Résumé
Objective:The immunogenicity of DNA vaccine immunogenicity can be improved by fusing antigenic genes to IgG Fc fragment.It is critical for enhancing immunogenicity of DNA vaccine to construct the secreting eukaryotic expressing vector.The purpose of this study is to construct a secreting eukaryotic expressing vector ligated with IgG Fc gene by amplifying human IgG Fc fragment and fusing the fragment with human CD5 signal peptide sequence for highly efficient expression.Methods:Human lymphocytes were isolated from the tonsil obtained by removal surgery.The total RNA of lymphocytes was extracted using Trizol reagent.Human IgG Fc cDNA was amplified by RT-PCR from lymphocytes and then inserted into pMD-18T vector.The Fc encoding sequence fused with CD5 signal peptide(sp) sequence was constructed by cross PCR using Fc fragment and CD5sp sequence and cloned in pcDNA-CD5 plasmid as the template.CD5sp-Fc fragment was inserted into pcDNA3.1 expressing vector to construct the pcDNA-CD5sp-Fc plasmid.The plasmids were transfected into HEK293T cells mediated by calcium phosphate.Western blot was used to detect expressed Fc protein in cultural supermatant of the cells.Results:The amplified sequence of human IgG Fc was consistent with that previously published.The secretory expression of Fc in HEK293T cells was achieved and the expressing level reached 50 ?g/106 cells at 48 h culture after transfection.Conclusion:The human IgG Fc gene is amplified by RT-PCR methods and the secretory expression of Fc gene mediated by CD5 signal peptide in 293T cells is achieved.The results provide a experimental basis for further construction of antigen genes fused to IgG Fc fragment and investigation on DNA vaccines and Fc as a biological adjuvant.