鸡 NK-lysin在毕赤酵母中的高效表达及活性鉴定

High-level expression and activity identification of chicken NK-lysin in Pichia pastoris

摘要:

目的 高效表达并纯化9 kDa和15 kDa鸡NK-lysin重组蛋白(cNKL9和cNKL15)，评估其体外抗菌活性及免疫调节活性。 方法 将 cNKL9和 cNKL15目的基因分别克隆至pPIC9K载体中，构建重组表达质粒进行异源表达。经SDS-PAGE分析及Western blotting特异性验证，确认重组蛋白正确表达后，通过镍亲和层析法获得高纯度的cNKL9和cNKL15重组蛋白。以鼠伤寒沙门氏菌和大肠埃希氏菌O157:H7为靶标菌株，采用平板活菌计数法(CFU法)评估重组蛋白的体外抗菌活性；通过RT-qPCR技术检测mRNA表达水平，分析重组蛋白处理HD11鸡巨噬细胞系后相关细胞因子的转录水平变化。在15 L发酵罐放大培养体系中，通过优化高密度发酵工艺实现重组蛋白的高效表达。 结果 成功构建毕赤酵母重组表达质粒pPIC9K- cNKL9和pPIC9K- cNKL15。SDS-PAGE及Western blotting验证结果显示，培养上清中可检测到重组cNKL9和cNKL15蛋白的特异性条带，证实目标蛋白已实现正确分泌表达。抑菌活性检测结果表明，重组cNKL9蛋白对鼠伤寒沙门氏菌及大肠埃希氏菌O157:H7均具有显著抗菌活性，而重组cNKL15蛋白对上述2株病原菌无明显抑制作用。RT-qPCR结果显示，重组cNKL15蛋白显著上调HD11细胞中CCL4、CCL20等细胞因子的基因转录水平，重组cNKL9蛋白则无此免疫调节作用。此外，在15 L发酵罐放大培养体系中，cNKL9和cNKL15的异源表达量分别可达0.64 g/L和0.53 g/L。 结论 本研究在毕赤酵母系统中实现了cNKL9和cNKL15蛋白的高效重组表达。其中，cNKL9蛋白对鼠伤寒沙门氏菌和大肠埃希氏菌O157:H7具有抗菌活性，cNKL15蛋白能够通过上调鸡巨噬细胞HD11中细胞因子的mRNA表达水平发挥免疫调节功能。上述结果为cNKL9和cNKL15蛋白作为抗菌或免疫调节产品的开发提供了实验依据，具有良好的应用前景。

Abstract:

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 activities of 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 and pPIC9K- 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.