Construction And Functional Identification Of Degue Virus Replicon Vector

Dengue virus belongs to the Flaviviridae Flavivirus virus. It’s a kind of the enveloped RNA virus,trans missed by mosquitoes. In China, almost every year hundreds of cases that infected with dengue virus have been reported. The virus can infect humans and animals, with high prevalence and low cure rate after the infection, which is easy to leave some serious sequel. It is a great threat to human health.And dengue disease clinically also lack of effective antiviral drugs, so the class in the early diagnosis of virus infection and the new type of vaccine research is particularly important to effectively control the spread of dengue type yellow virus outbreak.Existing research results show that on the basis of dengue and yellow virus genome RNA replicon vector technology has been widely used in various applications such as new type of vaccine research and development, the antiviral drug screening research field, in viral replication and translation of the molecular mechanism and so on basic research also has been widely used. The genome of dengue virus is about11kb, enveloped single-stranded positive strand RNA virus. It is constructed by the5’and3’untranslated region(UTR) and the intermediate single open reading frame(ORF), and the latter codes three structural proteins(C,prM/M,E) and seven non-structural proteins(NSl,NS2A,NS2B,NS3,NS4A,NS4B and NS5).The structural proteins play an important role during the assembly of the virus,and the nonstructural proteins and5’and3’UTR mainly involve in the initiation and regulation of viral RNA replication and translation. RNA replicon,based on the corresponding full-length infectious clone technology,retains important cis-acting elements (including the5’and3’UTR, all of the viral non-structural protein genes and parts of necessary viral structural protein genes) that is necessary in the viral replication and translation.The majority of its structural gene is deleted or substituted by the exogenous gene, constructing the viral replicon. In this study dengue virus type4infectious cDNA cloning p4remove most structure gene prM-E of the virus, construting the dengue virus replicon vector p4-△prME. To test and verify the packaging function of replicon vector, the red protein report gene mCherry cloning to replicon vector p4-△prME construting engineering vector p4-△prME-mCherry, at the same time construting missing RNA dependent RNA polymerase (RDRP)core sequence GDD defect type replicon vector p4-△prME-mCherry-△GDD. Vitro transcription and cell transfection experiment results show that p4-△prME-mCherry transcriptional RNA transfect Vero cell sustainable present specific red fluorescence, and24h～96h post transfection can form stable signal peak. By contrast, the defect of GDD replicon vector missing RDRP core sequence show no specific red fluorescence post transfection cell. The research results show that we are building replicon vector p4-△prME could successfully express red fluorescent protein reporter gene, which lay a solid foundation on the target gene expression and preparation of recombinant virus particles.The promoter of RNA replicon vector is prokaryotic promoter T7,SP6,etc.In the process of practical application we need vitro transcription operations, but in vitro transcription kit has the disadvantages such as complicated operation and expensive price. By contrast, plasmid DNA replicon vector can start transcription of vector within the host cell by eukaryotic promoters, such as CMV, SV40, etc. Therefore, plasmid DNA replicon vector need no vitro transcription like RNA replicon vector,and replicon vector ectors to cells, efficiently and stable expressing exogenous genes.In view of this, this study is on the basis of dengue virus type4infectious clone p4plasmid, insert the modified virus replication and translation required cis element and part NS5key sequence into CMV promoter downstream of the eukaryotic expression vector pcDNA3.1(+), constructing the DNA micro replicon vector pcDEN-△prME. To test and verify the packaging function of micro replicon vector, the qualitative EGFP reportor gene and quantitative sea renal luciferase gene is respectively cloned to pcDEN-△prME, constrcuting engineering vector pcDEN-△prME-EGFP and pcDEN-△prME-GLUC. Transfection experiment results show that the micro replicon vector could successfully express EGFP reportor gene and sea renal luciferase reportor gene.In this study, based on dengue virus type4infectious cDNA cloning p4, respectively construt the RNA replicon vector p4-△prME and DNA micro replicon vector pcDEN-△prME, both can express exogenous genes successfully. Especially DNA micro replicon vector on reservation virus necessary role cis element and part NS5key sequence missing most of the structure of genes at the same time, makes the vector in simplifying experiments and greatly improve the capacity of the viral vector. The construction of the dengue virus replicon vector provides a powerful tool for further research on Flavivirus virus antiviral drug screening and new vaccine development, and provides a new technical platform for molecular regulation mechanism of viral replication and translation.