To construct a vector for DNA vaccine and protein expression by using chromosome-plasmid balanced lethal system which was based on the thyA+ gene/deltathyA Escherichia coli. The thyA genes from Escherichia coli and Vibrio cholerae were amplified by polymerase chain reaction and cloned into pCDNA3 by replacing ampilicilin resistant gene. Multiple cloning sites, the prokaryotic replicon, CMV promoter and the boving growth hormone polyA signal were also included in the vectors. Two new non-antibiotic recombinant plasmids renamed as pcDNATE and pcDNATC which had the nutritional marker as thyA were constructed and were transformed respectively into the deltathyA derivative of E. coli K-12 strain DY330-TI, then two chromosome-plasmid balanced systems for E. coli based on the thyA were developed. To test the efficiency and stability of the newly constructed chromosome-plasmid balanced lethal system, a reporter gene--red fluorescent protein (DsRed2) gene was cloned into pcDNATE, pcDNATC and expressed as fusion to the c-myc. The two recombinant plasmids, pcDNATE-DsRed2, pcDNATC-DsRed2, were transfected into HEK293 solely and DsRed2-myc was detected by the fluorescence microscope assay and western-blot. Meanwhile, the loss of recombinant plasmids were not seen in cultures without thymidine after 20 generations. The chromosomal-plasmid balanced lethal system is proved to be an effective vector system for the expression of target genes and share the same stability with the antibiotic-resistant plasmid vector system. It holds great potential in gene vaccine vector because obviating the weakpoints of the drug resistance marker during application.