Objective:To construct an adeno-associated vector expressing Snail "tough decoy" (TUD).Methods:The nucleotide sequence of Snail gene were obtained from GenBank,then 2 cDNAs were designed and synthesized coding expression of small hairpin RNAs for Snail genes.The pAAV-U6-shRNA-Snail-TUD-EGFP expression vector was constructed by molecular biological techniques.Then pAAV-U6-shRNA-Snail-TUD-EGFP was co-transfected with Helper Free adeno-associated virus system pAAV-RC and pAAV-Helper into AAV-293 cell line to form rAAY-U6-shRNA-Snail-TUD(shRNA-Snail).The silencing effect of shRNA-Snail in Tca8113 and Cal-27 cells was detected by RT-PCR.The titer of the virus was measured.The expression level of green fluorescent protein in AAV-293 cells was monitored by the fluorescent microscopy.Results:The result of gene sequencing showed that shRNA-Snail was successful constructed in pAAV-U6-shRNA-Snail-TUD-EGFP vector.The titer of the recombined virus was 4.4 × 1010/ml.The Snail mRNA and protein expression level was significantly reduced in Tca8113 and Cal-27 cells by rAAY-U6-shRNA-Snail-TUD-EGFP transfection.Conclusion:rAAY-U6-shRNA-Snail-TUD-EGFP may inhibite Snail gene expression in cells.%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pAAV-U6-shRNA-Snail-TUD-EGFP表达质粒和pAAV-RC及pAAV-Helper质粒共转染至AVV-293细胞中,包装生成携带shSnail-TUD的重组腺相关病毒rAAV-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shSnail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rAAV-U6-shRNA-Snail-TUD-EGFP可抑制细胞Snail基因表达.
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