Construction of pSUPER-H1 RNAi System to Inhibit the Expression of NGF

Objective To construct the expressing vector of siRNA in order to inhibit nerve growth factor expression.Methods We constructed the plasmid that contained PolⅢ H1 promotor(pSUPER-H1).Two pair of oligonucleotides comprised of 64 bases were chemically synthetized and annealed.pSUPER-H1 vectors were linearized with BglⅡand HindⅢ.The annealed oligonucleotides were inserted into downstream of H1 promotor to constructed the shRNA plasmid(pSUPER-H1-NGF).Recombinant pSUPER-H1-NGF vectors were then transformed into competent E.coli.The positive clones were selected and recombinant pSUPER-H1-NGF plasmids were extracted.The plasmids were digested with EcoRⅠand HindⅢ and loaded in agarose gel electrophoresis.Results Recmbinant pSUPER-H1-NGF plasmids were transformed into competent E.coli. successfully.Agarose gel electrophoresis demonstrated that 64bp oligonucleotides were inserted into the expected sites.Sequencing analysis showed the inserted sequences were exactly correct.Conclusion Successful construction of recombitant pSUPER-H1-NGF plasmids provides the precondition of further study of NGF and develops the tools to express siRNA in vivo mediated by vectors.