Cloning and identification of shRNA recombinant plasmid targeting on COX-2 gene
- VernacularTitle:靶向COX-2基因短发夹RNA重组表达载体的构建及鉴定
- Author:
Yiming YANG
;
Yu LIU
;
Zhiguang TU
;
Yuqian ZHANG
- Publication Type:Journal Article
- Keywords:
cyclooxygenase-2;
short hairpin RNA;
recombinant plasmid
- From:Journal of Third Military Medical University
1988;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone shRNA (short hairpin RNA) recombinant plasmid targeting on COX-2 gene and analyze the nucleic acid sequence of the recombinant plasmid. Methods One pair of 21 bp reverse repeated sequence targeting on COX-2 mRNA spaced by 9 bp nucleotides were synthesized. The complement double strands was formed by annealing and inserted into plasmid pGenesil-1 to generate eukaryotic expression plasmid. The recombinant plasmid was transformed into Jm109 strain, and the recombinant plasmid extracted was identified by restriction enzyme and sequence analysis. Inhibition effects of COX-2 mRNA and protein were detected by RT-PCR and Western blotting respectively. Results The target DNA was directly cloned to vector and the result was correct by sequence analysis. Compared to untransfected group, recombinant expression plasmid pshRNA-COX-2 resulted in reduction of COX-2 mRNA and protein expression to 69.9% and 50.3% respectively. Conclusion The recombinant plasmid targeting on COX-2 gene was successfully constructed, and it inhibited the expression of COX-2 gene significantly.
