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目的:构建DD3启动子调控并携带SATB1基因shRNA溶瘤腺病毒,研究其对前列腺癌的特异抗肿瘤作用。方法:以pSilencer3.1-SATB1为模板,通过PCR扩增出SATB1-shRNA表达框并克隆至pZD55载体,构建重组质粒pZD55-SATB1-shRNA。EcoRⅤ和XbaⅠ分别酶切pZD55-SATB1-shRNA和pZXC2-DD3-E1A,将目的片段连接重组,获得质粒pDD3-ZD55-SATB1,将其与腺病毒包装质粒pBHGE3共转染293细胞。PCR鉴定正确者即为溶瘤腺病毒DD3-ZD55-SATB1。扩增,纯化,测病毒滴度。结晶紫染色观察对前列腺癌细胞毒性;Western印迹检测E1A在列腺癌细胞中的表达;RT-PCR和Western印迹检测列腺癌细胞中SATB1基因沉默效果。结果:成功构建DD3启动子调控同时携带SATB1-shRNA的溶瘤腺病毒DD3-ZD55-SATB1,初步证实DD3-ZD55-SATB1复制具有高度的前列腺癌靶向性和特异的SATB1基因沉默效果。结论:成功构建的溶瘤腺病毒DD3-ZD55-SATB1为进一步体内外研究其对前列腺癌的治疗作用奠定基础。
OBJECTIVE: To construct the DD3 promoter to regulate and carry the SATB1 gene shRNA oncolytic adenovirus to study its specific anti-tumor effect on prostate cancer. Methods: The sTB1-shRNA expression cassette was amplified by PCR using pSilencer3.1-SATB1 as a template and cloned into pZD55 vector to construct recombinant plasmid pZD55-SATB1-shRNA. PZD55-SATB1-shRNA and pZXC2-DD3-E1A were digested with EcoRV and XbaI respectively. The recombinant plasmid pDD3-ZD55-SATB1 was co-transfected into 293 cells with adenoviral packaging plasmid pBHGE3. PCR identification is the oncolytic adenovirus DD3-ZD55-SATB1. Amplification, purification, virus titer. The expression of E1A in prostate cancer cells was detected by crystal violet staining and the SATB1 gene silencing effect in prostate cancer cells by RT-PCR and Western blot. Results: DD3 promoter was successfully constructed to control the oncolytic adenovirus DD3-ZD55-SATB1 carrying SATB1-shRNA, which demonstrated that DD3-ZD55-SATB1 replication has a high degree of prostate cancer targeting and specific SATB1 gene silencing. Conclusion: The constructed oncolytic adenovirus DD3-ZD55-SATB1 lays the foundation for further study of its therapeutic effect on prostate cancer in vitro and in vivo.