Soluble expression and purification of human neuroprotein α-synuclein related to Parkinson's disease and its antibody preparation

Objective To express human α-synuclein protein(SNCP) in prokaryotic cells with genetic engineering technique and prepare SNCP specific rabbit polyclonal antibody.Methods Total RNA of a human neuroblastoma cell line SHSY5Y was extracted and the single-stranded cDNA was synthesized.With PCR technique,the cDNA sequences encoding SNCP was amplified.After being confirmed by DNA sequence analysis,the full-length SNCP cDNA was cloned into glutathione S transferase(GST) fusion protein expression vector pQE-30-GST,and GST-SNCP fusion protein was expressed in E.coli.New Zealand rabbits were immunized by the purified GST-SNCP proteins to prepare specific antiserum and detect its antibody.Results A 420 bp α-synuclein cDNA has been amplified and verified by sequence assay.SDS-PAGE and Western blot assays revealed that the GST-SNCP protein was expressed in soluble form with a rough 45 000 relative molecule weight.The immunoreactive titer of the prepared α-synuclein rabbit's antiserum was evaluated up to 1:32 000 with ELISA.Western blot confirmed that this antibody could specifically recognize the endogenous SNCP in the brain tissues of BALB/c mice.Conclusions Human SNCP has been successfully expressed in soluble form with the prokaryotic expression system and the SNCP polyclonal antibody with reliable immunospecificity has been elicited by immunization of the purified SNCP,which provides foundation for further study of SNCP's biological functions and pathogenesis of neurodegenerative disorders.