摘要
目的 构建含有人survivin基因的重组腺病毒载体,为转染树突状细胞构建DC疫苗和基因治疗奠定基础。方法 自行设计一对分别含有KpnⅠ和XholⅠ酶切位点的survivin基因上下游引物,以质粒pCITE/survivin为模板,通过PCR扩增获得survivin基因全部序列。片段回收后经酶切,定向插入腺病毒穿梭质粒pAdTrack CMV ,获得重组质粒pAdTrack CMV survivin。通过KpnⅠ和XholⅠ双酶切、PCR及插入片段测序鉴定后,将正确重组体pAdTrack survivin转化包含腺病毒骨架质粒pAdEasy 1的BJ5 183菌。同源重组后用选择性培养基筛选阳性克隆,提取质粒用脂质体介导转染2 93细胞,通过观察绿色荧光蛋白(GFP)的表达及PCR扩增目的基因等方法鉴定重组的腺病毒。同时将病毒上清转染树突状细胞,通过观察绿色荧光蛋白的表达以及Westernblot分析观察survivin蛋白表达。结果 成功构建了含有人survivin基因的重组腺病毒,病毒滴度为1 65×10 8PFU/ml。在19×10 3 频段附近可见survivin蛋白表达为16 5×10 3 。结论 该重组腺病毒载体的构建及成功转染到树突状细胞内表达,为下一步研究人survivin作为靶抗原及基因治疗奠定了基础。
Objective To construct the recombinant adenovirus vector containing human survivin, and transfect it into dendritic cells. Methods Full length survivin cDNA was obtained from recombinant plasmid pCITE-survivin by PCR. The PCR product was double-digested with restriction endonucleases KpnⅠ and XholⅠ, and inserted orientationally into pAdTrack-CMV. The plasmid of pAdTrack-survivin was lined with PmeⅠ, and the fragment containing survivin was reclaimed and transfected into E. coli. BJ5183. After having been screened, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by the green fluorescence protein (GFP) expression and by PCR method. The virus was transfected into dentritic cells, and the expression of survivin was proved by the GFP expression and Western blot analysis. Results The recombined adenovirus-survivin was constructed successfully and the titer was about 1.65×10 8 pfu/ml. A band was observed by Western boltting and its relative molecular mass was about 16.5×10 3. Conclusion A recombinant adenovirus vector containing human survivin was constructed successfully, and the survivin protein was expressed by dendritic cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第9期821-824,共4页
Journal of Third Military Medical University
