새로운 dual vector 개발을 통한 Edwardsiella tarda ghost bacteria 백신의 안전성 향상

Abstract

Using a newly constructed vector containing both the ghost inducing PhiX174 E gene cassette and the bacterial DNA degrading staphylococcal nuclease A(SNA) gene cassette, Edwardsiella tarda ghost (ETG) devoid of intact genomic DNA and plasmids containing antibiotic resistance gene was generated. The secretion signal sequence and the amino terminus of nuclease B sequence deleted SNA did not show any intracellular nuclease activity, whereas the SNA fused with N-terminal 26 amino acids of the lambda phage Cro gene showed successful degradation of bacterial nucleic acids. Furthermore, the nuclease activity of SNA in E. tarda was enhanced by codon optimization of SNA gene. Via coexpression of SNA gene and lysis gene E under the control of the λPR promoter, a safety-improved ETG was generated in the respect of minimizing the existence of bacterial genomic DNA and plasmids in the final products of the ETG. The present ghost bacteria generation system would be advantageous to produce ghost bacteria vaccines against fish pathogens in that readiness (not use two plasmid combinations but use one kind of plasmid), safety (limit the residual genetic content of ghost bacteria), and production cost (simply induced by upshift of temperature).