Abstract: Objective To construct eukaryotic expression vector expressing short hairpin RNA(shRNA) section targeting human MCHR2 and to observe their transfection efficiency. Methods According to the sequence of human MCHR2 gene, the oligonucleotides of shRNA were designed and synthesized and directionally cloned into plasmid pGenesil-1.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing. The recombinant vectors were transfected into HEK293 cell line by lipofectamineTM2000，the expression of fluorescence and efficiency of transfection were detected by fluorescence microscopy and flow cytometry. Results Four shRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into HEK293 cells successfully and the transfection efficiency achieved about 50%. Conclusion The construction of eukaryotic expression vector expressing shRNA section targeting human MCHR2 and transfection in vitro successfully established a favourable foundation for further study on the function of MCHR2.