Studying On The Secreted Expression Of Dengue Virus PrM/E Protein In Eukaryotic Cells

Dengue viruses(DV) belong to the flavivirus genus of the Flaviviridae family. There are four antigenically distinct but related serotypes of dengue virus, types1,2,3and4. Infection with any one of the four viruses can produce a broad spectrum of clinical illness, including classic dengue fever, and the lethal dengue hemorrhagic fever and shock syndrome. As a result of the lack of effective cross-protection between the serotypes of dengue virus and the existence of antibody dependent enhancement of infection, there is no safe and effective vaccine approved currently.Dengue virus contains a positive-strand RNA genome of about11kb in size, which is translated into a single polyprotein encoding three structural proteins (capsid (C),envelope (E) and precursor membrane (PrM) proteins). The envelope (E) glycoprotein and the matrix (M) protein are embedded in the lipid bilayer. The E protein is the principal envelope glycoprotein and the major structural protein exposed on the surface of mature dengue virion. It is composed of495amino acids with a molecular weight of60kDa. It contains many B and T cell epitopes. The E glycoprotein is the primary antigen that induces protective immunity. The prM is a precursor to the M protein. It is well established that dengue virus prM and M actively induce a protective immune response. It also contributes to the E protein correct folding and stability. A common feature in the replication of flaviviruses is the assembly of structural proteins, PrM and E, into subviral particles. Co-expression of PrM and E proteins of dengue virus can generate recombinant subviral particles. Thus, the PrM and E proteins becomes the important target for the vaccine research of dengue virus.In recent years, the largest epidemic of dengue virus type1in Guangzhou is the dengue virus type1strain GZ01/95. After analyzing the evolution of the E proteins CDS, it has been found that the strain is popular in Southeast Asia, Oceania, the Indian Ocean region and South Africa. It is the important representation of dengue virue type1. In this study, we selected the dengue virus type1strain GZ01/95as candidate, and expressed the PrM and E proteins in CHO cell and insect cell. The PrM and E proteins were modified by adding the signal peptide preceding the prM gene or replaying the carboxyl-terminal20%of DEN-1E protein. The expression and secrection of PrM and E proteins were identified by indirect immuno-fluorescence assay(IFA) as well as SDS-PAGE and Western blot. It will lay a foundation for the futher research about the immunogenic function and properties.1.Secreted Expression of Dengue virus type1prM/E protein in mammalian cellsThe full-length prM/E gene of dengue virus type1strain GZ01/95was obtained by RT-PCR.. Through gene synthesis and overlapping PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal20%of DEN-1E was replaced with the corresponding JE sequence in the meanwhile. Then the three of the constructions were cloned into the pcDNA5/FRT. They were transfected into293T cells by lipofectamine respectively.The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot. In the cytoplasm of293T cells transfected with all the recombinant plasmids DNA, the expressed products of dengue virus type1were confirmed by IFA. The secreted expression products of dengue virus type1were confirmed by Western blot existing in the cell supernatants transfected with the modified recombinant plasmids DNA. Therefore, the prM/E protein of dengue virus type1were expressed in293T cells transfected with all the three recombinant plasmids DNA.The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal20%of E with the corresponding JE sequence.2. Secreted Expression of Dengue virus type1prM/E protein in insect cellsThe full-length C42prM/E gene of dengue virus type1strain GZ01/95was obtained by RT-PCR. We obtained the D1JSSprME gene and prME gene by PCR. Then the constructions were cloned into the pAcUW51-M, pAcGP67-B,pAcSecG2T to get The recombinant clones D1C42prME-W51,D1JSSprME-W51, D1prME-W51, D1prME-67B, D1prME-G2T. They were co-transfected into sf9cells with linearized baculovirus DNA. The high titer recombinant viruses were obtained by plaque purified. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot. In the cytoplasm of sf9cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type1were confirmed by IFA as well as SDS-PAGE and Western blot. The secreted expression products for gene of dengue virus type1specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with D1JSSprME-W51, but the protein band is weak. Therefore, the prM/E protein of dengue virus type1were expressed in sf9cells transfected with all the five recombinant plasmids DNA. The prM/E protein was not secreted or secreted lowly in baculovirus/insect system.In summary, the prM/E protein has been successfully secreted expression in mammalian systems after modifying the prME gene. This study provides a theoretical basis for the prME protein secreted expression research of dengue virus other serotypes on how to choose the optimization condition and secretory expression system. It creates the condition for studying the immunological function and features of prM/E protein. It also lays a foundation for the tetravalent dengue virus-like particles vaccine research.