OBJECTIVETo clone and express human neuron-specific enolase (HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis.

METHODSHuNSE gene was amplified by RT-PCR and subcloned into a HIS-tagged expression vector pQE30 after sequence verification. HIS-NSE fusion protein expression was obtained in E. coli M15 after IPTG induction followed by purification of the fusion protein by Ni-NTA affinity chromatography. Two male rabbits were immunized for 4 times with the purified protein, and the antiserum against NSE protein was collected and evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemistry.

RESULTSSDS-PAGE assay yielded an approximately 22 kD HIS-NSE fusion protein. The prepared antiserum could recognize both recombinant NSE protein and native NSE protein extracted from the brain tissues of different mammalian species as shown by Western blotting and immunohistochemistry.

CONCLUSIONHigh expression of HuNSE is obtained in E. coli and the prepared antiserum against HuNSE can be used potentially for diagnosis of prion-associated diseases and other nervous degeneration diseases.