该研究将增强型绿色荧光蛋白（EGFP）基因片段与抗菌肽牛乳铁蛋白肽（LfcinB-His）基因片段的N末端连接，构建以EGFP为报告基因的胞内表达载体，融合表达重组蛋白EGFP-LfcinB-His，并对其进行活性检测。将重组表达质粒电转入毕赤酵母GS115中，筛选阳性转化子，甲醇诱导其发酵表达，488 nm的激发光下检测到较强的荧光。当酵母细胞培养达到最高密度后，收集菌体细胞，破壁，用酵母细胞蛋白裂解液进行裂解。裂解液经过超滤浓缩、Ni-NTA亲和层析、甲酸裂解等纯化步骤后，Tricine-SDS-PAGE检测到相对分子质量为4.1 ku的目标蛋白（LfcinB-His）条带。通过液质联用测定与分析，最终获得了纯度为90.32%的LfcinB-His超滤浓缩液，对测试的6株致病菌均有不同程度的抑制作用，抑菌浓度范围在16~64 μg/mL。综上，该研究为毕赤酵母表达小分子多肽提供了较好的方法学参考，也为进一步研究LfcinB的生物活性及高密度发酵奠定了基础。

In this study, the enhanced green fluorescent protein (EGFP) gene fragment was ligated with the N-terminus of the antimicrobial peptide bovine lactoferrin peptide (LfcinB-His) gene fragment to construct an intracellular expression vector with EGFP as the reporter gene. The recombinant protein EGFP-LfcinB-His was fused and expressed, and its activity was determined. The recombinant expression plasmid was electroporated into Pichia pastoris GS115, and the positive transformants were screened. The fermentation expression was induced by methanol, and strong fluorescence was detected under the excitation light of 488 nm. When the yeast cell culture reached the highest density, the cells were collected, the cell walls were broken, and the cells were lysed with a yeast cell protein lysate. The target protein (LfcinB-His) band with a relative molecular mass of 4.1 ku was detected by Tricine-SDS-PAGE, after the lysate was purified by steps such as ultrafiltration concentration, Ni-NTA affinity chromatography and formic acid lysis. Finally, a LfcinB-His ultrafiltration concentrate with a purity of 90.32% was obtained through the determination and analysis by liquid chromatography-mass spectrometry, which had different degrees of inhibition on the six strains of pathogenic bacteria tested, and the inhibitory concentration range was 16~64 μg/mL. In summary, this study provides a good methodological reference for the expression of small molecular peptides in Pichia pastoris, and also lays a foundation for further research on the biological activity and high-density fermentation of LfcinB.

四川省科技计划项目-科研院所科技成果转化项目（2022JDZH0036）；四川省科研院所科技成果转化基金项目（22YSZH0016）；江苏大学高水平大学-师资建设-科研启动基金（4111360007）；江苏大学高级专业人才科研启动基金（12JDG069）