Construction and identification of a vector expressing short hairpin RNA targeting annexin II gene / 第二军医大学学报

ABSTRACT

Objective: To construct a vector encoding short hairpin RNA(shRNA) targeting annexin H and to observe its influence on annexin II expression. Methods: Annexin II-targeted hairpin RNA1 and RNA2 were devised according to the Gen-Bank annexin II sequence. Meanwhile, a nonspecific sequence was taken as negative control. The oligonucleotide strands of DNA fragments encoding the above shRNAs were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into human pGenesil1 plasmid followed by amplification in E. coli and sequencing. The 3 pGenesil1 constructs were then transfected into HEK293 cells and the expression of annexin II was examined by real-time fluorescence quantitative RT-PCR and Western bolt. The non-transfected cells were taken as blank control. Results: Restrictive enzyme digestion and sequencing verified that the 3 recombinants (pGenesil1-annexin II shRNA1, pGenesil1-annexin II shRNA2, and pGenesil1-negative shRNA) were correct. The annexin II expression was significantly lower in HEK293 cells transfected with pGenesil1-annexin II shRNA2 compared with those transfected with pGenesil1-annexin II shRNA1, pGenesil1-negative shRNA, and blank control (P<0.05); and there was no significant difference between the latter 3. Conclusion: We have successfully generated an annexin II-targeted shRNA construct, which can significantly inhibit the expression of annexin II.