Abstract: Objective To develop an effective and rapid method for screening recombinant hairpin RNA expression plasmids through reconstructing pSilencer3.1 -H1 vector. Methods The double-strand DNA fragement containing a NotⅠrestriction endonuclease site, the flanking sequences of which were not complementary, was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer3.1-H1 to construct the pSilencer3.1-H1/NotⅠvector. With BamHⅠand Hind III, the pSilencer3.1-H1/NotⅠwas digested and ligated with siRNA expression cassette targeting JAK2 gene. The plasmid DNA of the positive clones was extracted and digested with NotⅠ. The un-digested vector was selected to identify by sequence analysis to construct the vector pSilencer3.1-H1/JAK2. After transfection of pSilencer3.1-H1/JAK2 into gastric cancer AGS cells, the JAK2 protein was detected by Western blotting. Results The sequencing results showed that pSilencer3.1-H1/NotⅠand pSilencer3.1-H1/JAK2 vectors were successfully constructed. The JAK2 protein was inhibited in transfected AGS cells. Conclusion The reconstructed pSilencer3.1-H1/ NotⅠvector could rapidly and efficiently screen shRNA expression vecteors.