大鼠ZnT1基因重组腺病毒载体的构建及鉴定
发布时间：2019-05-24 23:57
【摘要】：目的利用GatewayTM技术构建含大鼠锌转运体1(ZnT1)基因重组腺病毒载体,鉴定外源基因在真核细胞中的良好表达。方法采用化学合成的方法,合成ZnT1/RES/EGFP目的基因片段,通过PCR方法在基因片段两端加入attB重组位点,与含有attP重组位点的pDonr221供体载体BP反应形成入门载体pDown-ZnT1。将含有attL重组位点的入门载体与含有attR位点目的载体Ad/CMV/V5-DEST通过LR反应形成腺病毒表达载体pAd-ZnT1。酶切和测序鉴定后,由PacⅠ酶切线性化转染HEK293A细胞包装,提取病毒颗粒。采用终点稀释法测定重组腺病毒滴度;Western blotting技术分析目的蛋白表达情况。结果目的基因按正确的方向重组入克隆载体中,重组腺病毒表达载体在HEK293A细胞中包装成功,获得成熟的腺病毒颗粒,病毒滴度为1.6×108 pfu/L,在HEK293A细胞中高表达。结论该实验采用GatewayTM技术构建了含有大鼠ZnT1基因的重组腺病毒载体,为下一步研究该基因在脊髓神经细胞中的表达以及与BDNF/TrkB信号调节通路的关系奠定了基础。
[Abstract]:Objective to construct recombinant adenoviral vector containing rat zinc transporter 1 (ZnT1) gene by GatewayTM and identify the good expression of foreign gene in eukaryotic cells. Methods the target gene fragment of ZnT1/RES/EGFP was synthesized by chemical synthesis. AttB recombination site was added to both ends of the gene fragment by PCR, and the entry vector pDown-ZnT1. was formed by reaction with pDonr221 donor vector BP containing attP recombination site. The entry vector containing attL recombination site and the target vector Ad/CMV/V5-DEST containing attR site were reacted with the target vector Ad/CMV/V5-DEST to form adenoviral expression vector pAd-ZnT1. by LR. After restriction endonuclease digestion and sequencing, the virus particles were extracted from HEK293A cells and packaged by HEK293A cells by restriction endonuclease digestion and sequencing. The expression of the target protein was analyzed by end-point dilution method. The recombinant adenoviral titer; Western blotting technique was used to analyze the expression of the target protein. Results the target gene was recombined into the clone vector in the right direction. The recombinant adenoviral expression vector was successfully packaged in HEK293A cells, and the mature adenoviral particles were obtained. the virus titer was 1.6 脳 108 pfu/L, and the expression of the recombinant adenoviral expression vector was high in HEK293A cells. Conclusion the recombinant adenoviral vector containing rat ZnT1 gene was constructed by GatewayTM technique, which laid a foundation for further study of the expression of the gene in spinal cord nerve cells and its relationship with BDNF/TrkB signal regulation pathway.
【作者单位】： 辽宁医学院附属第一医院骨科;辽宁医学院附属第一医院中心实验室;
【基金】：国家自然科学基金资助项目(81171799)
【分类号】：R651.2
[Abstract]:Objective to construct recombinant adenoviral vector containing rat zinc transporter 1 (ZnT1) gene by GatewayTM and identify the good expression of foreign gene in eukaryotic cells. Methods the target gene fragment of ZnT1/RES/EGFP was synthesized by chemical synthesis. AttB recombination site was added to both ends of the gene fragment by PCR, and the entry vector pDown-ZnT1. was formed by reaction with pDonr221 donor vector BP containing attP recombination site. The entry vector containing attL recombination site and the target vector Ad/CMV/V5-DEST containing attR site were reacted with the target vector Ad/CMV/V5-DEST to form adenoviral expression vector pAd-ZnT1. by LR. After restriction endonuclease digestion and sequencing, the virus particles were extracted from HEK293A cells and packaged by HEK293A cells by restriction endonuclease digestion and sequencing. The expression of the target protein was analyzed by end-point dilution method. The recombinant adenoviral titer; Western blotting technique was used to analyze the expression of the target protein. Results the target gene was recombined into the clone vector in the right direction. The recombinant adenoviral expression vector was successfully packaged in HEK293A cells, and the mature adenoviral particles were obtained. the virus titer was 1.6 脳 108 pfu/L, and the expression of the recombinant adenoviral expression vector was high in HEK293A cells. Conclusion the recombinant adenoviral vector containing rat ZnT1 gene was constructed by GatewayTM technique, which laid a foundation for further study of the expression of the gene in spinal cord nerve cells and its relationship with BDNF/TrkB signal regulation pathway.
【作者单位】： 辽宁医学院附属第一医院骨科;辽宁医学院附属第一医院中心实验室;
【基金】：国家自然科学基金资助项目(81171799)
【分类号】：R651.2
【二级参考文献】
相关期刊论文 前1条
1 徐小良,戴克戎,汤亭亭,郁朝锋,徐e，
本文编号：2485302
本文链接：https://www.wllwen.com/xiyixuelunwen/2485302.html
最近更新
教材专著
