| Summary: | 碩士 === 國防醫學院 === 微生物及免疫學研究所 === 98 === Dengue virus nonstructural protein 1 (DNS1) is expressed on the cell-surface of and secreted from the infected cells. The DNS1 protein is immunogenic and is functionally associated with viral replication. Here we described the identification of a previously unknown tDNS1 protein deficient in N-terminal 1 ~ 88 amino acid residues of DNS1 expressed from internal DNS1 gene sequence in bacteria. Plasmid harboring full-length DNS1 gene under the control of the T7 promoter was found producing two variable proteins namely DNS1 (42 kDa) and tDNS1 (32 kDa) in E.coli BL21 recognized by anti-DNS1 mAb respectively, compared to tDNS1 alone in E. coli DH5α cells. The results suggested that tDNS1 expression differed independently on T7 promoter from DNS1. Consistently, only tDNS1 protein was observed when DNS1 gene was cloned downstream of a promoter-less plasmid. Further RT-PCR analysis revealed tDNS1 but not DNS1 mRNA transcribed, indicating tDNS1 expression is regulated at transcriptional rather than translational level. The putative promoter region of tDNS1, DNS1IP, located upstream of tDNS1 corresponding 5’end from 1 to 264 bp of DNS1 gene significantly promoted the expression when transcriptionally fused to a melanin reporter, suggesting DNS1IP functional as a promoter. Based on the structural feature in tDNS1 gene and the observation of tDNS1 protein exclusively in bacteria but not in infected cells, it is likely that tDNS1 may be of importance functionally a role on dengue pathogenesis.
Secondly, the immune response of recombinant Salmonella producing tDNS1 protein (Sal-tDNS1) following oral immunization in BALB/c mice was investigated. Recombinant Sal-tDNS1 was constructed based on a “balanced lethal host-vector system” in which Salmonella asd- mutant harboring a complemented asd+ plasmid to produce the tDNS1 antigen. The results showed that Sal-tDNS1 appeared stable in vivo in lymphoid organs, and was able to elicit a detectable sera DNS1-specific antibody titer following oral immunization. The sera cytokine levels of IFN-γ and IL-2 were significantly induced. Noticeably adverse effects including body weight loss and hemorrhagic signs were observed in mice group following given Sal-tDNS1. All the immunized mice died after challenged with virulent DEN2V via intracerebral injection, similar to the control group mice. These results demonstrated that oral immunization with Sal-tDNS1 vaccine did not tend to provide effectively protective immune responses against Dengue virus infection.
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