[The wild-type alpha-synuclein over-expression to induce the protein aberrant aggregation of alpha-synuclein in HEK293 cells in vitro].

ABSTRACT

OBJECTIVE: To investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro. METHODS: The cDNA encoding the human alpha-synuclein without the stop code was cloned into PGEM T-easy vector. Using enzyme map and DNA sequencing analyzed and determined the recombinant plasmid, and then sub-clone the alpha-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids alpha-synuclein-pEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence, anti-alpha-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining. RESULTS: The restriction enzyme map suggested that eukaryotic expression vector for human wild-type alpha-synuclein gene was constructed successfully. By EGFP fluorescence, anti-alpha-synuclein immunocytochemistry, it could be observed that the alpha-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells. CONCLUSION: The over-expression of wild-type alpha-synuclein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.