Preparation of an Ag85A-CD226 DNA vaccine and evaluation of its immunogenicity in a mouse mod-el

Abstract

Objective To construct a eukaryotic expression plasmid of pcDNA 3.1-Ag85A-CD226, and to use it as DNA vaccine then further evaluate its immunogenicity through oral administration in a mouse model.Methods The CD226-PCR2.1-ToPo plasmid was used as the template to clone CD 226 gene by PCR.The CD226 gene was then inserted into pcDNA 3.1-Ag85A plasmid to construct the recombinant plas-mid of pcDNA3.1-Ag85A-CD226.After identified by restriction enzyme analysis and sequencing , the re-combinant plasmid was transfected into HEK 293 cells by using lipofection .The expression of Ag85A-CD226 gene in HEK293 cells was detected by RT-PCR, Western blot and indirect immunofluorescence assay .The purified recombinant plasmid was used to prepare the Ag 85A-CD226 DNA vaccine by liposomal encapsula-tion.The vaccine was administered intragastrically to mice .The activities of NK cells , the cytokine levels in the supernatants of spleen cell cultures and the mRNA level of cytokines in the intestines were evaluated to analyze the immunogenicity of Ag85A-CD226 DNA vaccine.Results The Ag85A-CD226 DNA vaccine was prepared successfully .The expression of Ag85A-CD226 fusion protein was detected in HEK293 cells.The activities of NK cells from mice vaccinated with Ag 85A-CD226 DNA vaccine were higher than those from other control groups （P〈0.01）.The level of TNF-α, IFN-γand IL-2 in the supernatants of spleen cell cul-tures and in the intestines were significantly up-regulated in comparison with other control groups （ P 〈0.01）.The level of IL-4 in the supernatants of spleen cell cultures was down-regulated in the experimental group （P〈0.01）, but the level of IL-4 in intestines showed no significant difference among the five groups （P〉0.05）.Conclusion The Ag85A-CD226 DNA vaccine could significantly enhance Th1 type immune esponses systemically and in the intestine as in comparison with those vaccinated with single dose of Ag 85A DNA vaccine or CD226 DNA vaccine. Key words: Ag85A; CD226; DNA vaccine; Immune function