人Stathmin基因siRNA腺病毒载体的构建及病毒鉴定

Construction of human Stathmin gene siRNA adenovirus vector and Virus identification

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 construct replication-defective adenoviral vectors with Stathmin specific siRNA,and to seek a new way to resolve non-specific killing effect of gene therapy for astrocytoma.Methods：（1）Two target siRNA gene segments were synthesized and cloned into pSilencer4.1-CMV neo vector respectively to construct two recombinant eukaryotic expres-sion vectors：pSilencer4.1-CMV neo-S1 and pSilencer4.1-CMV neo-S2.（2）After amplified with EcoR I and BamH I I I enzyme action site and cloned into pShuttle,the two shuttle plasmids pShuttle-CMV neo-S1 and pShuttle-CMV neo-S2 were constructed.Dealing with the unique restriction endonucleases PI-Sce I and I-Ceu I digestion,the two shuttle plasmids were in vitro ligated with Adeno-XTM Viral DNA and the recom-binant DNA were identified with unique PCR methods.（3）Transfer Ad-pShuttle-CMV neo-S1 and Ad-pShut-tle-CMV neo-S2 after Linearization,when CPE appears,test the adenoviral DNA using PCR.Results：（1）Enzyme digestion analysis and DNA sequencing showed that the target siRNA segments was cloned into pSilencer4.1-CMV neo vector respectively.（2）Enzyme diges-tion analysis and showed that the target genetic fragments were cloned into pShuttle vector respectively.（3）The unique PCR of adenoviral DNA identified the successful in vitro ligation of recombinant adenovirus.A series of freeze-thaw cycles guaranteed a relative high titer of recombinant adenoviruses.Conclusion：Adeno-XTM System is a simplified and efficient expressin system for rapid generation of recombinant adenoviruses,which ensures the target gene＇s efficient expression.It may be a useful pathway through which Astrocytoma can be treated with high efficiency and safety.