CAJ | 학술논문

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/V5DEST-TRESK.转化DH5α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PacⅠ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781 bp,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-TRESK gDNA为模板的PCR扩增,目的片段781 bp,鉴定结果与预期相符.腺病毒滴度为1.31×109 TU/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.
목적 구건대서TRESK기인중조선병독재체.방법 종대서배근신경절세포중극륭TRESK전장cDNA,진행PCR감정급DNA측서험증,구건이CMV계동자전록조공적pAd/CMV/V5DEST-TRESK.전화DH5α대장간균,도선양성중조극륭행PCR감정병행DNA측서.장측서정학적질립경PacⅠ매절선성화,전염포장293T세포,포장산생선병독,축공희석적도법측정병독적도.결과 종대서배근신경절세포중극륭적TRESK전장cDNA위781 bp,DNA측서험증DNA서렬여GeneBank 중수록적대서TRESK서렬완전일치.이pAD-GFP공재체위대조,pAD/CMV/V5-DEST-TRESK gDNA위모판적PCR확증,목적 편단781 bp,감정결과 여예기상부.선병독적도위1.31×109 TU/ml.결론 본연구성공지구건료대서TRESK기인중조선병독재체: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. DH5α 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/V5-DEST-TRESK gDNA was matched with pAD-GFP blank vector as anticipated. The titer of the concentrated virus was 1.31×109 TU/ml. Conclusion Rat TRESK gene recombinant adenovirus vector is constructed successfully.