NGC strain of dengue virus type 2 partial sequence of E gene expression in the original nuclear protein.doc

 PAGE \* MERGEFORMAT 13 NGC strain of dengue virus type 2 partial sequence of E gene expression in the original nuclear protein [Abstract] Objective: To construct NGC strain of dengue 2 virus E gene region of 1 ~ 476 bp of the prokaryotic expression vector for prokaryotic expression. Methods: NGC strain of dengue virus type 2 partial sequence of E gene region was cloned into the prokaryotic expression vector pET28a (), named as pET28a ()-En; by restriction enzyme digestion, PCR and sequencing identified and converted into BL21 (DE3) strains, with IPTG inducible expression by SDS, Western blot identification of expressed protein; expression protein purification, and titration of the C6/36 cells TCID50. Results: (1) successfully constructed pET28a ()-En prokaryotic expression recombinant plasmid, SDSanalysis showed that, E gene region of partial sequence was highly expressed, the relative molecular weight of about 23 kD, the expression of the 29 total bacterial protein %; Western blot shows that the target protein with dengue virus type 2 mouse monoclonal antibody binding; (2) with Ni column affinity chromatography purification of prokaryotic expression of proteins, purity 90%; (3) DEN-2 NGC strain E gene sequences of prokaryotic expression of proteins on C6/36 cells TCID50 of 10-4.88/0.1 ml. Conclusion: pET28a ()-En may BL21 (DE3) strains highly expressed, DEN-2 NGC strain of E gene sequences of prokaryotic expression of a certain protein on the C6/36 cell cytotoxicity. [Keywords:] dengue virus; gene, E; gene sequence; gene expression [Abstract] Objective: To construct expression vector of gene E partial sequence of dengue virus type 2 strain NGC for prokaryotic expression. Methods: Gene E partial sequence of dengue virus type 2 strain NGC was amplified by RT-PCR, inserted into prokaryotic vector pET28a (), and transformed E. coli BL21 cells. The gene partial sequence was expressed under the induction of IPTG. The expressed products was identified by SDSand W