重组腺病毒载体Ad5-hTRX-EGFP的构建及其表达

Construction and Expression of Recombinant Adenovirus Vector Ad5-hTRX-EGFP

本研究旨在构建并制备人硫氧还蛋白(hTRX)和增强型绿色荧光蛋白(EGFP)基因腺病毒载体Ad-hTRX-EGFP,感染HEK293细胞,为基因治疗提供实验基础。设计含有NotⅠ和EcoRⅤ酶切位点的引物,PCR扩增hTRX,将扩增产物连接到带有EGFP标记的pDC316-mCMV穿梭质粒上,构建重组穿梭质粒pDC316-hTRX-EGFP,利用Lipofectamine2000脂质体的方法将AdMax腺病毒包装系统的骨架质粒pBHG lox_E1,3Cre和穿梭质粒pDC316-hTRX-EGFP共转染入HEK293细胞,进行同源重组,得到腺病毒重组质粒pAd-hTRX-EGFP,并在其中包装扩增病毒,用氯化铯高速梯度离心、纯化病毒,测定病毒颗粒数及滴度。采用PCR方法对重组腺病毒进行鉴定,用流式细胞仪测定感染HEK293细胞的效率,Western blot方法验证细胞表达hTRX蛋白。结果显示,重组腺病毒质粒经PCR和NotⅠ、EcoRⅤ酶切鉴定,证实含有hTRX基因,测序结果和设计片段的序列一致。重组腺病毒载体构建成功,病毒滴度达5.558×1010pfu/ml。病毒成功感染HEK293细胞,MOI=100时,感染效率达92.25%。经Western blot方法验证表明,感染后的HEK293细胞高表达hTRX蛋白。结论:应用细胞内同源重组方法成功构建了含hTRX基因的重组腺病毒载体,制备获得高滴度的病毒,能高效感染HEK293细胞并表达目的蛋白,为后续研究奠定了基础。

This study was purposed to construct and prepare the recombinant adenovirus vector carrying human thioredoxin（hTRX） and enhanced green fluorescence protein（EGFP）,and transfect it into HEK293 cells,so as to lay a foundation for further gene therapy.The PCR-amplified products of hTRX with a pair of primers containing Not Ⅰ and EcoR Ⅴ restriction sites were subcloned into shuttle plasmid pDC316-mCMV.HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC316-hTRX-EGFP and large adenovirus-helper plasmid pBHGlox（delta）E1,3Cre in mediation of liposome.The obtained replication-defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells,purified by CsCl gradient centrifugation,counted for virus particles and determined for titer.The recombinant adenovirus was identified by PCR.The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry.The expression of hTRX was confirmed by Western blot.The results showed that according to PCR and restriction endonuclease assay,the target gene was inserted into recombinant adenovirus vector successfully.The sequence of fusion gene was the same as that of designed fragments.The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5.558×1010pfu/ml.A transfection efficiency of 92.25% could be achieved at MOI=100.Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells.It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells,which laid a foundation for further investigation.