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Objective To achieve high-level expression and purification of the recombinant 9 kDa and 15 kDa chicken NK-lysin proteins (designated as cNKL9 and cNKL15) and evaluate their in vitro antibacterial and immunomodulatory activities. Methods The target genes—cNKL9 and cNKL15—were individually cloned into the pPIC9K vector to construct recombinant expression plasmids for heterologous expression in Pichia pastoris. After confirming the correct expression of the recombinant proteins via SDS-PAGE and Western blotting, we used nickel affinity chromatography to obtain highly purified recombinant cNKL9 and cNKL15 proteins. The in vitro antibacterial activitiesof recombinant proteins against Salmonella typhimurium and Escherichia coli O157:H7 were assessed by colony forming unit (CFU) assays. We measured the mRNA levels by RT-qPCR and analyzed the transcriptional changes of relevant cytokines after applying the recombinant proteins to the chicken macrophage cell line HD11. High-level expression of the recombinant proteins was achieved in a 15 L fermenter for scale-up culture via optimization of the high-density fermentation process. Results The P. pastoris expression plasmids pPIC9K-cNKL9 andpPIC9K-cNKL15 were successfully constructed. SDS-PAGE and Western blotting results revealed specific bands of recombinant cNKL9 and cNKL15 proteins in the culture supernatant, confirming the successful secretory expression of the target proteins. Antibacterial activity assay demonstrated that the recombinant cNKL9 protein exhibited significant inhibitory activities against both S. typhimurium and E. coli O157:H7, whereas the recombinant cNKL15 protein showed no obvious inhibitory effects on either pathogenic strain. RT-qPCR analysis showed that the recombinant cNKL15 protein significantly upregulated the transcription levels of cytokines such as CCL4 and CCL20 in HD11 cells, while the recombinant cNKL9 protein did not exhibit immunomodulatory activity. Furthermore, in the 15 L fermenter, the heterologous expression yields of cNKL9 and cNKL15 reached 0.64 g/L and 0.53 g/L, respectively. Conclusion For the first time, we achieved high-level expression of recombinant cNKL9 and cNKL15 proteins in the P. pastoris system. Specifically, cNKL9 exhibited inhibitory activities against S. typhimurium and E. coli O157:H7, while cNKL15 exerted immunomodulatory effects by upregulating the mRNA levels of cytokines in the chicken macrophage cell line HD11. These findings provide experimental evidence for the development of cNKL9 and cNKL15 as antibacterial or immunomodulatory agents, indicating their considerable application potential.
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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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