Construction and identification of co-expression DNA vaccine containing GAD65 fragment gene and IL-4 gene

Objective To construct co-expression DNA vaccine containing GAD65 fragment gene and IL-4 gene, and identify the transient expression products in COS-7 cells. Methods The GAD190-315 and GAD490-570 cDNA and hIL-2 signal peptide cDNA were linked together through overlap PCR,respectively.Then,the two fusion gene and IL-4 gene were cloned into eukaryotic expression vector pBudCE4.1 in order.Recombinants were identified by restriction enzymes digestion and nucleotide sequencing of target gene.The expression of fusion protein was detected in COS-7 cells transfected with recombinants using Western blot and the expression of IL-4 was determined by ELISA. Results The size of the fusion gene and IL-4 gene amplified were in accordance with that we expected,Nucleotide sequence of the cloned gene was the same as the report sequence, and their open reading fragment was correct. Western blot or ELISA showed that products of these DNA vaccines were expressed successfully in COS-7 cells. Conclusions The co-expression DNA vaccines containing GAD65 fragment gene and IL-4 gene were successfully constructed, which is a foundation for further development of DNA vaccine against type 1 diabetes.