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表达.pdfVIP

生物技术通报 BIO TE CHN OLOG Y B ULLETIN 2010 10 / PR39 余雪梅 文阳安 李朴 孔飞飞 陈光辉 邱欣欣 涂植光 (, 400016) : 旨在构建由巨噬细胞特异性启动 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 RAW 2647 中的表达PCR扩增巨噬细胞启动 /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( pAdT rackSP /PR39)与腺病毒骨架( pAdEasy1) 共转化大肠杆菌 BJ 183, 通过细菌内同源重组产生重组腺病毒载体 ( pAdSP /PR39)pAdSP/PR39经Pac I线性化后转染 293细胞, 包装出重组腺病毒( AdSP /PR39)AdSP/PR39 感染小鼠 巨噬细胞 RAW 2647, 经RTPCR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 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 : 抗菌肽 PR39 腺病毒载体 胞内菌 Construction of an Adenovirus Expressing Vector of Antim icrobial Peptides PR39 andM odulated by M acrophagespecific Promoter and ts Target Expression in RAW 2647 Cell Yu Xuem ei W en Yangan L iPu Kong Feifei Chen Guanghui Q iu X inx in Tu Zh iguang (Departm ent of LaboratoryM edicine, Chongqing M edical University, Chongqing 400016) Abstrac:t Th is study w as to construct an adenovirus expressing vector of antmi icrob ial peptides PR39 m odu lated by macrophage specific prom oter, and observe its expression in m ouse m acrophage RAW2647 cells. PR39 and macrophagespecific promoter gene w as amp lified w ith PCR and inserted into adenovirus shuttle vector pAdT rack, w hich was cotransform ed intoE. coli BJ 183. InE. coli BJ 183, recomb ination occurred to obtain recomb inan t adenovirus vector ( pAdSP /PR39), w hich was used to transfect 293 cells after linearized byP ac I to obtain recomb inant adenovirus AdSP /PR39. RTPCR, mi m unocytochem istry and antmi icrob ial activity testw ere app lied to detect the expression of PR39 gene in m ousem acrophageRAW 2647. Recomb inant adenovirusm odulated by a macrophage specific prom oter and encod