The
enolase2 gene is usually expressed in mature neurons and also named neuron specific enolase (
NSE). In the present study, we first obtained the
NSE gene cDNA sequence by using the RACE method based on the expressed sequence tag (EST) fragment from the cDNA library of
Gekko japonicus and identified one transcript of about 2.2 kb in central nervous system of
Gekko japonicus by Northern blotting. The open reading frame of
NSE is 1305 bp, which encodes a 435 amino-acid protein. We further investigated the multi-tissue expression pattern of
NSE by RT-PCR and found that the expression of
NSE mRNA was very high in brain, spinal cord and low in heart, while it was not detectable in other tissues. The real-time quantitative PCR was used to investigate the time-dependent change in the expression of the
NSE mRNA level after gecko spinal cord transection and found it significantly increased at one day, reaching its highest level three days post-injury and then decreasing at the seventh day of the experiment. The recombinant plasmid of pET-32a-NSE was constructed and induced to express His fused NSE protein. The purified NSE protein was used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65536 determined by ELISA. Western blotting showed that the prepared antibody could specifically recognize the recombinant and endogenous NSE protein. The result of immunohistochemistry revealed that positive signals were present in neurons of the brain and the spinal cord. This study provided the tools of cDNA and polyclonal antibody for studying NSE function in
Gekko japonicus.
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