小鼠白细胞介素-17及其shRNA重组表达质粒的构建  

Construction and expression of shRNA plasmid for mouse interleukin-17

作　　者：万俊丽[1] 李秋[1] 刘玮[2] 阳海平[1] 崔晶晶[1] 施静[1] 

机构地区：[1]重庆医科大学附属儿童医院肾脏免疫科,重庆400014 [2]重庆医科大学附属儿童医院儿科研究所,重庆400014

出　　处：《中国生物制品学杂志》2013年第11期1556-1560,1566,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(81070563)

摘　　要：目的构建小鼠白细胞介素-17(interleukin-17,IL-17)及其shRNA重组表达质粒。方法以小鼠脾脏细胞总RNA为模板,PCR扩增IL-17基因,并亚克隆至质粒pLVX-IRES-ZsGreen1,构建重组表达质粒pLVX-IL-17-IRESZsGreen1;设计并合成3对针对IL-17基因的shRNA序列和1对阴性对照shRNA序列,分别插入质粒pLVX-shRNA,构建重组干扰质粒pLVX-shRNA1、pLVX-shRNA2、pLVX-shRNA3和阴性对照质粒shRNAC,进行测序;用脂质体法将各重组干扰质粒分别与表达质粒共转染293T细胞,分为空白对照组、过表达组、shRNA1组、shRNA2组、shRNA3组和shRNAC组,经荧光定量PCR和ELISA法分别检测各组细胞中IL-17基因mRNA的转录水平和蛋白的表达水平。结果 PCR扩增获得500 bp的目的基因,IL-17重组表达质粒及其shRNA重组质粒经酶切及测序鉴定证明构建正确;重组表达质粒转染293T细胞48 h后可见绿色荧光表达;过表达组IL-17基因mRNA及蛋白表达水平均明显高于空白对照组(P均<0.05),shRNA1组、shRNA2组和shRNA3组IL-17基因mRNA和蛋白表达水平均明显低于shRNAC组(P均<0.05)。结论成功构建了小鼠IL-17基因重组表达质粒及其特异性shRNA表达质粒,并筛选出shRNA1,其对IL-17的表达具有最佳的抑制效应。Objective To construct the shRNA plasmid for mouse interleukin-17 （IL-17）. Methods IL-17 gene was amplified by PCR using the reversely transcribed product of RNA from spleen cells of BALB/c mice as a template, and subcloned into expression vector pLVX-IRES-ZsGreenl to construct recombinant plasmid pLVX-IL-17-IRES-ZsGreenl. Three pairs of shRNA sequences targeting IL-17 gene and one pair of negative control shRNA sequence were designed and synthesized, then inserted into expression vector pLVX-shRNA to construct shRNA plasmids pLVX-shRNA1, pLVX-shRNA2, pLVX-shRNA3 and negative control plasmid shRNAC respectively, which were identified by sequencing. The 293T cells were divided into six groups. The cells in blank control group were untransfected, and those in overexpression group were transfected with overexpression plasmid, while those in shRNA1, shRNA2, shRNA3 and shRNAC groups were co-expressed with expression plasmid and plasmids pLVX-shRNA1, pLVX-shRNA2, pLVX-shRNA3 and negative control plasmid shRNAC, in mediation of liposome, respectively. The expressions of IL-17 at mRNA and protein levels in various groups were determined by fluorescent quantitative PCR and ELISA, respectively. Results The target gene at a length of 500 bp was amplified by PCR Restriction analysis and sequencing proved that the expression plasmid and shRNA plasmids for IL-17 were constructed correctly. Green fluorescence was observed in the 293T cells 48 h after transfection with the recombinant plasmids. Both the expression levels of IL-17 mRNA and protein were significantly higher in overexpression group than in blank control group （each P 〈 0. 05）, while were significantly lower in shRNA1, shRNA2 and shRNA3 groups than in shRNAC group（each P 〈 0. 05）. Conclusion The expression plasmid and shRNA plasmids for mouse IL-17 were constructed successfully, of which shRNA1 plasmid showed satisfactory inhibitory effect.

关 键 词：白细胞介素-17 短发夹RNA 原发性肾病综合征 

分 类 号：R392.11[医药卫生—免疫学] Q782[医药卫生—基础医学]