Protein expression of human neuron-specific enolase and its antiserum preparation.
OBJECTIVE
To clone and express human neuron-specific enolase (HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis.


METHODS
HuNSE 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.


RESULTS
SDS-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.


CONCLUSIONS
High 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.
Xiao
Xin-li
XL
Institue of Neurobiology, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, China. xxli-2001@126.com
Han
Jun
J
Zhang
Lin
L
Chen
Lan
L
Zhang
Jin
J
Chen
Xin-lin
XL
Zhou
Wei
W
Jiang
Hui-ying
HY
Zhang
Bao-yun
BY
Liu
Yong
Y
Dong
Xiao-ping
XP
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000097:blood / Q000276:immunology*; D007150:Immunohistochemistry; D008297:Male; D010751:Phosphopyruvate Hydratase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017096:Prion Diseases / Q000175:diagnosis* / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D010751:Phosphopyruvate Hydratase