Immunogenicity of N-terminal 158 amino acid motif of E-protein of a newly purified mutant dengue virus from a Chinese patient / 中国免疫学杂志

ABSTRACT

Objective:The envelope protein Domain I(DI) of type II mutated dengue virus from a DHF patient (B-E) was expressed,refolded and purified.Its immunogenicity was also identified.Methods:The 1-476 bp of B-E was cloned into pET28a(+),and it was transformed into E.coli after digestion and sequencing.The inclusion body was denatured and refolded after inducing with IPTG.Polyclone antibody was obtained by immunizing C57BL/6 and BALB/c mice with purified B-E,and identified with Western blot and ELISA.Results:The cDNA of B-E gene was inserted into pET28a(+) vector and transformed into Rosetta(DE3).The protein of B-E was mainly in inclusion body and the molecular weight was 20 kD as predicted.Western blot was used to identify the recommend protein with anti-his antibody.The soluble recombination protein was purified after denaturation and refolding.The polyclonal antibody was obtained by immunizing BALB/c and C57BL/6 mice and could be used in Western blot and ELISA assay.The titer of polyclonal antibody from C57BL/6 was 112 800 through ELISA assay,and titer of polyclonal antibody from BALB/c was 1500 through Western blot.Conclusion:All the results suggests that B-E is immunogenic in BALB/c and C57BL/6 mice.