The gene fragment coding for amino acids 281 to 395 of the E protein of DENV-2 (New Guinea C strain) was amplified by PCR, comprising Domain III (amino acids 295 to 395) of the E protein. The fragment was cloned into pMD18-T vector and subcloned to expression vector pET-28a and pMAL-c2X. The recombinant plasmid pET-28a-D2EIII was transformed into E.coli BL21(DE3) and the pMAL-c2X-D2EIII was transformed into E.coli TB1. The induced recombinant proteins were purified by His-tag and MBP-tag affinity chromatography, respectively. The purified protein His-D2EIII was used to immunize rabbit three times at two-week intervals, the immunized rabbit produced high titer anti-His-D2EIII polyclonal antibody. The result of western blot indicated that the expressed fusion protein could react with the polyclonal antibody against Domain III of E protein.
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