Construction and identification of eukaryotic expression vector containing GAD65 fragment and IL-10 gene

Objective To modify an established full-length GAD65 DNA vaccine for prevention of type 1 diabetes, and to construct and identify an eukaryotic expression vectors containing GAD65 fragment and IL-10 gene. Methods The cDNAs of GAD190-315 and GAD490-570 fragments, which were amplified from GAD65 plasmid, were linked with hIL-2 signal peptide cDNA respectively through overlapping PCR. Then, the two fusion genes, SGAD190-315 and SGAD490-570, and IL-10 gene were cloned into an eukaryotic expression vector pBudCE4.1 to establish two recombinant eukaryotic expression vectors, pBud-SGAD190-315/IL-10 and pBud-SGAD490-570/IL-10. After being identified by DNA sequencing, the two recombinant eukaryotic expression vectors were transfected into COS-7 cells respectively under mediation by liposome. Then the expression of the fusion proteins in the COS-7 cells transfected with recombinants were detected using Western blot and the expression of IL-10 was determined by ELISA. Results Nucleotide sequence of the gene cloned into expression vectors were in accordance with the reported sequence, and the products of these recombinant eukaryotic expression vectors were expressed in COS-7 cells as detected by Western blot or ELISA. Conclusions The two eukaryotic expression vectors containing GAD65 fragment and IL-10 gene can be successfully constructed, which is a foundation for further development of gene vaccine against type 1 diabetes.