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目的构建抗菌肽PR39的真核表达载体,转染后观察PR39在小鼠巨噬细胞RAW264.7中的表达及抗菌作用。方法采用拼接PCR法合成抗菌肽PR39基因,克隆到真核表达载体pIRES2-GFP中,经鉴定后用阳离子脂质体转染小鼠巨噬细胞RAW264.7,通过RT-PCR和免疫荧光法检测目的基因的表达,并对其抗菌活性进行初步检测。结果成功构建了抗菌肽PR39的真核表达载体pIRES2-GFP/PR39,并能在小鼠巨噬细胞RAW264.7中表达抗菌肽PR39。表达抗菌肽PR39的巨噬细胞杀灭和清除胞内菌的能力明显增强。结论抗菌肽PR39基因能够在小鼠巨噬细胞RAW264.7中表达并发挥抗菌作用。
Objective To construct the eukaryotic expression vector of antibacterial peptide PR39 and observe the expression and antibacterial activity of PR39 in macrophage RAW264.7 cells after transfection. Methods The antimicrobial peptide PR39 gene was synthesized by splicing PCR and cloned into the eukaryotic expression vector pIRES2-GFP. After identification, RAW264.7 macrophages were transfected with cationic liposome. The expression of PR39 gene was detected by RT-PCR and immunofluorescence Target gene expression, and its antibacterial activity for the initial detection. Results The eukaryotic expression vector pIRES2-GFP / PR39 was successfully constructed and the antimicrobial peptide PR39 was expressed in mouse macrophage RAW264.7 cells. The ability of macrophages expressing antimicrobial peptide PR39 to kill and clear intracellular bacteria was significantly enhanced. Conclusion The antimicrobial peptide PR39 gene can express in mouse macrophage RAW264.7 and exert antimicrobial activity.