Expression, Identification Of Dengue Virus Serotype â…¡ Envelop Protein Domain â… ,â…¡ And Primary Screening Of Receptors Of Dengue Virus In Mosquito

Dengue viruses (DENV) are the causative agent of the most common mosquito-borne virus which belong the yellow virus. Dengue viruses are distributied more than100countries, especially in tropical and subtropical areas. In the past ten years, the incidence of dengue fever has increased nearly30times.The world health organization estimates,50million to100million people suffer dengue virus infection around the world every year, what’s more, the two kinds or more types of virus are all in the cycles, and the sensitive people appear constantly, not only lead to speed up the frequency of dengue popular, but also make the alien dengue virus causes the secondary infection such as the dengue hemorrhagic fever/dengue shock syndrome which all have the high fatality rate of cases appear in large Numbers. At present there are about2.5billion people in the world threatde by the dengue virus, about50million people infected with dengue fever each year, of which about2.1million serious cases,500000cases of dengue hemorrhagic fever,20000deaths. Dengue virus infections disease range is very wide, from self-limited disease to dengue fever, dengue hemorrhagic fever and dengue shock which are threaten the life. As the global economic integration degree and the increase of the climate for warming, coupled with the speeding of the spread of the mosquito vectors, whats more announced by the wide range change, dengue fever/dengue hemorrhagic fever has become the most important mosquito spread-viral disease in the tropical, subtropical areas, which is the important public health problem.Dengue virus is a single RNA virus, including four serum types (DENV1-4). Genome RNA of dengue virus codes three structure proteins including capsid protein, glycolprotein, membrane protein and its precursor (C, E, prM/M) and seven non-structure proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). The glycoprotein E is the main virus particles that coatde glycoprotein, which is composed by the amino acid residues from494to501, and the mass of the molecular is about55-60KDa.And the homology of the coding sequence in arbovirus is higher (about40%) and all the conservative Cys base are highly conserved in the protein, and all of the E members protein with the similar between the structure and function. Dengue viruses exists in the surface of mature virus particle by the three homologous body form which get together.Crystallography studies show that dengue virus glycoprotein E of coating, divided into three structure domain (Ⅰ-Ⅲ). In the three structures, the secondary structure which formed by the β chain is its main structure. The domain I located in the centra of the E protein, the domain Ⅱ of E protein fold the sample area, the structure of domain Ⅲof the E protein are constitutes by the amino acid residues in the C orientation.Because in all of the mature virus the infectious proteins are all made of the fusion of homologous dimers form, so we concluded that all of the structure of E protein may related the cell fusion process. The CD rings which composed by the98～111bit amino acid residues in the area of domain II of E protein rich in glycine and has great hydrophobic,and the sequence composition in almost all the worm virus is highly conservative. Recently found, the CD rings can plug in the membrane when the E protein transferred from the dimer to tripolymer and participate the fusion of cell membrane with host and virus, so is considered to be the virus fusion targets, or thought is the potential virus fusion peptides. So we inferred that the domain Ⅰ, Ⅱ of the E protein of the dengue virus might be related to the pathogenesis. The virus receptor can be defined as the infection of complex molecules or the adsorbent protein (virus attachment protein, VAP) in the host cell surface to identify and bond with the virus, the chemical nature is glycoprotein, proteins, lipids or glycolipid which are mostly belong to the protein. The process of the infection that the virus interact cells is essentially virus acts the host cells, virus binds the cell surface receptors together is the precondition of the virus infection. The combination reflects the corresponding relation between the virus structural protein and the space conformation of the receptor objectively. In the initial infection process, the structural protein located on the surface of the virus particles combine with the receptors on the cell surface, and through the membrane integrated or endocytosis, starting a series of biological events. And in the second infection of process, dengue virus also can exist in the body and form the immune complex with the virus specific and non-neutralization antibody (such as IgG), and via the Fc receptors on the cell through the cells, the antibody dependence enhanced infection (antibody-dependent enhancement, ADE) may revelant with the pathogenesis of the dengue hemorrhagic fever, dengue shock syndrome. The specific binding of the virus and the cell receptors is the result of the interaction of the host with virus in the long-term evolution. In order to get more chance of the infection, virus can acquire the ability identifying various cell receptors according to different target cells and invasion pathways through evolution, so as to meet the needs of their proliferation. At present we think that the combination of the virus and the receptors is more than complex process, involving different virus adsorption protein and multiple target cell receptors, the virus receptors on the cells surface can be specific, also can be nonspecific, at the same time the virus could incorporate more than one of the cell surface molecules, and the same virus may connect different receptor. At present, the identification of the potential dengue virus receptors more than a dozen kinds in the laboratory. As the typical mosquito-borne viral, dengue virus can replicate in the cell, also could infect the human and lead to disease, the researcher always pay attention to the receptors in the mosquito.With the receptor of research to develop, the pathogenic mechanism will finally able to clarify, which will play an immeasurable role in the choose of the antiviral drugs targets and the design of new vaccine.Objective:1. Expression, purification and identification of the domain Ⅰ, Ⅱ of envelop protein in Escherichia coli;2. The research about the function of the receptors of the BHK-21cell connected DENV-2;3. To screen potential receptors of dengue virus in different stages of the Aedes albopictus.Methods:1. Inoculate live dengue virus in the brain of suckling mice and extract total RNA from the brain of infected mice;2. The envelope protein DNA fragment was amplified by RT-PCR through the specific primers;3. PCR amplify the domain Ⅰ,Ⅱ DNA fragment of the envelope protein with the DV2-E;4. Constructing the restructuring plasmid pET28a-DV2-E-D Ⅰ, Ⅱ by double digests of the PCR product and the plasmid pET-28a (+);5. Screening the positive colonies, and transformed it into the E.coli BL21;6. The restructuring plasmid induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blotting;7. The cultivate of the BHK-21cell;8. The expansive cultivation and purification of dengue virus;9. Detect the infection of the cells by the RT-PCR and indirect immunofluorescence (IFA);10. Screen the binding molecules in BHK-21cell by virus overlay protein binding assay;11. The rearing of the Aedes albopictus;12. To screen the potential receptors of dengue virus in different stages of the Aedes albopictus.Results: 1. The fragment of the target have got after RT-PCR and the length is900bp though the extraction of total RNA from the sucking mice;2. After the double digests with the Xho1and Nde1enzyme,a900bp fragment have got from the restructuring plasmid pET28a-DV2-E-D1&2;3. After induction with IPTG, a specific inclusion body protein was expressed in the precipitation. from Escherichia coli which contains the expression plasmid BL21(DE3)/pET-28a (+)-DV2-E-D1&2;4. Western blot demonstrated the specific reactivity of the recombinant protein with His-Tag McAb and DENV(Ⅰ-Ⅲ).McAb;5. A specific30and60kDa binding molecule in BHK-21cell was found;6. A specific35binding molecule in the larvae, pupa, adult mosquitoes of the Aedes albopictus was identified.Conclusion:1. The recombinant plasmid can be highly expressed in E.coli BL21(DE3) in a soluble form and the recombinant protein can react with DENV (Type Ⅰ-Ⅳ) McAb;2. A specific30and60kDa binding molecule in BHK-21cell was found by VOPBA,and the further research is go on;3. The35kDa binging molecules are specific in the larvae, pupa and adult mosquito of the Aedes albopictus.