Construction and Identification of Human Proinsulin DNA Vaccine

To construct the DNA vaccine of human Proinsulin, which is recognized as the crucial and specific autoantigen oftype 1 diabetes, and confirm the transient expression product in eukaryotic cells. Human Proinsulin complete cDNA was ampli-fied with half net-PCR method from human pancreas cDNA library, and cloned into PGEM-T vector. Then the target gene wassubcloned into eukaryotic expression vector pcDNA3. 1 (+) to generate DNA vaccine-recombinant plasmid pcDNA3. 1 (+)/Proinsulin. Afer the DNA vaccine being confirmed to contain the correct target gene sequence with fluorescence sequencing,COS-7 was transfected with it in vitro by DOTAP liposome, and the expression of protein was detected with Western blot. Thesequencing results for cloned human Proinsulin gene showed that the insertion of cDNA is correct and the sequence is similar tothat of human Proinsulin cDNA in Cenbank, except for the site 63 amino acid (C peptide region)where there is a mutation[ CAG (Glu ) → CGG (Arg) ]. Western blot confirmed that the products of DNA vaccine pcDNA3. 1 (+) / Proinsulin was efficient -ly expressed in eukaryotic cells in vitro. It is conclused that eukaryotic cells transfected with human Proinsulin DNA vaccineobtained by the method of gene engineering can express recomhinant Proinsulin antigen efficiently. An experimental basis forimmune intervention of Type 1 diabetes is provided.