大鼠TRESK基因重组腺病毒载体的构建

Contruction of rat TRESK gene recombinant adenovirus vector

目的构建大鼠TRESK基因重组腺病毒载体。方法从大鼠背根神经节细胞中克隆TRESK全长cDNA，进行PCR鉴定及DNA测序验证，构建以CMV启动子转录调控的pAd／CMV／V5-DEST-TRESK。转化DH5a大肠杆菌，挑选阳性重组克隆行PCR鉴定并行DNA测序。将测序正确的质粒经Pac Ⅰ酶切线性化，转染包装293T细胞，包装产生腺病毒，逐孔稀释滴度法测定病毒滴度。结果从大鼠背根神经节细胞中克隆的TRESK全长cDNA为781bp，DNA测序验证DNA序列与GeneBank中收录的大鼠TRESK序列完全一致。以pAD-GFP空载体为对照，pAD／CMV／V5-DEST-TRESKgDNA为模板的PCR扩增，目的片段781bp，鉴定结果与预期相符。腺病毒滴度为1．31×10^9Tu／ml。结论本研究成功地构建了大鼠TRESK基因重组腺病毒载体：pAD／CMV／V5-DEST-TRESK。

Objective To construct rat TRESK gene recombinant adenovirus expression vector. Methods TRESK full-length cDNA was cloned from rat dorsal root ganglion cells and confirmed by RT-PCR and sequencing. pAd/CMV/V5-DEST-TRESK was then constructed under the control of CMV-promotor. DH5a colibacillus was translated and the positive recombinants were subsequently identified by PCR and DNA sequencing. 293T cells were cotransfected and packed to produce adenovirus. Results The titer of virus was tested using Hole-by-dilution titer method. The full length of TRESK from rat dorsal root ganglion cells is 781 bp. It was demonstrated that the DNA sequencing was completely consistent with TRESK sequencing of rat recorded in GeneBank. The PCR amplification of the pAd/CMV/VS-DEST-TRESK gDNA was matched with pAD-GFP blank vector as anticipated. The titer of the concentrated virus was 1.31 × 10^9 TU/ml. Conclusion Rat TRESK gene recombinant adenovirus vector is constructed successfully.