携带SLC基因的Birc5-siRNA质粒载体的构建及鉴定

摘要

Objective To construct the Birc5‐siRNA vector carrying SLC in order to provide a basis for the research on tumor biological targeted therapy.Methods Interfering sequences of Birc5 were designed by using Ambion Target Finder.The Birc5‐siRNA was inserted into PEGFP6‐1 ,and then transformed and cultured with E. coli. Afterwards ,the plasmid was extrac‐ted and identified.SLC was inserted into pGenesil‐8 ,which was transformed ,cultured with E. coli ,followed by the extraction and identification of the plasmid. The bacterial transformation solution was selected after identification with enzyme diges‐tion. The plasmid that silenced Birc5 and carried SLC was constructed by molecular cloning technology and named pgsiRNA‐Birc5+SLC. Results The plasmid Birc5 was cleaved by EcoRⅠ,resulting in a 400 bp DNA strip ,indicating that Birc5‐siRNA was insert‐ed into PEGFP6‐1. Plasmid pGenesil‐8‐SLC was cleaved by BglⅡ+ XbaⅠto form a 430 bp DNA strip ,which suggested that SLC was in‐serted into pGenesil‐8.The sequences of the recombinant plasmid were proved to be completely correct.Restriction enzyme analysis showed the Birc5‐siRNA plasmid carrying SLC was successfully constructed.Conclusion The Birc5‐siRNA plasmid carrying SLC was successfully constructed ,which provides a tool and basis for further study of tumor targeted therapy aimed at SLC and Birc 5.%目的：构建携带SLC基因的Birc5‐siRNA质粒载体，为肿瘤靶向治疗研究提供基础。方法使用Ambion公司siRNATarget Finder设计的Birc5 mRNA干扰序列，将Birc5‐siRNA插入载体 PEGFP6‐1后转化大肠埃希菌扩增培养，提取质粒；将SLC插入质粒pGenesil‐8后转化细菌扩增培养，提取质粒；挑取酶切鉴定正确的质粒转化菌液测序鉴定；采用分子克隆技术构建特异性沉默Birc5且携带SLC基因的质粒，命名为pgsiRNA‐Birc5＋SLC。结果质粒Birc5能被 EcoRⅠ酶切出1条约400 bp的DNA条带，说明Birc5‐siRNA 已插入质粒载体PEGFP6‐1；pGenesil‐8‐SLC能被BglⅡ＋ XbaⅠ酶切出1条约430 bp的DNA条带，说明SLC插入正确；酶切鉴定正确的质粒转化菌液Birc5‐siRNA和SLC测序结果显示均为插入正确的克隆质粒；经酶切鉴定分析显示pgsiRNA‐Birc5＋SLC符合酶切鉴定结果，表明携带SLC基因的Birc5‐siRNA质粒载体构建成功。结论成功构建了携带SLC基因的Birc5‐siRNA质粒，为进一步研究靶向沉默肿瘤Birc5基因并趋化淋巴细胞抗肿瘤研究提供了工具和基础。