摘要
目的:利用pEGFP6-1和pGenesil-1质粒构建针对Fas的2个短发夹RNA(short hairpin RNA,shRNA)串联表达的栽体．方法:设计表达2个shRNA结构的互补DNA序列,经退火成双链,分别克隆至带有U6启动子的质粒载体pEGFP6-1和pGenesil-1中,构建重组质粒,转化大肠杆菌DH5α菌株,扩增,提取质粒,酶切鉴定后测序分析．分别用SacI+ SalI双酶切重组质粒,胶回收酶切pEGFP6- 1-siFas1大片段和酶切pGenesil-1-siFas2小片段,T4 DNA Ligase连接酶切大、小片段,得到pEGFP6-1-siFas1+siFas2重组质粒．转化感受态细胞DH5α,扩增,提取串联重组质粒进行酶切鉴定．结果:成功构建靶向Fas的2个shRNA串联重组质粒载体pEGFP6-1-siFas1+siFas2．酶切鉴定和测序分析重组质粒,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功．结论:表达2个靶向Fas的shRNA表达框成功构建在一个重组质粒载体上．
AIM： To construct the recombinant plasmid expressing two Fas-targeted short hairpin RNA （shRNA） by pEGFP6-1 and pGenesil-1 plasmids vector.METHODS： Two pairs of DNA sequences were designed, and then synthesized into complementary chains by annealing, respectively. Then the obtained products containing short hairpin structure were inserted into plasmid vector pEGFP6-1/pGenesil-1 with U6 promoter. The recombinant plasmids were transformed into Escherichia coli strain DHSα for screening and amplifying. The sequence analysis of the plasmids identified by restriction enzyme were carried out. The two plasmids were first digested with Sac I, and then with Sal I. A long segment from pEGFP6-1-siFas1 and a short segment from pGenesil-1-siFas2 were reclaimed, and connected with T4 DNA Ligase. The recombinant plasmid series was identified by restriction endonuclease digestion.RESULTS： The two Fas-targeted shRNAs were successfully inserted into the plasmid vector pEGFP6-1, and the coding sequences of the obtained shRNAs were consistent with the designed fragments. CONCLUSION： The recombinant plasmid series of two Fas-targeted shRNAs is successfully constructed in one pEGFP6-1 plasmid.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第22期2174-2179,共6页
World Chinese Journal of Digestology
基金
山西省自然基金资助项目
No.20051114
关键词
FAS
RNAI
SHRNA
串联质粒
Fas, RNA interference, Short hairpin RNA, Plasmid series
