Objective    To construct a plasmid expressing a short hairpin RNA (shRNA) targeting a cyclin-dependent kinase inhibitor (CKI)

p27Kip1

and evaluate its effect on the proliferation of bovine corneal endothelial cells (bCEC).      Methods    Three p27Kip1-shRNA template DNA sequences were designed and synthesized as experimental groups

a plasmid expressing irrelevant shRNA with a random combination was used as negative control. The products were inserted into the pGenSil-1 plasmid containing U6 promoter and EGFP. The recombinant plasmids of pGenSil-1/p27Kip1-1

pGenSil-1/p

27Kip1-2

pGenSil-1/p27Kip1-3 and pGenSil-1/HK were constructed respectively. Then the recombinant plasmids were expanded in DH5α cells and identified by restriction enzyme and sequence analysis. The 4 plasmids were transfected into bCEC respectively by Lipofectamine 2000

TM

. The level of p27Kip1 protein was detected by Western blotting and the proliferation of bCEC were detected by MTT 48 h after transfection.      Results    The 4 recombinant plasmids was confirmed to be constructed successfully. The inhibitory rates of p27Kip1 protein were 66.41%

61.51％ and 71.32％ in the 3 experimental groups respectively

and the proliferation of bCEC was promoted significantly.      Conclusion    shRNA- p27Kip1 RNAi system has been constructed successfully. It facilitates the experimental study of the promotion of bCEC by the method of RNAi.