含PR结构域的锌指蛋白5特异性小干扰RNA载体构建及干扰效果评价  

Construction and evaluation of PR domain containing 5 specificity small interfering RNA vector

作　　者：丁琼琼[1] 张彬彬[1] 张亚平[1] 杨海杰[1] 王磊[1] 冯志伟[1] 

机构地区：[1]新乡医学院生命科学技术学院,河南新乡453003

出　　处：《新乡医学院学报》2016年第3期174-177,共4页Journal of Xinxiang Medical University

基　　金：河南省高等学校重点科研计划项目(编号:15A180010)

摘　　要：目的构建含PR结构域的锌指蛋白5(PRDM5)特异性小干扰RNA(siRNA)干扰载体并鉴定其干扰效果。方法根据小鼠PRDM5基因全长c DNA序列,设计合成6条siRNA干扰片段(si PRDM5-1、si PRDM5-2、si PRDM5-3、si PRDM5-4、si PRDM5-5和si PRDM5-6)和2条对照序列(si CTL-1和si CTL-2),并将前3条干扰片断和si CTL-1克隆插入p Silencer3.1-U6,另3条干扰片断和si CTL-2插入p Silencer4.1-CMV干扰载体中,构建小鼠PRDM5基因的siRNA真核表达载体si PRDM5-1、si PRDM5-2、si PRDM5-3、si PRDM5-4、si PRDM5-5和si PRDM5-6,2个对照序列si CTL-1和si CTL-2,并通过双酶切和测序进行验证,构建成功后转染小鼠黑色素瘤细胞B16F10并采用实时荧光定量聚合酶链反应和Western blot法检测其干扰效率。结果成功构建真核siRNA表达载体,并且所构建的6个干扰载体均可降低小鼠黑色素瘤细胞中PRDM5基因mRNA表达(P<0.05)。其中si PRDM5-2和si PRDM5-6载体干扰效果最好。结论成功筛选出PRDM5特异性siRNA载体,为之后研究锌指蛋白基因PRDM5在小鼠黑色素瘤转移过程中功能及分子机制打下基础。Objective To construct PR domain containing 5（ PRDM5） specificity small interfering RNA（ siRNA） interference vector and identify its interference effect. Methods According to the whole coding c DNA sequence of mouse PRDM5 gene,six siRNA interference fragments（ si PRDM5-1,si PRDM5-2,si PRDM5-3,si PRDM5-4,si PRDM5-5 and si PRDM5-6） and two control sequence（ si CTL-1 and si CTL-2） were designed. The first three interference fragments and si CTL-1 were inserted into p Silencer3. 1-U6 interference vector,and the other three interference fragments and si CTL-2 were inserted into p Sliencer4. 1-CMV interference vector. The six siRNA eukaryotic expression vectors（ si PRDM5-1,si PRDM5-2,si PRDM5-3,si PRDM5-4,si PRDM5-5 and si PRDM5-6） of PRDM5 gene and two control sequence（ si CTL-1and si CTL-2） were constructed. The recombinant plasmid was verified by double enzyme digestion and sequencing. Then the above vectors were transfected into mouse B16F10 cells and the interference effect was determined by real-time quantitative polymerase chain reaction（ q PCR） and Western blot method. Results The siRNA eukaryotic expression vectors were constructed successfully,and all of the six interference vectors could reduce the PRDM5 gene mRNA expression（ P〈 0. 05）. The interference effect of si PRDM5-2 and si PRDM5-6 vector was best. Conclusion This study successfully constructed efficient PRDM5 specificity siRNA vector,which provide experimental basis for further studying the function of PRDM5 gene in mouse melanoma cells B16F10.

关 键 词：含PR结构域的锌指蛋白5 小干扰RNA载体 干扰 

分 类 号：Q78[生物学—分子生物学]