The Expression Of Dengue Virus Type 1 To 4 Envelope Domain Ⅲ And Its Application In Serological Diagnosis And Protective Immunity

In recent years, with the increase of dengue infection cases, the prevention of dengue transmission has become a common concern in the world. In this study, four recombinat protein antigens based on dengue virus type 1 to 4 envelope domainâ…¢were expressed in Escherichia coli and applicated in serological diagnosis. Meanwhile, we have constructed three dengue vaccines for protective immunity assay.1. Expression of dengue virus type 1 to 4 envelope domainâ…¢and the application of recombinant proteins in serological diagnosisDengue virus type 1 to 4 envelope domainâ…¢s were expressed in E.coli. The type 1 to 4 recombinant proteins were purified by electroelution and used to develop an indirect ELISA to detect anti-dengue type 1 to 4 IgG antibodies respectively. Sensitivities of 100% were obtained, by compared with IFA. A captured ELISA showed that the DEN1 protein not only recognized all IgM positive samples identified by IFA but also identified 2 samples missed by the latter test. However, a sensitivity of 82.6% was obtained by a sandwiched ELISA using DEN1 protein as the captured antigen to detect anti-dengue type 1 antibodies. The specificities of three tests were 100% by compared with IFA. We also developed an immunochromatographic test that incorporated 4 recombinant antigens to test serum from patients infected with dengue virus. The results were in excellent agreetment with those obtained by using a commercially available diagnostic kit, Dengue Duo rapid strip test from PanBio (P>0.05).2. Construction of dengue vaccines and analysis of their protective immunity in mice2.1 Construction of a sub-unit dengue vaccine and analysis of its immunogenicity in mice EDâ…¢s of dengue virus type 1 and 2 as well as type 3 and 4 were spliced by a flexible peptide linker (Gly-Gly-Ser-Gly-Ser)3, and cloned into prokaryotic expression plasmid pET30a (+), then expressed in E.coli. The recombinant protein was purified by high-performance liquid chromatography (HPLC) and used to immunize BALB/c mice. The neutralizing antibodies were tested by neutralizing assay, as well as in newborn mice challenged intracranially with dengue virus type 1 to 4. The neutralizing assay showed neutralizing antibody titers of 1:34.9, 1:45.3, 1:24.7 and 1:38.4 for DEN-1 to 4 respectively. 100% of newborn mice challenged with DEN-1 or 2 in combination with sera from mice immunized with the recombinant proteins were protected, whereas 83% protection was obtained when challenged with DEN-3 or 4.2.2 Construction of chimeric eukaryotic plasmids carrying bivalent EDâ…¢s fusion gene and observation of its immunity in miceWe constructed two chimeric enkaryotic plasmids encoding EDâ…¢s fusion gene. The recombinant protein expression was tested in BHK-21 cells by indirect immunofluorescent assay and Western blot, which suggested that the bivalent proteins were expressed correctly in mammalian cells. DNA plasmids were used to immunize BALB/c by intramuscular route. The results of neutralizing assy showed neutralizing antibody titers of 1:24.7, 1:26.9, 1:13.4 and 1:16.0 for DEN-1 to 4 respectively.2.3 Analysis of antibody responses in mice immunized combinantions of DNA and recombinant proteinsMice were immunized with DNA plasmids and/or EDâ…¢s fusion proteins to observe their immunogenicity and compare their ability to induce neutralizing antibodies. The results showed that the genometric mean neutralization titers was significantly higher for mice immunized with combination than that for mice immunized with proteins or DNA. Meanwhile the neutralization titers from mice immunized with recmbinant proteins was also significantly higher than that of DNA-immunized mice.2.4 Construction and identification of recombinant adenovirus vectors encoding EDâ…¢s fusion genesThe recombinant replication-defective adenovirus vectors encoding EDâ…¢s fusion genes were constructed and transfected into 293A cells, which generated recombinant adenoviruses with a titer of 109 pfu/ml. The ability of the recombinant adenoviruses to express protein was examined in cultured mammalian cells. The results of indirect immunofluorescent assay and Western blot indicated that the chimeric adenoviruses could express the recombinant protein in BHK-21 cells.