人miR-16真核表达载体的构建与鉴定  

Construction and Identification of Eukaryotic Expression Vector of miR-16

作　　者：张小勇[1] 余冰[1] 倪明[2] 陈姗姗[1] 吕婷婷[1] 江敏[1] 杨银柯 陆蒙吉[1] 杨东亮[3] 

机构地区：[1]华中科技大学同济医学院病原生物学系微生物室,武汉市430030 [2]华中科技大学同济医院感染科,武汉市430030 [3]华中科技大学同济医院临床免疫室,武汉市430030

出　　处：《医学分子生物学杂志》2008年第1期31-34,39,共5页Journal of Medical Molecular Biology

基　　金：湖北省科技攻关项目(No.2006AA301C22)~~

摘　　要：目的构建人miR-16真核表达载体,为研究其基因表达调控机制建立实验基础。方法从人类基因组DNA中扩增miR-16的前体序列,引入酶切位点和polyT转录终止序列,酶切后插入siRNA表达载体pSuper中,构建miR-16真核表达载体pmiR-16,然后进行酶切和测序鉴定;同时构建含miR-16完全互补序列及乙肝表面抗原HBsAg的报告质粒pXF3H-HBs。将pmiR-16与pXF3H-HBs共转染肝癌细胞系HepG2,通过HBsAg ELISA检测试剂盒检测表达产物HBsAg的改变,鉴定真核表达载体pmiR-16的生物学活性。结果成功构建了人miR-16真核表达载体pmiR-16及其报告质粒。pmiR-16与pXF3H-HBs共转染HepG2后,pmiR-16组HBsAg的基因表达与对照组相比显著降低,证实pmiR-16转染真核细胞后具有生物学活性。结论miR-16真核表达载体的成功构建为进一步研究其基因表达调控机制建立了实验基础。Objective To construct eukaryotic expression vector of miR-16. Methods miR-16 precursor sequence was amplified from the human genomic DNA, and was introduced into the restriction sites and polyT transcription terminate sequence. The target gene was inserted to the digested pSuper siRNA expression vector. Restriction digestion, ligation and sequencing were performed to evaluate the recombinant. Then pXF3H-HBs expression vector containing miR-16 complementary sequence was constructed the report plasmid-HBs were constructed. The pmiR-16 and report vector pXF3H-HBs were co-transfected into HepG2 cells to detect the biological activity of pmiR-16. Resuits The miR-16 expression vector and its report vector pXF3H-HBs were successfully construc- ted. pmiR-16 and pXF3H-HBs were co-transfected into HepG2 cell line, HBsAg gene expression, as compared with the control group, was decreased significantly. Co-transfection confirmed the biological activity of pmiR-16. The expression of HBsAg was obviously decreased. Conclusion pmiR- 16 expression vector has been successfully constructed.

关 键 词：基因的表达与调控 MICRORNA miR-16 报告质粒 

分 类 号：Q939.93[生物学—微生物学]