摘要
目的 在克隆IκBα基因并构建IκBαM的基础上 ,构建重组腺病毒AdIκBαM。方法 将IκBαM克隆至穿梭质粒 ,线性化后与骨架质粒共同转染BJ5 183菌 ,构建重组腺病毒质粒。酶切及PCR法鉴定 ;将其线性化后转染 2 93细胞 ,观察绿色荧光以确定转染结果 ,以Westernblot法检测目的基因表达。结果 凝胶电泳及行酶切鉴定表明同源重组成功 ;PCR产物测序证明确为IκBαM ;以重组腺病毒质粒转染 2 93细胞 ,见绿色荧光 ;感染 2 93细胞得到大量病毒。结论 成功构建了IκBαM重组腺病毒 。
Objective To construct the adenoviral vector that expresses IκBαM gene for further study of the tumor gene therapy on the bases of successfully cloning Chinese IκBα gene and constructing IκBαM the super repressor form of the NF κB, and to study the roles of NF κB/ IκBα system. Methods The IκBαM gene was cloned to the shuttle plasmid Track CMV. The shuttle plasmids were linearized with Pme Ⅰ. Shuttle plasmid was co transform digested with adenoviral backbone vector to E.coli BJ5183 cells. The candidate clones were further tested by restriction nuclease digestion. Before transfection, recombinant adenoviral plasmids were digested with Pac Ⅰ. Then, the digested recombinant adenoviral plasmid was transfected to 293 cells with FuGENE TM 6 transfection reagent. Transfection viral productions were monitored by GFP(green fluorescent protein) expression. Presence of the recombinant adenoviruses was confirmed by PCR. Results The results show that we have successfully cloned the IκBαM gene to the plasmid Track CMV. The restriction analysis and the sequencing analysis of PCR product confirmed that correct recombinant adenoviral plasmid was constructed. 2 days after transfection, the fluorescence was observed in 293 cells. The expression of the IκBα gene was detected by Western blot. Conclusion We have constructed the recombinant adenovirus AdIκBαM. It will provide a solid basis for the study of IκBα mediated antitumor gene therapy and the roles of the NF κB /IκBα system.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2004年第1期65-69,73,共6页
Immunological Journal
基金
国家"8 63"计划项目 (20 0 1AA2 1 71 2 1)
国家自然科学基金面上资助项目 (30 0 70 2 97)
