Cloning, expression, and antibody preparation of nestin with immunohistochemical analysis

OBJECTIVE: To obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development. METHODS: The nestin cDNA was cloned from human neural stem cells by RT-PCR and ligated to prokaryotic expression plasmid pQE30 for construction of the recombinant vector pQE30-nestin. After sequencing, the recombinant vector was transformed into E.coli M15 and His-tagged nestin was induced by IPTG. The nestin was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blotting. BALB/c mice were immunized with the purified recombinant protein to prepare the antiserum, which was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: The nestin gene was successfully cloned from human neural stem cells, which was identical to that reported in GenBank. After IPTG induction, the E.coli transformed with pQE30-nestin plasmid expressed a 25,000 His-tagged protein, which was successfully purified and identified as nestin by Western blotting. Western blotting, ELISA and immunohistochemistry demonstrated that the antiserum could specifically bind to the recombinant nestin as well as to nestin in fetal human and rat brains. CONCLUSION: We successfully cloned the nestin gene and expressed the nestin, and nestin mAb prepared can specifically recognize not only the recombinant nestin, but also nestin from human and rats brain tissues.