressed.After purification,it was confirmed by animal immunization that the mixture of E protein and adjuvant can stimulate the body to produce significant neutralizing antibodies.On the other hand,the E protein secreting expression vector and E protein dimer expression vector were successfully constructed,and the target protein was successfully expressed by WB experiment,which laid the foundation for the development of subunit vaccine for dengue fever in the future.