Construction,identification and pharmikinetic research on a therapeutic double plasmid of anti-HBV DNA vaccine / 中国免疫学杂志

ABSTRACT

Objective:To develop an effective therapeutic double HBV DNA vaccine with two eukaryotic expressing motif,namely pS2.S and pIIF,a fusion ORF of hIL-2/hIFN-?.Methods:Two genes were amplified,which separately encoded HBV preS2.S middle envelope protein as a vaccine and human IL-2/IFN-? fusion protein as an adjuvant by PCR technique from plasmids pcDNA3.1/preS2.S and pcDNA3.1/hIL-2/IFN-?.Then the genes subcloned into eukaryotic vector pVAX1.The new plasmid was analysed by restriction endonuclease and DNA sequencing.The constructed plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000.The expressing products in supernatants were quantified by ELISA.Space structure of fusion hIL-2/IFN-? expressed by the adjuvant plasmid was simulated by CE software.The double plasmid association were injected into BALB/c mice by in situ electroporation method,and specific humoral and cellular immune responses were examined.Results:The gene segments and inserting direction of pVAX1-S2.S(pS2.S)and pVAX1-IL-2IFN-?(pIIF)were both correct by genetic analysises.At 48 h after transfection,HBsAg and the cytokines of IL-2 and IFN-? were respectively 45.1 ng/ml,10.03 ng/ml and 11.5 ng/ml by ELISA.The activity domains of the two cycokines in fusion protein were entirely exposed on surface by CE software.Experiments were pefrormed in mice by injection of plasmid pS2.S.Protective antibodies of anti-HBs were produced in terms of dose-dependment pattern for the efficacy.Co-injection of plasmid pS2.S together with adjuvant pIIF resulted in more evident immune responses and dose of the plasmid pS2.S needed was diminished.There was strongly specific humoral and cell immunity by pS2.S and pIIF co-injection with EP technique.Conclusion:The double plasmids pS2.S and pIIF of anti-HBV are constructed succeedly and induce strongly specific immune activity in vitro and vivo.