人Toll样受体4重组质粒的构建与鉴定

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-REPORTTM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.

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

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.