中文摘要：

目的构建RelB基因siRNA慢病毒载体及检测其沉默效率。方法针对已经筛选确定的RelB基因RNAi有效靶序列，合成5对靶序列的Oligo DNA，退火形成双链DNA，与经XhoI和KspAI双酶切后的pLentiLox3．7质粒载体[含u6启动子和绿色荧光蛋白（GFP）]连接形成pLentiLox—sh—RelB慢病毒载体，PCR筛选阳性克隆，酶切测序鉴定。然后用脂质体包裹转染小鼠RM—1前列腺癌细胞，运用Western blot法检测小鼠前列腺癌细胞RelB蛋白的表达，以鉴定sh—RelB的沉默效率。用pVSYG，plp1，plp2慢病毒包装系统质粒在磷酸钙介导F共转染包装细胞293T细胞，包装产生慢病毒，以293T细胞GFP蛋白的表达水平测定病毒滴度。结果聚合酶链反应（PCR）和测序证实，成功构建RelB—shRNA的慢病毒载体pLentiLox—sh—RelB。Western blot鉴定最有效的siRNA序列为5’-TGTCGTCAGGATCTGCTTC-3’，病毒液过滤后测定滴度为8×10^10 pfu／L。结论成功构建小鼠RelB基因RNAi慢病毒载体。

英文摘要：

Objective To construct a recombinant lentivirus plasmid of RNA interference （RNAi） of RelB gene. Methods According to the GenBank information of RelB ,5 complementary DNA containing both sense and antisense Oligo DNA of the targeting sequences were designed, synthesized, and cloned into PlentiLox 3.7 which contained U6 promoter and green fluorescent protein （GFP）. The positive clones were confirmed by PCR, sequencing, and authentication with KspA I and Hind Ⅲ. RM-1 cells were transfected and detected by Western-blot. 293T cells were cotransfected with lentiviral vector and virus packaging plasmids. All virus stocks were produced by calcium phosphate mediated transfection. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of RelB was constructed successfully. The most effective siRNA sequence was 5＇ -TrGGAAATCATCGACGAAT-3 ＇. The titer of concentrated virus was 8 ×10^10 pfu/L. Conclusion The lentivirus RNAi vector of RelB was constructed successfully. The lentivirus RNAi vector of RelB significantly inhibited the RelB expression.