Cloning, Expression And Subcellular Localization Of RdRp Gene Of Duck Hepatitis Virus TypeⅠ

[Objective] To prepare the specific polyclonal antibody against RNA-dependent RNA polymerase(RdRp) of the duck hepatitis virus type1(DHV-1), the coding gene of RdRp was cloned and insertedinto the prokaryotic expression vector pET-32a (+). Besides, the recombinant eukaryotic expressionplasmid pEGFP-C3was also constructed for exploring the expression and the subcellularlocalization of RdRp in eukaryotic cells.[Methods](1) A pair of specific primer, according to the complete genome of duck hepatitis virus1(DHV-1) ZJ-Ⅴ strain, was designed to amplify RdRp gene. The sequence of RdRp was analyzed withsome reference sequences of DHV-1published in GenBank using DNAstar soft. Then RdRp gene wasinserted into pET-32a (+) vector, and the recombinant plasmid pET-RdRp was transfected into BL21(DE3) cells. The expression of RdRp was analyzed with SDS-PAGE and Western blot assay.(2) The recombinant plasmid pEGFP-RdRp, containing RdRp fused with the green fluorescentprotein (GFP), was constructed and transfected into MDCK cells. The expression of RdRp wasdetected with fluorescence microscope and Western blot. The subcellular localization of RdRp inMDCK cells was determined with focusing microscope.[Results](1)The length of RdRp gene is1359bp, which encodes453amino acids. The amino acididentity of the predicted ZJ-Ⅴ RdRp protein when compared to R85952, was97.1%, indicatingthat RdRp is high conserved among DHV-1.(2)The RdRp fusion protein was successfullyexpressed in E.coil cells; the results of SDS-PAGE showed that the molecular weight of RdRp wasabout65ku. Western blot assay disclosed that the protein could react to the specific positive serumagainst DHV, indicating that the expression product had well antigenecity. The specific polyclonalantibody against RdRp was obtained, when rabbit was immunized with RdRp protein.(3)Therecombinant plasmid pEGFP-RdRp was constructed successfully, after transfected into MDCK cells;the expression of RdRp was detected with western blot. The localization of RdRp in cells was alsodetermined with focusing microscope, and the result indicated that RdRp was mainly distributed inthe nucleus of MDCK cells, suggesting that RdRp may be translated in cytoplasm and transferredinto nucleus to play its replicase function.[Conclusion] The RdRp gene of DHV-1was successfully cloned and expressed both in prokaryoticand eukaryotic cells, and expressed efficiently in eukaryotic cells, and the subcellular localization ofRdRp in eukaryotic cells was also first observed. These results are helpful for further study on thethe structure and function of RdRp and the replication mechanism of DHV-1.