重组质粒pSUPER-HBs RNAi的构建及筛选 |
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作者姓名: | 罗祥基 程庆保 徐峰 谭蔚锋 姜小清 张柏和 王红阳 吴孟超 |
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作者单位: | 第二军医大学东方肝胆外科医院胆道一科,上海,200438 |
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基金项目: | 上海市科委重点基金(54119519). |
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摘 要: | 目的:利用pSUPER-RNAi载体系统进行HBs RNAi序列的初步筛选和干扰效果鉴定。方法:设计并合成针对HBs基因区的3条siRNA(HBs-siRNA1、HBs-siRNA2、HBs-siRNA3),构建3种重组载体pSUPER-HBs-siRNA,与HBV质粒同时瞬转人胚肾293T细胞,72 h后观察转染效果,实时定量PCR和ELISA法鉴定3种干扰序列对HBs的干扰效果,筛选最佳干扰序列。结果:成功构建3种干扰质粒pSUPER-HBs-siRNA,能高效转染人胚肾上皮细胞293T,转染效率在70%以上;实时定量PCR和ELISA法结果均显示HBs-siRNA2可有效抑制HBs的表达,抵制率可达80%,而HBs-siRNA1、HBs-siRNA3干扰效果不显著,差异具有统计学意义(P<0.05)。结论:成功构建3种干扰质粒pSUPER-HBs-siRNA,筛选出其中能有效抑制HBs基因表达的pSUPER-HBs-siRNA2序列,为后续研究奠定了基础。
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关 键 词: | pSUPER载体 HBs基因 小RNA干扰 筛选 |
收稿时间: | 7/25/2008 9:48:00 AM |
修稿时间: | 2008/9/22 0:00:00 |
Construction and screening of pSUPER-HBs-siRNA efficiently targeting HBs gene |
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Authors: | LUO Xiang-ji CHENG Qing-bao XU Feng TAN Wei-feng JIANG Xiao-qing ZHANG Bai-he WANG Hong-yang WU Meng-chao |
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Institution: | Department of Biliary Surgery,Eastern Hepatobiliary Hospital,Second Military Medical University,Shanghai 200438,China |
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Abstract: | Objective:To develop a system to screen for the effective siRNA sequence targeting HBs gene and to identify the interference efficiency.Methods:Three HBs-targeting siRNA segments (HBs-siRNA1,HBs-siRNA2,and HBs-siRNA3) were designed,synthesized and cloned into pSUPER vector to construct three recombinant plasmids pSUPER-HBs-siRNA,which were then transfected into human embryonic kidney 293T cells together with HBV plasmid.The transfection efficiency was observed 72 h later,the interference efficacies of the 3 segments were identified by real-time PCR and ELISA analysis,and the best one was identified.Results:Three recombinant plasmids of pSUPER-HBs-siRNA were constructed successfully and effectively transfected into 293T cells to induce RNAi,with a transfection rate higher than 70%.The results of real-time PCR and ELISA analysis showed that HBs-siRNA2 silenced the HBs gene expression by more than 80%.Compared with HBs-siRNA2,HBs-siRNA1 and HBs-siRNA3 did not demonstrate obvious interfering effect (P<0.05).Conclusion:We have successfully constructed 3 siRNA sequences targeting HBs,and pSUPER-HBs-siRNA2 can effectively silence HBs genes,which paves a way for future study. |
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Keywords: | pSUPER vector HBs gene small interfering RNA screening |
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