Cloning and expressions of NS1 gene encoding non-structure 1 protein of Dengue virus serotype 2 and preparing its antibodies

Objective To clone and express the ns1 gene encoding the non-structural 1 protein of Dengue virus serotype 2(DV2),then to prepare monoclonal antibodies against DV2 non-structual 1 protein.Methods The NS1 cDNA of DV2 was amplified with R T-PCR from the C6/36 cells infected with DV2 and inserted into pMD18-T vector.Then the NS1 gene was inserted to the multi-cloning sites of plasmid pQ E30 and expressed with induction of IPT G.After purification under denatured condition and renaturated,the immunogenicity of the recombinant protein was identified with anti-His monoclonal antibody and anti-NS1 monoclonal antibody with four sera types by Western blot assay.Balb/c mice were immunized by recombinant DV2-NS1 protein or inactive DV2 virus.Spleenocytes of immunized mice were fused with myeloma cells NS1 to produce hybridoma cell line with anti-DV2-NS1 protein antibodies.Enzyme-linked immunosorbent assay (ELISA),immnofluorescence assay(IFA)to identify specificity of antibodies.Results The recombinant NS1 protein was highly expressed in E.coli M15 and the purified NS1 protein could be recognized by anti-His monoclonal antibody and anti-NS1 monoclonal antibody with four sera types.Ten hybridoma cell lines with anti-DV2-NS1 protein antibodies were obtained.Three cell lines with anti-DV2-NS1 protein antibodies were specially to DV2-NS1 protein,and other seven cell lines crossed with DV other sera types.Conclusion The expressed DV2-NS1 renaturational protein showed good immunogenicity and ten hybridoma cell lines with anti-DV2-NS1 protein antibodies were successfully obtained,which may make foundations to study the function and serology detection of the NS1 protein.