携带小鼠Brcc3基因重组腺病毒的包装及鉴定

发布时间：2018-01-27 17:06

本文关键词： 重组腺病毒载体 Gateway技术限制性酶切和连接 Brcc3　出处：《扬州大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：构建能够在体外真核细胞中过表达目标基因Brcc3的重组腺病毒真核表达载体,并进而使用HEK293细胞包装重组腺病毒,从而为后续转染小鼠心脏成纤维细胞(cardiofibroblasts, cFs)探讨细胞内Brcc3基因过表达对TGF-β信号通路各环节的影响做准备。方法： 1、目的基因片段的扩增(扩增包括双酶切位点的目标基因序列) 分别以Ndel-Sacl-Brcc3-F和Brcc3-R为上下游引物扩增Sacl-Brcc3;以Brcc3-GST-F和Xhol-Spel-GST-R扩增GST-Xhol及以Ndel-Sacl-Brcc3-F和Xhol-Spel-GST-R扩增Sacl-Brcc3-GST-Xhol。 2、利用酶切连接构建pDown-Brcc3/GST/IRES/EGFP并测序鉴定 Sacl/Xhol双酶切骨架载体pDown-MCS-IRES/EGFP和PCR回收产物Sacl-Brcc3-GST-Xhol,并将骨架片段pDown-MCS-IRES/EGFP和目的基因Brcc3-GST连接,接着转化连接产物到感受态细胞大肠杆菌stbl3,进行菌落PCR鉴定后挑取单克隆摇菌提取质粒pDown-Brcc3/GST/IRES/EGFP送测序。 3、利用Gateway Technology构建pAV.Ex1d-CMVBrcc3/GST/IRES/EGFP pDown-Brcc3/GST/IRES/EGFP和pAV.Des1d进行LR反应,将目标基因克隆到腺病毒载体pAV.Des1d,于(反应产物转化到)大肠杆菌感受态细胞stbl3中进行(复制),PCR鉴定阳性克隆,挑单克隆摇菌提取质粒送检测序。 4、将重组腺病毒转染293A细胞,进行病毒的包装和扩增,并提取转染的CFs细胞蛋白行Western blot检测Brcc36蛋白表达水平。主要结果： 1、经琼脂糖电泳鉴定表明目的片段Sacl-Brcc3-GST-Xhol扩增成功。 2、经PCR检测、琼脂糖电泳及基因测序表明载体pDown-Brcc3/GST/IRES/EGFP构建成功。 3、经PCR检测、琼脂糖电泳、基因测序及western blot分析表明重组腺病毒真核表达载体pAV.Exld-CMVBrcc3/GST/IRES/EGFP构建成功。主要结论：本研究利用Gateway技术成功构建重组腺病毒真核表达载体pAV.Exld-CMVBrcc3/GST/IRES/EGFP,并成功包装获得过表达Brcc3基因的腺病毒。
[Abstract]:Objective: To construct the recombinant adenovirus eukaryotic expression vector which can over-express the target gene Brcc3 in eukaryotic cells in vitro, and then use HEK293 cells to package the recombinant adenovirus. Therefore, it can be used to transfect the cardiac fibroblasts of mouse heart fibroblasts. CFS) to investigate the effect of overexpression of Brcc3 gene on TGF- 尾 signaling pathway. Methods: 1. Amplification of target gene fragments (amplification of target gene sequences including double restriction sites) Sacl-Brcc3 was amplified with Ndel-Sacl-Brcc3-F and Brcc3-R as upstream and downstream primers, respectively. Amplification of GST-Xhol with Brcc3-GST-F and Xhol-Spel-GST-R and Ndel-Sacl-Brcc3-F and Xhol-Spel-GS with Ndel-Sacl-Brcc3-F. T-R amplification of Sacl-Brcc3-GST-Xhol. 2. PDown-Brcc3/GST/IRES/EGFP was constructed by restriction endonuclease ligation and identified by sequencing Sacl/Xhol double enzyme digestion framework carrier pDown-MCS-IRES/EGFP and PCR recovery product Sacl-Brcc3-GST-Xhol. The skeleton fragment pDown-MCS-IRES/EGFP was ligated with the target gene Brcc3-GST, and then the ligation product was transformed into the competent Escherichia coli stbl3. After the colony PCR identification, the clone pDown-Brcc3/GST/IRES/EGFP was selected and sequenced. Using Gateway Technology to build pAV.Ex1d-CMVBrcc3/GST/IRES/EGFP The target gene was cloned into adenovirus vector pAV.Des1d by LR reaction with pDown-Brcc3/GST/IRES/EGFP and pAV.Des1d. The positive clones were identified by PCR in the stbl3 of E. coli competent cells, and the plasmids extracted from monoclonal bacteria were selected and sequenced. 4Recombinant adenovirus was transfected into 293A cells for packaging and amplification. The expression of Brcc36 protein was detected by Western blot. Main results: 1. The fragments of the epidermis were amplified successfully by agarose electrophoresis. 2. By PCR detection, agarose electrophoresis and gene sequencing showed that the vector pDown-Brcc3/GST/IRES/EGFP was successfully constructed. 3. Agarose electrophoresis was performed by PCR. Gene sequencing and western blot analysis showed that the recombinant adenovirus eukaryotic expression vector pAV.Exld-CMVBrcc3/GST/IRES/EGFP was successfully constructed. Main conclusions: In this study, the recombinant adenovirus eukaryotic expression vector pAV.Exld-CMVBrcc3/GST/IRES/EGFP was successfully constructed by using Gateway technique. The adenovirus expressing Brcc3 gene was successfully packaged.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R3416

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