人Toll样受体4重组质粒的构建与鉴定  

Construction and identification of recombinant plasmid of Toll-like receptor 4

作　　者：钱钧[1] 郭彩玲[1] 崔洪波[1] 宋武琦[1] 李爱梅[1] 

机构地区：[1]哈尔滨医科大学微生物学教研室、黑龙江省感染与免疫重点实验室、黑龙江省高校医学病原学重点实验室、伍连德研究所,150081

出　　处：《国际免疫学杂志》2016年第6期543-546,共4页International Journal of Immunology

基　　金：黑龙江省教育厅科学技术研究项目（12521289）

摘　　要：目的 构建人Toll样受体4（TLa-4）3＇非编码区（UTR）野生型/突变型基因重组表达质粒.方法 在293T细胞中提取基因组DNA为模板,扩增并获得TLR4基因的3＇UTR片段.根据TLR43＇UTR与miR-122的靶点预测,设计TLR4 3＇UTR突变型TLR4 3＇UTR-m1.将pmiR-RB-REPORT^TM质粒和TLR4 3＇UTR/TLR4 3＇UTR-m1 PCR产物双酶切,将酶切后的载体与PCR产物连接,转化入DHSα细胞,在含Amp抗生素的LB固体培养基上培养,筛选阳性菌株,进行PCR验证及序列测定.结果 成功构建人TLR4 3＇UTR野生型/突变型重组质粒.结论 构建的质粒可以用于TLR4与miR-122相互作用的研究,为研究miRNA的作用提供依据.Objective To construct Toll-like receptor 4 （TLR4） 3＇untranslated region （UTR） wild/ mutant type recombinant plasmids.Methods C enomic DNA was extracted in 293T cells as a template,and TLR4 3＇UTR gene fragment was amplified.According to the predicted binding of miR-122 and TLR4 3＇UTR,mutant types of TLR4 3＇UTR were designed as TLR4 3＇UTR-m1.The vector plasmid pmiR-RB-REPORTTM and TLR4 3＇UTR/TLR4 3＇UTR-m1 PCR product were digested and connected.The product was transformed to DH5αt cells and the bacterial with the recombinant plasmid were cultured overnight in Amp^＋ LB solid media.The colonies were picked for PCR validation and sequencing.Results TLR4 3＇UTR/TLR4 3＇UTR-m1 recombinant plasmids were successfully constructed and identified.Conclusion The recombinant plasmid can be used in the study of miR-122 function in cells.

关 键 词：TOLL样受体4 基因重组 质粒 

分 类 号：R392.3[医药卫生—免疫学]