携带KGF基因重组腺病毒载体的构建与鉴定  

Construction and identification of recombinant adenovirus vector of keratinocyte growth factor

作　　者：贾庆华[1] 哈小琴[1] 吕同德[1] 刘春杰[1] 

机构地区：[1]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,兰州730050

出　　处：《重庆医科大学学报》2011年第1期64-67,共4页Journal of Chongqing Medical University

摘　　要：目的:构建携带角化细胞生长因子(Keratinocyte growth factor,KGF)的腺病毒载体并观察其在细胞中的表达。方法:通过PCR从pcDNA3-KGFpIRES2-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,纯化后病毒滴度达1.6×1010 pfu/ml。Ad-KGF转染A549细胞后24 h在荧光显微镜下即可观察到绿色荧光蛋白,48 h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,为下一步研究KGF用于肺纤维化治疗研究奠定了实验基础。Objective：To construct and identify a recombinant adenovirus vector expressing keratinocyte growth factor（KGF） gene.Methods：KGF gene was amplified from the plasmid pIRES2-EGFP-KGF by polymerase chain reaction（PCR） and then inserted into the plasmid pShuttle-CMV to construct pShuttle-KGF.After being confirmed by restriction enzyme digestion and DNA sequencing,the DNA encoding KGF in the new structure was inserted into the vector of recombinant 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,and the virus titer was detected using 50% tissue culture infective dose（TCID50） assay.The expression of the green fluorescent protein and KGF were detected by fluorescent microscope and ELISA after the recombinant adenovirus transfected human lung cancer A549 cell line.Results：The recombinant adenovirus pAdxsi-KGF was constructed and amplified with titer of 1.6×1010 pfu/ml;the green fluorescence proteins could be observed under fluorescent microscope in A549 24 h after transfection and with a stronger degree after 48 h,and KGF expression was detected by ELISA.Conclusion：The recombinant adenovirus pAdxsi-KGF was constructed successfully,which will be a good foundation for the research on the therapy of KGF for pulmonary fibrosis.

关 键 词：角化细胞生长因子 腺病毒 构建 表达 

分 类 号：R392[医药卫生—免疫学]