Construction of a chromosome-plasmid balanced lethal system based on murI in the attenuated live vaccine strain of Pseudomonas plecoglossicida

Abstract:

【Objective】 To construct a chromosome-plasmid balanced lethal system based on the glutamate racemase (MurI) gene for the expression of exogenous antigens in the attenuated vaccine strain of Pseudomonas plecoglossicida (Pp ΔtssD-1), so as to provide new ideas and methods for the development of multi-component live vaccines. 【Methods】 We constructed a murI-deleted strain from Pp ΔtssD-1 by homologous recombination. First, we replaced the kanamycin resistance gene of the pBBR1MCS-2 plasmid with murI to construct a balanced lethal plasmid. Subsequently, we inserted the green fluorescent protein gene into the multicloning site of the plasmid to examine the expression stability of the exogenous antigen. Finally, we characterized the recombinant strain in terms of the growth curve, plasmid stability, and expression of the exogenous antigen. 【Results】 The murI-deleted strain was unable to grow in the lysogeny broth medium without D-glutamate. The non-resistant complemented strain regained growth capability in the lysogeny broth medium without D-glutamate. However, its growth was slower than that of the starting strain. Exogenously introduced antigens were identified as stable in the absence of antibiotic selection, and distinct green fluorescence signals were observed under a fluorescence microscope. Additionally, the balanced lethal plasmid exhibited high genetic stability within the recombinant strain. 【Conclusion】 A novel chromosome-plasmid balanced lethal system targeting murI was developed in this study. It enabled the expression of exogenous antigens in Pp ΔtssD-1 without the need for antibiotic selection. This system provides a new method for the development of multi-component live vaccines, with no need of antibiotic resistance markers and high plasmid stability.