CONSTRUCTION AND IDENTIFICATION OF THERAPEUTIC DOUBLE-PLASMID HBV DNA VACCINE / 解放军医学杂志

ABSTRACT

To develop an effective therapeutic HBV DNA vaccine, two eukaryotic expressing plasmids, namely pcDNAS 2 ?S and pcDNAIIF , which respectively encoding HBV middle envelope protein preS 2 ?S and human IL 2/IFN ? fusion protein, were constructed by DNA recombinant technique. After identification by restriction analysis and DNA sequencing, the constructed recombinant plasmids were transfected into COS 7 cells in vitro with transfection reagent Lipofectamine. The expressed product in supernatant was quantified by ELISA , and the biologic activities of IL 2 and IFN ? were assayed by CTLL 2 cell line proliferation and cytopathogenic inhibition, respectively. The results showed that secretive expression of preS 2 ?S and hIL 2/hIFN ? fusion protein peaked at 48h after transfection, and the expression levels were HBsAg(P/N)=7 63, IL 2=10 35ng/ml, and IFN ?=7 90ng/ml. The IL 2 and IFN ? activities in the culture supernatant of COS 7 cells transfected with pcDNAIIF were 998U/ml and 249U/ml, respectively. These results suggested that the recombinant plasmids pcDNAS 2 ?S and pcDNAIIF were correctly constructed and the transfected cells could express secretive active protein.