靶向survivin的siRNA表达载体的构建及生物活性鉴定  

Construction and identification of survivin-targeting siRNA-expressing plasmid

作　　者：王小林[1] 侯建全[1] 何军[1] 杨慎敏[1] 温端改[1] 

机构地区：[1]苏州大学附属第一医院泌尿外科,江苏苏州215006

出　　处：《临床检验杂志》2007年第1期28-31,共4页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省重点人才基金资助项目(RC2003094);江苏省卫生厅科技基金资助项目(H200517);江苏省教育厅科技基金资助项目(Q1122031);江苏省社会发展基金资助项目(BS2005619)

摘　　要：目的构建靶向survivin的siRNA表达载体。方法设计合成靶向survivin的siRNA相应DNA模板单链,将其克隆入空质粒pRNAT-U6.1/Neo,模板链两端分别设计两个不同的酶切位点,退火形成siRNA载体插入片段。用限制性酶切将空载体线性化,T4DNA连接酶将siRNA片段插入空载体中,对该重组表达载体进行鉴定,获得RNA干扰质粒载体pRNAT-U6.1/Neo-sur-vivin。脂质体法将pRNAT-U6.1/Neo-survivin导入膀胱癌T24细胞株,实时定量PCR法检测对T24细胞survivin基因表达的抑制情况。结果PCR、酶切鉴定、DNA测序证实表达质粒构建成功,无碱基突变。重组载体能干扰T24细胞survivin基因的表达,并具有新霉素抗性,能够用G-418筛选稳定转化的细胞株。结论成功构建了靶向survivin基因表达的RNA干扰质粒载体pR-NAT-U6.1/Neo-survivin,并转染膀胱癌细胞株,为进一步运用RNA干扰技术进行survivin基因功能研究奠定了基础。Objective To construct survivin-targeting siRNA-expressing plasmid. Methods DNA sequence correspond to siRNA targeting survivin was designed and synthesized,and cloned into plasmid pRNAT-U6.1/Neo to produce survivingtargeting plasmid. Two ohgos in the template with cohesive BamH Ⅰ and HindⅢ sites were prepared and anneallcd to form the insert fragment for siRNA vector. The vector was cut with BamH Ⅰ and HindⅢ and ligated with the insert fragment using T4 ligase. The recombinant vector was confirmed by restriction digestion and DNA sequencing, and then was transfected into T24 cells with Lipofectamine^TM2000 and the expression of survivin was detected by real-time quantitive PCR. Results DNA sequencing for the PCR product showed that the recombinant vector pRNAT-U6.1/Neo-survivin was successfully constructed without any base pair mutation. The plasmid pRNAT-U6. 1/Neo-survivin could efficiently reduce the expression of survivin and confer G-418 resistance in T24 cells. Conclusion The siRNA-expressing plasmid which were successfully constructed and transfected into T24 cells in this study may facilitate the application of RNA interference technique, and lay foundation for further studies on the function of survivin.

关 键 词：SURVIVIN基因 小干扰RNA 遗传载体 

分 类 号：Q78[生物学—分子生物学]