Expression, Identification Of Dengue Virus Serotype Ⅱ Envelop Protein Domain Ⅲ And Primary Screening Of Receptors Of Dengue Virus In C6/36 Cell Line

Dengue virus (DENV) belongs to the family Flaviviridae, genus Flavivirus. There are four antigenically related serotypes referred to as DENV1, DENV2, DENV3 and DENV4. Virus transmission occurs through the infective bite of Aedes aegypti and Aedes albopictus. Dengue is the most important arboviral disease of humans. The consequences of DENV infection in humans ranges from a self-limiting illness known as dengue fever (DF) to a severe dengue hemorrhagic fever (DHF) which can progress to dengue shock syndrome (DSS).An estimated 50 million dengue infections and 500,000 DHF cases occur annually, particularly in south-east Asia, the western Pacific and the Americas. At present, however, there is no clinical vaccine against dengue virus and no effective antiviral drugs available. Dengue has became a serious public health problem in tropical and subtropical regions.The dengue viral genome consists of a single positive sense RNA of 11 kb. This RNA encodes three structural proteins (C, M/prM and E) that form the components of the virion, and seven nonstructural proteins (NS1, NS2a/b, NS3, NS4a/b, NS5) involved in viral RNA replication.The envelope glycoprotein of dengue virus consists of about 500 amino acid residues, is the major structural protein exposed on the surface which mediated viral infection into host cells. The crystal structure analysis of envelope protein revealed that it includes three domains (Ⅰ,Ⅱ, andⅢ) that exhibit significant structural conservation when compared to other flaviviruses. DomainⅠis organized into an eight-stranded centralβ-barrel structure. DomainⅡcontains the highly conserved hydrophobic fusion peptide which is involved with dimerization of envelop protein monomers on the surface of mature virion. DomainⅢhas an immunoglobulin-like fold which has been implicated in receptor binding.DomainⅢof can fold independently into an immunoglobulin-like module which makes it can function as a receptor binding site. DomainⅢof dengue virus envelop protein lies exposed and accessible on the virion surface. These structural features make it may be the main receptor binding site. Also, domainⅢof the dengue virus envelope protein contains epitopes which can induce type-specific and subtype-specific neutralizing antibodies that may block the entry of virus into host cells. Therefore, domainⅢhas emerged as a promising important target for vaccine candidate and interactions with host cell receptors.The life cycle of dengue virus is in host cells. Dengue virus is internalized by receptor-mediated endocytosis into host cell and complete a series of replication, translation, assembly and packaging at the endoplasmic reticulum membrane. Overall, binding between dengue virus and receptors of host cell surface is the most important initial step for virus infection. As a typical mosquito-borne virus, researchers have paid more attentions on receptors of dengue virus in mosquito cells and tissue. Identify these receptor molecules and explain its mechanism is very helpful for blocking virus spreading in mosquito, developing effective mosquito control strategies and antiviral drugs.Objective:1. To amplify and clone DENVⅡenvelope protein gene and construct pMD 18-T-DV2-E plasmid;2. To amplify and clone domainⅢgene and construct expression plasmid pET-28a(+)-DV2-EDⅢand pET-32a(+)-DV2-EDⅢ;3. To induce the expression of domainⅢin Escherichia coli; 4. To purify recombinant protein and immunize rabbit to obtain polyclonal antibody;5. To screening receptors of Dengue virus in C6/36 cell by VOPBA;Methods:1. DomainⅢof envelope protein of DENV, JEV, TBEV, WNV and YFV were analysed by bioinformatics software, to search conserved regions and construct the phylogenetic tree;2. Analyze the rare codons in the encoding sequence of DENVⅡenvelope protein domainⅢ;3. Inoculate live dengue virus in the brain of suckling mice and extract total RNA from the brain of infected mice, the envelope protein DNA fragment was amplified by RT-PCR and ligated into pMD 18-T to construct pMD 18-T-DV2-E plasmid;4. PCR amplify the domainⅢDNA fragment of the envelope protein with pMD 18-T-DV2-E as the template and cloned into pET-28a(+) and pET-32a(+) to construct the expression plasmid pET-28a(+)-DV2-EDⅢand pET-32a(+)-DV2-EDⅢ;5. Transform the recombinant plasmids into BL21 (DE3) and induce by IPTG, analyze the expressed products by SDS-PAGE;6. Lysis the induced bacteria by sonication to analyze the solubility of recombinant protein;7. Optimize expression conditions such as induce temperature, induce time and IPTG concentration;8. Analyze the immunoreactivity of the recombinant protein with His·Tag McAb and DENV(Ⅰ-Ⅳ) McAb by Western blot;9. Purify the recombinant protein with Ni-NTA and electroelution from SDS-PAGE gels;10. Immunize rabbit sith purafled 'ebombinant pr+teij to obtaij poly#lonal antibody;11. Det%rmine the titer of polyclonal antibody by ELISA;12. Analyze th(?) specif caty of polyclojal anti"ody by Wectern blot and IFA; 13. Passage dengue virusi' C6/36 cell, purify virus and determine TCID50;14. Prepare total protein and membrane protein of C6/36 cell;15. Screen the binding molecules in C6/36 cell by Virus overlay protein binding assay;Results:1. Conserved regions exist in domainⅢof envelope protein of DENV, JEV, TBEV, WNV and YFV;2. Rare codons exist in the encoding sequence of DENVⅡenvelope protein domainⅢ;3. The plasmid pMD 18-T-DV2-E was successfully constructed;4. The expression plasmid pET-28a(+)-DV2-EDⅢand pET-32a(+)-DV2-EⅢwere successfully constructed;5. The bacteria with recombinant expression plasmid pET-28a(+)-DV2-EDⅢwas no expressed products after induction with IPTG;6. After induction with IPTG, a specific soluble protein was expressed from Escherichia coli which contains the expression plasmid pET-32a(+)-DV2-EDⅢ;7. The optimize expression condition were induced with lmmol/L IPTG at 25℃for 6h;8. Western blot demonstrated specific reactivity of the recombinant protein with His·Tag McAb and DENV(Ⅰ-Ⅳ) McAb;9. The purity of recombinant protein purified can reach 98.6%;10. The titer of polyclonal antibody was 1:12800 by ELISA and it has some specificity;11. Dengue virus was successfully passaged in C6/36 cell and the purified virus titer was 4.52×107PFU/ml;12. A specific 67kDa binding molecule in C6/36 cell was found;Conclusion:1. There exists conserved regions in domainⅢof envelope protein of DENV, JEV, TBEV, WNV and YFV; 2. Successfully constructed pET-32a(+)-DV2-EDⅢplasmid and it can be highly expressed in a soluble and fusion form in Escherichia coli. Western blot demonstrated the recombinant protein had specific immunoreactivity with His·Tag McAb and DENV(Ⅰ-Ⅳ) McAb;3. The obtained polyclonal antibody against domainⅢof envelope protein has some specificity;4. A specific 67kDa binding molecules maybe receptor of dengue virus in C6/36 cell.