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[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 ofPseudomonas plecoglossicida (Pp ΔtssD-1), so as to provide new ideas and methods for the development of multi-component live vaccines. [Methods] We constructed amurI-deleted strain fromPp ΔtssD-1 by homologous recombination. First, we replaced the kanamycin resistance gene of the pBBR1MCS-2 plasmid withmurI 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] ThemurI-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 targetingmurI was developed in this study. It enabled the expression of exogenous antigens inPp 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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