针对人caspase8基因的siRNA的载体的构建及其表达.DOCVIP

临床医学论文-针对人caspase8基因的siRNA的载体的构建及其表达 ?【摘要】? 目的 构建针对人caspase8 基因mRNA的siRNA表达载体，并观察其对转染细胞中caspase8的抑制作用。方法 化学合成针对caspase8发夹状的RNA寡核苷酸，将其退火形成双链，连接入经HindⅢ和BglⅡ双酶切后的pSUPER真核表达载体。对重组质粒进行酶切分析和测序鉴定。通过脂质体介导，把重组质粒瞬时转染入HeLa细胞，RTPCR测其对mRNA和蛋白表达的干涉效果。结果 成功地构建了针对人caspase8基因的RNA干涉真核表达载体pSUPERC1和pSUPERC2，并在人HeLa细胞中有效发挥了对caspase8基因的干涉作用，而且pSUPERC1对于caspase8基因的抑制作用要优于pSUPERC2。结论 成功地构建了针对人caspase8基因的siRNA载体，转染HeLa细胞后可抑制caspase8基因的表达。 【关键词】? caspase8；RNA干涉；真核表达载体；HeLa细胞 ??? Abstract:Objective? To construct the eukaryotic expression vector for RNA interferencing human caspase8 gene and detect its interference effect HeLa cell line. Methods? Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors：pSUPERC1 and pSUPERC2．The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing Then HeLa cells were transfected with pSUPERC1 or pSUPERC2， the interference effect was detected by RTPCR. Results? Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSUPER vector respectively．The results of RTPCR indicated that both vectors could knock down the transcription and expression of caspase gene，and that pSUPERC1 had better interference effect than pSUPERCS2. Conclusion? The transcription and expression of caspase8 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the HeLa cells. ??? Key words:caspase8；RNA interference；eukaryotic expression vector；HeLa cells ??? 细胞凋亡(apoptosis)是细胞在受到生理或病理刺激后发生的自发性、无炎症的死亡过程。 近年研究表明，在众多的凋亡相关蛋白中，caspase是细胞凋亡的关键执行者［1］。caspase即天冬氨酸特异性半胱氨酸蛋白酶(cysteinyl aspartate specific protease，caspase)，是一类进化上高度保守的半胱氨酸蛋白酶家族。caspase8 全长有479个氨基酸， 在Fas、TNFR1、DR4、DR5等死亡受体介导的细胞凋亡中，caspase8? 经“诱导接近模式”活化，活化的caspase8 激活下游效应caspase，如caspase3、6、7等，诱发细胞凋亡［24］。 ??? RNA干扰（RNA interference,RNAi）是由双链RNA引发的转录后基因沉默现象，这一机制能够特异性地降低目的基因的表达，是生物体内普遍存在的生理现象。通过对这一现象的深入研究，从利用体外合成双链RNA，