shRNA-Snail载体的构建及其病毒的包装

发布时间：2018-06-16 01:13

本文选题：腺相关病毒 + shRNA-Snail　；参考：《实用口腔医学杂志》2017年06期

【摘要】：目的:构建一种抑制Snail基因表达的腺相关病毒载体系统。方法:在GenBank中获取Snail基因的核苷酸序列,设计并合成2条Snail基因的shRNA,应用分子生物学方法将腺相关病毒(AAV)载体与shRNA(TUD)相结合,构建p AAV-U6-shRNA-Snail-TUD-EGFP表达质粒和p AAV-RC及p AAV-Helper质粒共转染至AVV-293细胞中,包装生成携带sh Snail-TUD的重组腺相关病毒r AAV-U6-shRNA-Snail-TUD-EGFP。将重组病毒感染至口腔鳞癌细胞Tca8113和Cal-27中,实时定量PCR检测sh Snail对细胞内Snail基因的沉默效果,并测定病毒感染滴度。结果:DNA测序证明构建的shRNA-Snail基因沉默子序列正确并成功克隆到腺病毒表达载体上;AAV-293细胞表达绿色荧光蛋白提示共转染成功。感染口腔鳞癌细胞Tca8113和Cal-27细胞后,能显著下调口腔鳞癌细胞Tca8113和Cal-27细胞内的Snail mRNA和蛋白的表达水平。重组AAV感染滴度为4.4×1010/ml。结论:包装的重组腺相关病毒r AAV-U6-shRNA-Snail-TUD-EGFP可抑制细胞Snail基因表达。
[Abstract]:Aim: to construct an adeno-associated virus vector system that inhibits Snail gene expression. Methods: the nucleotide sequence of Snail gene was obtained from GenBank, and two shRNAs of Snail gene were designed and synthesized. The adeno-associated virus (AAVV) vector was combined with shRNA-TUD by molecular biology. The pAAV-U6-shRNA-Snail-TUD-EGFP expression plasmid and the pAAV-RC and pAAV-Helper plasmids were co-transfected into AVV-293 cells, and the recombinant adeno-associated virus r AAV-U6-shRNA-Snail-TUD-EGFPwas generated by pAAV-U6-shRNA-Snail-TUD-EGFP. The recombinant virus was infected into oral squamous cell carcinoma Tca8113 and Cal-27. The silencing effect of sh Snail on Snail gene was detected by real-time quantitative PCR, and the titer of virus infection was measured. Results the sequence of shRNA-Snail gene silencer was correct and cloned into adenovirus expression vector. The expression of green fluorescent protein in AAV-293 cells indicated that the gene was cotransfected successfully. The expression of Snail mRNA and protein in oral squamous carcinoma Tca8113 and Cal-27 cells was significantly decreased after infection with Tca8113 and Cal-27 cells. The titer of recombinant AAV infection was 4.4 脳 10 ~ (10) / ml. Conclusion: recombinant adeno-associated virus rAAV-U6-shRNA-Snail-TUD-EGFP can inhibit Snail gene expression.
【作者单位】：海南医学院第一附属医院;
【基金】：海南省重点研发计划项目(编号:ZDYF2016113)
【分类号】：R739.8

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