巨噬细胞特异性启动子/抗菌肽PR39基因腺病毒载体的构建及其表达  

Construction of an Adenovirus Expressing Vector of Antimicrobial Peptides PR39 andModulated by Macrophage-specific Promoter and Its Target Expression in RAW264.7 Cell

作　　者：余雪梅[1] 文阳安[1] 李朴[1] 孔飞飞[1] 陈光辉[1] 邱欣欣[1] 涂植光[1] 

机构地区：[1]重庆医科大学医学检验系,重庆400016

出　　处：《生物技术通报》2010年第10期201-204,209,共5页Biotechnology Bulletin

基　　金：国家自然科学基金(30600790)

摘　　要：旨在构建由巨噬细胞特异性启动子调控的抗菌肽PR39基因腺病毒表达载体,检测目的基因在小鼠巨噬细胞RAW264.7中的表达。PCR扩增巨噬细胞启动子/抗菌肽PR39成熟肽基因序列,插入腺病毒穿梭载体pAdTrack中,重组穿梭质粒(pAdTrack-SP/PR39)与腺病毒骨架(pAdEasy-1)共转化大肠杆菌BJ5183,通过细菌内同源重组产生重组腺病毒载体(pAd-SP/PR39)。pAd-SP/PR39经PacI线性化后转染293细胞,包装出重组腺病毒(Ad-SP/PR39)。Ad-SP/PR39感染小鼠巨噬细胞RAW264.7,经RT-PCR和免疫细胞化学检测PR39的靶向表达特异性。成功构建了由巨噬细胞特异性启动子调控的抗菌肽PR39基因腺病毒表达载体。包装出的重组腺病毒仍然能感染小鼠巨噬细胞且高效表达抗菌肽PR39。构建的重组腺病毒能够在巨噬细胞中表达抗菌肽PR39基因,为靶向表达抗菌肽在胞内菌感染治疗中的应用奠定了基础。This study was to construct an adenovirus expressing vector of antimicrobial peptides PR39 modulated by macrophage- specific promoter, and observe its expression in moose maeropbage RAW264.7 cells. PR39 and macropbage-specific promoter gene was amplified with PCR and inserted into adenovirus shuttle vector pAdTrack, which was co-transformed into E. coli BJ5183. In E. coli BJ5183, recombination occurred to obtain recombinant adenovirus vector （pAd-SP/PR39） , which was used to transfect 293 cells after linearized by Pac I to obtain recombinant adenovirus Ad-SP/PR39. RT-PCR, immunocytochemistry and antimicrobial activity test were applied to detect the expression of PR39 gene in mouse macrophage RAW264. 7. Recombinant adenovirus modulated by a macrophage- specific promoter and encoding antimicrobial peptide PR39 were successfully constructed, which could infect RAW264.7 cell efficiently, and expressed antimicrobial peptides PR39 gene. Recombinant adenovirus Ad-PR39 is macrophage-specific, which can express antimicrobial peptide PR39 in mouse macrophage RAW264.7 cell. Our study has provided a foundation of intracellular bacterial infection therapy with target expression of antimicrobial peptides.

关 键 词：抗菌肽 PR39 腺病毒载体 胞内菌 

分 类 号：Q789[生物学—分子生物学]