含hITF基因cDNA的腺病毒载体的构建及其感染肠上皮细胞的研究  

Construction of recombinant adenovirus vector containing hITF and infection of intestinal epithelial cells

作　　者：孙勇[1,2] 陈宝君[1,2] 勒娟[1,2] 潘晓峰[1,2] 张方[1,2] 

机构地区：[1]徐州医学院附属淮海医院烧伤科,江苏徐州221004 [2]中国人民解放军第九七医院烧伤科,江苏徐州221004

出　　处：《西安交通大学学报（医学版）》2014年第6期795-799,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(81100252);南京军区医学科研课题重点项目(12Z10)~~

摘　　要：目的构建含有人肠三叶因子基因cDNA的腺病毒载体,并观察其对肠上皮细胞的感染能力。方法利用PCR技术扩增hITF基因cDNA全长序列,测序后亚克隆至腺病毒穿梭质粒,转化至含腺病毒骨架质粒pAdEasy-1大肠杆菌株BJ5183感受态细胞中进行同源重组,内切酶PmeⅠ线性化后转染HEK293细胞进行病毒包装及扩增,并进行滴度测定。将重组腺病毒感染肠上皮细胞(HT-29),荧光显微镜观察感染效果。结果成功构建出重组穿梭质粒pAdTrack-CMV-hITF,PmeⅠ线性化后与骨架质粒pAdEasy-1同源重组,PacⅠ酶切鉴定后命名为Ad-hITF;重组腺病毒载体在HEK293细胞中包装,获得重组腺病毒颗粒;根据Karbers公式计算病毒颗粒滴度为2×109 pfu/mL;重组腺病毒可以感染肠上皮细胞(HT-29),感染效率较高。结论成功构建含hITF基因cDNA的重组腺病毒载体并获得大量子代重组腺病毒,为腺病毒介导hITF基因治疗的应用打下基础。Objective To construct a recombinant adenovirus vector of human intestinal trefoil factor （hITF） and to determine its infection efficiency in human intestinal epithelial cells （HT-29）. Methods The genomic fragment of hITF was amplified by PCR and subeloned into adenovirus shuttle plasmid to construct recombinant vector. Homologous recombination was performed by transferring the recombinant vector to E. coli BJ5183 containing the backbone pAd-Easy. The correct recombinant was selected and linearized with Pme I , and then transfected into 293 cells for packaging and amplification. The titration of the recombinant adenovirus was determined. Human colon adenocarcinoma cell line （HT-29） was infected with recombinant adenovirus and infection efficiency was determined. Results The recombinant vector pAdTrack-CMV-hlTF was constructed. After digestion with restriction enzyme Pine I , linearized recombinant vector was transformed into BJ5183 bacteria and the adenoviral plasmid Ad-hITF carrying hITF was generated with homologous recombination. The mature viruses would be packaged in 293 cells. The titer of the viruses was 2 × 109 pfu/mL by TCID50 method. Transfeetion efficiency in HT-29 was high. Conclusion Recombinant adenovirus vector containing hITF gene has been successfully constructed and this research lays foundations for further application of hITF gene therapy.

关 键 词：肠三叶因子 腺病毒 肠上皮细胞 基因治疗 

分 类 号：R379[医药卫生—病原生物学] R394.3[医药卫生—基础医学]