Prokaryotic Expression of Gene E Partial Nucleotide Sequence of Dengue Virus Type 2 Strain NGC
［Key words］ dengue virus; genes, E; gene order; gene expression
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 SDS-PAGE and Western-Blot and purified. The cytotoxicity (indicated as TCID50) of the purified protein on C6/36 was detected. Results： Prokaryotic expression recombinant vector, pET28a(+)-Eb, was successfully constructed. SDS-PAGE assay showed that the gene E partial sequence could be highly expressed in BL21,and the yields were 29% of total bacterial proteins. The expressed protein had a relative molecular weight of 23KDa, and Western-Blot indicated that the expression products could specifically react with monoantibody against dengue virus type 2. Cytotoxic experiments in vitro suggested that the protein had relatively cytotoxicity to C6/36, and the TCID50 was 10~4.88/0.1ml. Conclusion： pET28a(+)-Eb containing gene E partial sequence can be highly expressed in BL21. The antigenicity of the produced proteins provide a potential source of reagent for dengue virus diagnosis；The expressed proteins have some cytotoxicity to C6/36.
