Screening and identification of dengue virus-specific antigens and the establishment of ELISA detection method for dengue antibody / 中华微生物学和免疫学杂志

ABSTRACT

Objective To screen and identify the dengue virus-specific antigens,then establish the ELISA detection method for dengue virus antibody.Methods Using bioinformatic software DNAStar and ANTHEPROT to analyze the hydrophilicity,flexibility,surface probability and antigenicity of dengue virus type 1-4,Japanese encephalitis virus and Yellow fever virus M,E and NSI protein amino acid sequence and also consider the influence of secondary structure.Then in accordance with epitopes localion and amino acid sequence similarity,forecast the share and specific epitopes.Reference the sequence information of different dengue virus strains in GenBank to analyze the epitopes conservative.Based on the results of bioinformatic analysis,5 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-C2x or pET32a.Then the vectors was transferred into E.coli Rosetta( DE3 ).lsopropyl-β-D-thiogalactoside(IPTG) was used to induce the expression of gene segments.SDS-PAGE were used to identify the expression proteins,and the antigenicity were tested using Western blot.Using the antigen selected by Western blot,ELISA method for dengue virus antibody detection was established.Results Eighty shared epitopes and 25 specific epitopes were forecasted,and 5 antigenic fragments encloude analyzed epitopes from dengue virus type 2 and 3 were expressed in E.coli successfully.One dengue virus type 1-4 shared antigens (Den-Ag5),one dengue virus type 2 and 4 shared antigens( DenAg3),one dengue virus type 1-3 shared antigens(Den-Ag2) and two dengue virus type 1,2 and 4 shared antigens( Den-Ag1,Den-Ag4)were conformed using Western blot.Using antigens Den-Ag5,Den-Ag1 and DenAg2,the ELISA method for dengue virus antibody detection were established.Conclusion Based on the bioinformatic analysis and Western blot verification,5 dengue virus specific antigen were conformed,and the ELISA detection method for dengue virus antibody were established.