大鼠半胱氨酸蛋白水解酶-3基因真核表达质粒和RNA干扰质粒的构建及鉴定  

Construction and Identification of Eukaryotic Expression Plasmids of Caspase-3 and RNA Interfer- ence Plasmids of Rats

作　　者：吴靖平[1] 朱斌[1] 丁磊[1] 李德芳[1] 

机构地区：[1]复旦大学附属金山医院骨科,上海200540

出　　处：《中华实验外科杂志》2011年第9期1564-1567,共4页Chinese Journal of Experimental Surgery

基　　金：上海市卫生局基金资助项目（2009-35市卫生局）

摘　　要：目的构建SD大鼠半胱氨酸蛋白水解酶（Caspase）-3的真核表达质粒和干扰质粒，探究干扰质粒对Caspase-3表达的干预效应。方法从SD大鼠软骨终板细胞中获得模板cDNA并扩增，约834bp。克隆至载体pEGFP—N1，获得重组质粒pEGFP-N1-Caspase-3。针对Caspase-3设计4对RNAi靶点序列，克隆至载体pcDNA6．2。成功构建pcDNA6．2-caspase．3．1、pcDNA6．2-caspase-3-2、pcDNA6．2-Caspase-33、pcDNA6．2-caspase-3-4，构建对照质粒pcDNA6．2-HIiRNA。将5个干扰质粒分别与pEGFP．N1．Caspase_3（用量比为7：1）共转染至293T细胞，qPCR检测转染48h后对caspase．3表达的干预效应。结果通过菌落聚合酶链反应（PCR）、酶切及测序表明pEGFP-N1-Capase-3和干扰质粒构建成功；共转293T细胞后，qeCR示pcDNA6．2-Caspase-32、pcDNA6．2-Caspase-3-3干预组的caspase-3的表达高于对照组，分别为33％和8％，而pcDNA6．2-Caspase-3-1、pcDNA6．2-Caspase-34干预组的caspase-3的表达与对照组比较明显减少，抑制效率分别为24％和67％（P〈0．05）。结论成功构建了Caspase．3的表达质粒和干扰质粒，并证实pcDNA6．2-Caspase-34对Caspase-3的表达具有明显的抑制效应。Objective To construct the eukaryotic expression plasmids and RNA interference plasmids of Caspase-3 in SD rats, and to investigate their inhibitory effects on Caspase-3. Methods The cDNA was obtained from the endplate cartilage cells of SD rats, and the full-length fragment of Caspases-3 was constructed, about 834 bp. The target segment was cloned into the expression vector pEGFP-N1 and the recombinant plasmid pEGFP-N1-Caspases-3 was obtained. Four pairs of pre-microRNA （pre-miRNA） sequences were designed according to Caspases-3 gent, and pre-miRNA was cloned into the expression vector pcDS!A6. 2, while non-related interference plasmid was constructed, pcDNA6. 2-Caspases-3-1, pcDNA6, 2- Easpases-3-2, pcDNh6. 2-Caspases-3-3, pcDNh6. 2-Caspases-3-4 and pcDNA6. 2-rniRNC were constructed successfully, and they were co-transfected into 293T cells with pEGFP-N1-Caspases-3 （7：1 ）. The inhibition of Caspases-3 expression was analyzed by qPCR 48 h after transfection. Results pEGFP-N1-Caspases-3 and RNA interference plasmids were successfully constructed and confirmed by colony PCR, Western blotting and sequence analysis, qPCR results showed that in the intervention group of pcDNA6. 2-Caspase-3-2 and pcDNA6. 2-Caspases-3-3, Caspases-3 expression levels were increased as compared with the control group （33% and 8%, respectively）. But in the intervention group of pcDNA6. 2-Caspase-3-1 and pcDN6. 2- Caspases-3-4, Caspases-3 expression levels were significantly reduced as compared with the control group, and the inhibition efficiency reached 24% and 67% （P 〈0. 05）. Conclusion Caspases-3 eukaryotic expres- sion vector and the RNA interference plasmids were constructed successfully, and it was verified that pcD- NA6. 2-Caspase-3-4 could specifically inhibit the Caspases-3 expression at the cellular level.

关 键 词：RNA干扰 半胱氨酸蛋白水解酶-3 质粒 

分 类 号：R681.53[医药卫生—骨科学]