大鼠ZnT1基因重组腺病毒载体的构建及鉴定

摘要

Objective To establish a recombinant adenovirus vector containing rat ZnT1 gene and to express the gene efficiency in eukaryotic cells. Methods Using chemosynthesis method to synthesize the interest gene Znt1/IRES/EGFP. Adding attB recombination site to the interest gene both ends thought PCR method,the later and pDONR221 vector with attP site was create into a entry vector pDown-ZnT1 through BP recombination. The entry vector with attL site and the target vector Ad/CMV/V5-DEST with attR site were recombined together to create the expression vector pAd-ZnT1 under LR reaction. Enzyme digesting and sequencing appraisal were used to prove the expression vector,digesting with Pac I enzyme,and transferred into HEK293A cells to be packaged, then the maturated adenovirus were extracted. The recombinant adenovirus titre was measured by endpoint dilution method. The interest protein s expression was analyzed by western blotting. Results Our interest gene was transferred into target vector Ad/CMV/V5-DEST correctly with right open reading frame by LR recombination reaction,and it was confirmed by restriction enzyme analysis and sequencing appraisal. The expression clone was packaged into HEK293A cells successfully. Finally, we got maturated adenovirus particles, and virus titre was 1. 6 × 108 pfu/L. It was confirmed with high level expression by western blotting method in HEK293A cell. Conclusion In this experiment, we successfully and efficiently constructed the rat ZnT1 gene s recombinant adenovirus vector,and laid a good foundation for the ZnT1 gene to express in spinal cord nerve cell and the relationship with BDNF/TrkB signal regulation channel.%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.