抗肿瘤的PF4及相关肽基因重组腺病毒的构建  

Construction of PF4(1-70) and PF4(17-70) Gene Recombinant Adenovirus

作　　者：吴立华[1] 刁世勇[1] 蔡英林[1] 顾洁[1] 任倩[1] 李彬[1] 杨仁池[1] 韩忠朝[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所中法实验室,天津市300020

出　　处：《中国肿瘤临床》2003年第8期551-555,共5页Chinese Journal of Clinical Oncology

基　　金：国家攀登计划项目(编号:95-专-10);长江学者计划以及天津市科技发展计划项目资助(编号:003114311)

摘　　要：利用细菌内同源重组法构建含PF4(血小板第四因子)全长1-70及PF4的小肽17-70基因的重组腺病毒。方法:将连有MCP-1信号肽的PF4全长及其C末端的改构体PF4-17-70两个基因片段克隆至腺病毒穿梭质粒pAdshuttle-CMV中,形成转移质粒pAdshuttle/PF4(1-70)和pAdshuttle/PF4(17-70),将之酶切线性化后与腺病毒骨架质粒pAdEasy-1共转化大肠杆菌BJ5183,抽提并鉴定含目的基因的重组体质粒,而后用磷酸钙共沉淀方法转染HEK293细胞,包装成重组腺病毒AdPF4(1-70)和AdPF4(17-70)。采用PCR方法对重组体腺病毒进行鉴定,扩增并纯化病毒,用TCID50方法测病毒滴度,利用带有CMV-LacZ的对照重组腺病毒(Ad-LacZ)对感染效率进行监测。结果:利用电穿孔法分别将质粒pAdshuttle/PF4(1-70),pAdshuttle/PF4(17-70)与质粒pAdEasy-1共转化大肠杆菌BJ5183获得70%以上的阳性重组体细菌克隆。PCR检测结果表明重组腺病毒已含有目的基因,滴度为1×1010pfu/ml。结论:利用细菌内同源重组法可以简便、快捷地构建含PF4(1-70)、PF4(17-70)cDNA的腺病毒载体,为两者在肿瘤基因治疗中的应用奠定了基础。Objective:To construct the recombinant adenovirus of PF4(1-70)and(17-70)by using the method of homogenouse recombination in bacteria.Methods:The cDNA of PF4(1-70)and PF4(17-70)was subcloned into the shuttle vector of pAdshuttle-CMV,forming transfer vector of pAdshuttle /PF4(1-70)and pAdshuttle /PF4(17-70).Then they were cotransformed into BJ5183cells with adenovirual geonomis plasmid of pAdEasy-1.The DNA of identified recombi -nant plasmid were digested with PacI and transfected to HEK293cells to package adenovirus.The PCR technique was used to detect the target gene.The titres of recombinant adenovirus were measured by TCID50method.The infection rate of recombinant adenovirus were same as Ad5.CMV-LacZ virus based on theβ-galactosidase.Results:There were over70%positive recombi -nant bacterial clones after cotransformation BJ5183bacterial cells with pAdshuttle /PF4(1-70)?pAdshuttle /PF4(17-70)and pAdEasy-1by electroporation techneque.PCR indicated each the re-combinant adenovirus contained the insert of PF4(1-70)and PF4(17-70).The titres of recombi -nant adenovirus were1×10 10 pfu/ml .Conclution:The method of homogenouse recombinantion in bacteria is convinent and efficient,Meanwhile the pepared AdPF4(1-70)and AdPF4(17-70)pave the sound foundation for tumor treatment study.

关 键 词：腺病毒 PF4 同源重组 细菌 肿瘤 

分 类 号：R73-36[医药卫生—肿瘤]