The Cloning and Characterization of the Enolase2 Gene of Gekko japonicus and Its Polyclonal Antibody Preparation

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.

Different Astrocytic Activation between Adult Gekko japonicus and Rats during Wound Healing In Vitro

“…All experimental protocols applied to the animals were approved by the Laboratory Animal Care and Use Committee of our medical school. Animal models of spinal cord injury were established using procedures described previously [ 11 ]. Briefly, rats were anesthetized with 1% pentobarbital sodium, and a laminectomy was applied at the T9 to T10 spinal segment level.…”

Depletion of C3orf1/TIMMDC1 Inhibits Migration and Proliferation in 95D Lung Carcinoma Cells

“…According to the methods followed in our previous study [ 27 ], total RNA was isolated from cells by using TRIzol reagent (Life Technologies, Grand Island, NY, USA). The first strand cDNA was synthesized using an Omniscript Reverse Transcription Kit (Qiagen, Venlo, Limburg, The Netherlands) in a 20-μL reaction system containing 2 μg total RNA.…”

“…The sequences of the universal control (Ctrl) siRNA and gene-specific c3orf1 siRNA are listed in the Table 4 . Protocols used for siRNA experiments were based on our previous study [ 27 ]. For studies involving siRNA in cultured cells, isolated and dissociated cells were transfected using Lipofectamine2000 (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.…”

Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system

“…It is possible that the refolding and aggregation speed was sometimes too fast when the recombinant protein refolded, leading to the random aggregation formation of certain protein species by the 2 disulfide bonds. The SDS-PAGE results demonstrated that some recombinant proteins were still present in the loaded liquid flow of the Ni 2+ column, indicating that the affinity of the Ni 2+ column on the recombinant His tag protein did not reach saturation, or that the recombinant protein structure itself limited the affinity of the Ni 2+ column on the His tag (20,21). Based on the above analysis, 500 mmol/l of imidazole was selected to directly elute the Ni 2+ column instead of gradient elution to facilitate subsequent protein purification.…”