携带KGF基因重组腺病毒载体的构建与鉴定.docVIP

携带KGF基因重组腺病毒载体的构建与鉴定 贾庆华 哈小琴﹡ 吕同德 刘春杰 （中国人民解放军兰州军区兰州总医院医学实验中心，甘肃省干细胞与基因药物重点实验室，甘肃兰州 730050；﹡通讯作者） 摘要 目的 构建携带KGF基因的腺病毒载体（Ad-KGF）并观察其在细胞中的表达。方法 通过PCR从pIRES2-EGFP-KGF质粒中扩增出目的片段KGF，定向克隆至穿梭质粒载体pShuttle-CMV中，构建pShuttle-KGF。经酶切及测序鉴定后，再将KGF定向克隆至重组腺病毒骨架载体pAdxsi，构建携带KGF基因表达盒以及GFP的重组腺病毒载体（pAdxsi-GFP-KGF）。酶切鉴定正确后，转染人胚肾细胞系HEK293细胞，进行重组腺病毒的包装、生产及纯化，半数组织感染量法（50% tissue culture infective dose，TCID50)测定重组腺病毒滴度。用重组腺病毒及空病毒Ad-null(作为阴性对照)转染人肺腺癌细胞A549，荧光显微镜下观察细胞绿色荧光蛋白的表达，ELISA鉴定KGF的表达。结果 成功构建了携带KGF的重组腺病毒（Ad-KGF），纯化后病毒滴度达1.6×1010pfu/ml。Ad-KGF转染A549细胞后24h在荧光显微镜下即可观察到绿色荧光蛋白，48h表达更强，ELISA实验证实细胞表达KGF。结论 成功构建了携带KGF的重组腺病毒（Ad-KGF），为下一步研究KGF用于肺纤维化治疗研究奠定了实验基础。 关键词：KGF（keratinocyte growth factor, KGF）；腺病毒；构建；表达 Construction and identification of recombinant adenovirus vector of kerationcyte growth factor JIA Qing-hua HA Xiao-qin LV Tong-de LIU Chun-jie Experimental Center of Medicine, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA, Key Labaratory of Stem Cell and Gene Drug in Gansu Province, Lanzhou 730050, Gansu Province, China Abstract Objective To construct and identified a recombinant adenovirus vector expressing keratinocyte growth factor(KGF) gene. Methods KGF gene amplified from the plasmid pIRES2-EGFP-KGF by polymerase chain reaction(PCR) and then inserted into the plasmid pShuttule-CMV to construct pShuttle-KGF. After confirmed by restriction enzyme digestion and DNA sequencing, the DNA encoding KGF in the new construct was inserted into the vector of recombine plasmid adenovirus and confirmed by restriction enzyme digestion. Then the human embryonic kidney cell line 293 was transfected with correctly identified pAdxsi-KGF and the recombinant adenovirus were amplified and purified, the virus titer was detected using 50% tissue culture infective dose (TCID50) assay. The expression of the green fluorescence protein and KGF were detected by fluorescent microscope and ELISA after the recombinant adenovirus transfecting human lung cancer A549 cell line. Re