AIMTo construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection.

METHODSThe specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting.

RESULTSThe DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1.

CONCLUSIONA RNAi eukaryotic vector containing prohibitin gene was successfully constructed.