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目的:为了寻求解决非特异性杀伤作用的星形细胞瘤的基因治疗问题的新途径。本研究构建带有StathminsiRNA的复制缺陷腺病毒载体并包装病毒。方法:(1)两个目标siRNA基因片段合成并分别插入pSilencer4.1-CMV载体中从而构建两个重组真核表达载体:pSilencer4.1-CMV-S1和pSilencer4.1-CMV-S2。(2)用EcoR I和BamH Ⅲ双酶切pSilencer4.1-CMV-S1和pSilencer4.1-CMV-S2,将目的片段分别装进穿梭质粒,从而构建pShuttle-CMV neo-S1和pShuttle-CMV neo-S2。以独特的限制性内切酶PI-Sce I合I-CeuI双酶切这两个穿梭质粒并重组至骨架Adeno-XTMViral DNA上。并以PCR方法来鉴定。(3)线性化Ad-pShuttle-CMV neo-S1和Ad-pShuttle-CMV neo-S2并转染入HEK293细胞,出毒后进行PCR毒种鉴定和滴度分析。结果:(1)酶切分析和DNA测序表明,小干扰RNA片段的成功连接到pSilencer4.1-CMV载体上分别。(2)酶切分析表明两个目的基因片段,都分别成功连接入穿梭载体pShuttle。PCR表明pShuttle-CMV neo-S1和pShuttle-CMV neo-S2分别与骨架重组成功。(3)腺病毒DNA进行PCR鉴定后表明成功地在体外重组并生产出腺病毒。且冻融产毒细胞保证了较高的重组腺病毒滴度。结论:Adeno-XTM系统是一个能简单而有效生产能表达目的基因腺病毒的系统。我们构建的腺病毒有望成为星形细胞瘤新的高效而安全的治疗方法。
OBJECTIVE: To find new ways to address the gene therapy of astrocytomas with nonspecific killing. This study constructed replication-deficient adenovirus vector with StathminsiRNA and packaged the virus. Methods: (1) Two target siRNA gene fragments were synthesized and inserted into pSilencer4.1-CMV vector respectively to construct two recombinant eukaryotic expression vectors: pSilencer4.1-CMV-S1 and pSilencer4.1-CMV-S2. (2) pSilencer4.1-CMV-S1 and pSilencer4.1-CMV-S2 were double digested with EcoR I and BamHIII, respectively, and the target fragments were put into shuttle plasmid respectively to construct pShuttle-CMV neo-S1 and pShuttle-CMV neo -S2. The two shuttle plasmids were digested with the unique restriction endonuclease PI-Sce I-I-CeuI and recombined into the framework Adeno-XTMViral DNA. And PCR method to identify. (3) Ad-pShuttle-CMV neo-S1 and Ad-pShuttle-CMV neo-S2 were linearized and transfected into HEK293 cells. Results: (1) Enzyme digestion analysis and DNA sequencing showed that small interfering RNA fragments were successfully ligated into pSilencer4.1-CMV vector respectively. (2) The digestion analysis showed that the two target gene fragments were successfully ligated into the shuttle vector pShuttle respectively. PCR showed that pShuttle-CMV neo-S1 and pShuttle-CMV neo-S2 were successfully recombined with the backbone respectively. (3) The adenovirus DNA was identified by PCR and was successfully recombined in vitro to produce adenovirus. And freezing and thawing toxic cells to ensure a higher recombinant adenovirus titer. Conclusion: The Adeno-XTM system is a simple and effective system for expressing adenovirus of interest. The adenovirus we constructed is expected to be a new efficient and safe treatment of astrocytoma.