Construction Of The Chromosome-Plasmid Balanced-Lethal System Based On E.coli Thymidylate Synthase Gene

Chromsome-plasmid balanced-lethal systems can provide safer slection markers for the expression vectors of gene therapy and gene vaccines. To construct a thymidylate synthetase deficient (ThyA-) E.coli, we inoculated wild type DH5αE. coli on agar plates containing thymidine and trimethoprim (TMP) and 21 ThyA- strains were obtained after selection for 20 passages. Sequence analysis showed that the mutant strains 4, 7 and 13 had 6-bp deletions which resulted in GLY25 and Thr26 deletions, while strain 6 mutant strain had a G→A transition at position 92 which resulted in Gly31 substitution with Asp. The thyA Open reading frame (ORF) was amplified from the wild type DH5αE. coli and subcloned into prokaryotic expression vector pQE-31. The expected size of His-ThyA fusion protein was revealed by SDS-PAGE of the cell lysate of the recombinant M15 E. coli after induction with IPTG. The anti-ThyA serum was produced by immunizing mice with the recombinant antigen and used as the antibody for detection the mutant gene expression by Western blotting, which showed the mutated thyA gene expression in the ThyA- E.coli.To construct the chromosome-plasmid balanced lethal system based on the thyA gene, the thyA gene including its promoter was amplified from Salmonella typhimurium, and inserted into a eukaryotic expression vector, resulting in non-antibiotic selection expression vector pDTA. The cDNA for human lysozyme was inserted into the vector and the recombinant vector was named as pDTALYZ. The pDTALYZ vector was transformed into the ThyA- DH5αE.coli and the recombinant E.coli could grow normally in LB medium. High density fermentation showed that the thyA-containing vector transformed E.coli had similar growth dynamics, stability and plasmid yield to that of wild type DH5αE.coli.