小鼠miR-125b慢病毒过表达载体构建与鉴定  

Construction and identification of lentivirus-mediated overexpression vector containing miR-125b in mouse

作　　者：顾玉青[1] 孙杰[1] 李占军[1] 高文涛[1] 钱祝银[1] 

机构地区：[1]南京医科大学第一附属医院胆胰外科,江苏省210029

出　　处：《江苏医药》2013年第14期1616-1618,F0002,共4页Jiangsu Medical Journal

基　　金：江苏省高校优势学科建设工程(PAPD)

摘　　要：目的构建小鼠胰腺β细胞NIT-1中miR-125b慢病毒过表达载体。方法从小鼠基因组DNA中用PCR扩增miR-125b的前体序列,经NheⅠ和PstⅠ酶切后连接到Lv-CMV-GFP载体中,并对重组质粒进行双酶切鉴定和测序分析。将miR-125b过表达载体、pVSV-G和delta8.91质粒共转染293T细胞,收获慢病毒感染NIT-1细胞,流式细胞术分选阳性细胞,RT-PCR鉴定稳转细胞株中miR-125b的表达。结果成功构建了miR-125b慢病毒表达载体,感染小鼠胰腺β细胞NIT-1后能够成功过表达miR-125b。结论成功构建miR-125b慢病毒过表达载体,为进一步研究其功能建立了实验基础。Objective To construct the lentivirus-mediated overexpression vector containing miR-125b in mouse pancreatic β cell line NIT-1.Methods Precursor sequence of miR-125b was amplified from the mouse genomic DNA and inserted into Lv-CMV-GFP vector with restriction enzymes NheⅠand PstⅠ.The recombinant plasmid was identified by double digestion and sequencing.miR-125b overexpression,pVSV-G and delta8.91 vectors were co-transfected into 293T cells,and NIT-1 cells were infected with recombinant lentivirus.The positive cells were separated by flow cytometry,and miR-125b expression was detected by RT-PCR.Results The lentivirus-mediated expression vector containing miR-125b was successfully constructed,which could be stably overexpressed in mouse pancreatic β cell line NIT-1.Conclusion miR-125b expression vector has been successfully constructed,which provides a foundation for further studying the function and mechanism of miR-125b.

关 键 词：miR-125b 慢病毒过表达载体 胰腺Β细胞 

分 类 号：R735.102[医药卫生—肿瘤]