SOCS3基因3’端非翻译区野生型与突变型载体的构建及活性鉴定  

Construction of the wildtype and mutant vectors of socs3 3＇ -UTR and activity determination

作　　者：牛立萍 寇艳波 王静 吴清源[2] 房贤达 周峰 郑葵阳 汤仁仙 刘晓梅 

机构地区：[1]徐州医科大学病原生物学与免疫学教研,江苏徐州221004 [2]江苏省镇江中学,江苏镇江212017 [3]徐州市第三中学,江苏徐州221004

出　　处：《徐州医学院学报》2016年第5期311-315,共5页Acta Academiae Medicinae Xuzhou

基　　金：江苏省科技厅面上项目（BK20151168,BK20141136）;江苏省脑病生物信息重点实验室开放课题（2015KF01）;徐州市科技计划项目（KC14SH074）;徐州医学院专项人才基金（2012KZJ02）

摘　　要：目的研究非编码小RNA-1896（miRNA-1896）和miRNA-409—3p对细胞因子信号抑制蛋白-3（suppressors of cytokinesignaling-3，SOTS3）基因的调控作用，构建floes3基因3’端非翻译区（3’-untranslated re-gion，3’-UTR）野生型和突变型重组荧光素酶报告载体。方法以原代培养的小鼠星形胶质细胞总eDNA为模板，通过点突变和缺失突变的方式分别对SOCS33’-UTR序列中miRNA-1896和miRNA-409—3p种子区的结合位点CAGAGA（897—902位）和AACATT（1425—1430位）进行突变，并将野生型的3’-UTR序列与突变的3’-UTR序列分别插入到虫荧光素酶表达质粒pGL3-Promoter获得重组质粒，命名为pGL3-SOCS3-WT、pGL3-SOCS3-M1和pGL3-SOCS3-M2。将上述重组质粒分别与miRNA-1896和miRNA-409—3p共转染至HEK293T细胞中，测定虫荧光素酶的活性。结果酶切验证及测序结果表明，重组质粒pGL3-SOCS3-WT、pGL3-SOCS3-M1和pGL3-SOCS3-M2构建成功。与对照组相比，转染miRNA-1896和miRNA-409—3p均显著降低了pGL3-SOCS3-WT虫荧光素酶的活性，但对pGL3-SOCS3-M1和pGL3-SOCS3-M2荧光素酶的活性并无明显影响。结论成功构建了soc33’-UTR野生型及突变型的虫荧光素酶重组表达质粒，确认CA-GAGA（897—902位）和AACATr（1425—1430位）分别是miRNA-1896和miRNA-409—3p种子区与socs33’-UTR结合的关键位点。Objective To investigate the regulation of micro RNA - 1896 （miRNA - 1896） and miRNA -409 -3p on suppressors of cytokine signaling - 3 （socs3） gene and construct a luciferase reporter plasmid carrying the wild - type and mutant sots3 3＇ - untranslated region （3＇ - UTR）. Methods The total eDNA of mouse primary astroeytes was used as templates. Then, the binding sites within miRNA - 1896 （ CAGAGA, 897 - 902） and miRNA - 409 - 3p （ AACATT, 1425 - 1430） of soes3 3＇ - UTR were mutated by point mutation or deletion. The wild - type and mutant 3＇ - UTRs were inserted into a lueiferase expressing vector pGL3 - promoter, resulting in recombinant plasmids namely pGL3 - SOCS3 - WT, pGL3 - SOCS3 - M1 and pGL3 - SOCS3 - M2. Furthermore, these plasmids together with miRNA - 1896 and miR- NA -409 -3p were co -transfected into HEK293T cells to determined luciferase activities. Results The recombinant plasmids pGL3 - SOCS3 - WT, pGL3 - SOCS3 - M1 and pGL3 - SOCS3 - M2 were successfully constructed through restriction enzyme digestion and sequencing. Compared with the control, miRNA- 1896 and miRNA -409 -3p could re- markably weaken the luciferase activity of pGL3 - SOCS3 - WT, rather not that of pGL3 - SOCS3 - M1 or pGL3 - SOCS3 - M2. Conclusion The recombination plasmids were successfully constructed. Meanwhile, CAGAGA （897 - 902） and AACArTT （1425 -1430） are the key binding sites of miRNA- 409- 3p.

关 键 词：微RNA 细胞因子信号抑制蛋白-3 3’端非翻译区 虫荧光素酶报告载体 

分 类 号：Q782[生物学—分子生物学]