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The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, Jiangsu, China
Clinical Laboratory, the Central Hospital of Huzhou, Huzhou 313000, Zhejiang, China
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed.
Received: 6 February 2013; in revised form: 7 April 2013 / Accepted: 16 April 2013 / Published: 24 April 2013
Abstract: 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.
Keywords: Gekko japonicus; Molecular cloning; Neuron specific enolase (NSE); polyclonal antibody
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MDPI and ACS Style
Li, J.; Wu, R.; Chen, H.; Zhou, Y.; Li, Y.; Wang, Y.; Liu, Y.; Liu, M. The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation. Int. J. Mol. Sci. 2013, 14, 8787-8800.
Li J, Wu R, Chen H, Zhou Y, Li Y, Wang Y, Liu Y, Liu M. The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation. International Journal of Molecular Sciences. 2013; 14(5):8787-8800.
Li, Jing; Wu, Ronghua; Chen, Haijiao; Zhou, Youlang; Li, Yan; Wang, Yongjun; Liu, Yan; Liu, Mei. 2013. "The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation." Int. J. Mol. Sci. 14, no. 5: 8787-8800.