抗菌肽PR39真核表达载体的构建及其在巨噬细胞中的表达和抗菌作用  

Construction of eukaryotic expression vector encoding antimicrobial peptides PR39 and its expression and antimicrobial function in RAW264.7 cells

作　　者：文阳安[1] 郭金英[1] 余晓林[1] 岑东[1] 陈彦[2] 张键[3] 罗淼[1] 涂植光[1] 

机构地区：[1]重庆医科大学医学检验系生物化学教研室,临床检验诊断学省部共建教育部重点实验室,重庆市重点实验室,重庆400016 [2]西南大学医院,重庆400715 [3]重庆医科大学附属第一医院骨科,重庆400016

出　　处：《第三军医大学学报》2008年第21期2044-2046,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金(30600790)~~

摘　　要：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 an and observe its expression and antimicrobial eukaryotic expression vector encoding antimicrobial peptide PR39, function in mouse macrophage RAW264.7 cells. Methods PR39 gene sequence was synthesized with PCR amplification and inserted into an eukaryotic expression vector plRES2-GFP. After identification, plRES2-GFP/PR39 vector was transfected into RAW264.7 cells with cationic liposome. The expression of PR39 was detected by RT-PCR and immunofluorescence method, and its antibacterial activity was also detected. Results The plRES2-GFP/PR39 vector was successfully constructed, and PR39 was expressed in RAW 264.7 cells with antimicrobial activity. RAW 264.7 cells expressing PR39 showed stronger antibacterial ability. Conclusion Antimicrobial peptide PR39 is expressed and shows antimicrobial function in RAW264.7 cells. The successful construction of the PR39 expressing vector have established a foundation for the gene therapy of antimicrobial peptides against intracellular bacterial infection.

关 键 词：抗菌肽 PR39 真核表达载体 巨噬细胞 胞内菌 

分 类 号：R392.11[医药卫生—免疫学] R394-33[医药卫生—基础医学]