人miR-146b慢病毒载体构建及人脂肪细胞中表达验证

中国儿童保健杂志 ›› 2013, Vol. 21 ›› Issue (11): 1155-1157.

人miR-146b慢病毒载体构建及人脂肪细胞中表达验证

陈玲^1,2,史春梅^1,2,杨蕾^1,2,朱璐¹,徐广峰¹,季晨博²,郭锡熔^1,2,张春梅²

1 南京医科大学儿科研究所,江苏南京 210029; 2 南京医科大学附属南京妇幼保健院,江苏南京 210004

收稿日期:2013-01-05 发布日期:2013-11-06 出版日期:2013-11-06
通讯作者: 张春梅,E-mail:zhangcm79@163.com
作者简介:陈玲(1987-),女,江苏人,博士在读,主要研究方向为儿童肥胖症。
基金资助:
国家自然科学基金(81100619);江苏省医学创新团队项目(LJ201108);南京市科技发展计划(201104013)

Construction of a recombinant lentiviral vector carrying miR-146b and validation of its transduction efficiency in human pre-adipocytes

CHEN Ling^1,2,SHI Chun-mei^1,2,YANG Lei^1,2,ZHU Lu¹,XU Guang-feng¹,JI Chen-bo²,GUO Xi-rong^1,2,ZHANG Chun-mei²

1 Institute of Pediatrics,Nanjing Medical University,Nanjing,Jiangsu 210029,China; 2 Nanjing Maternal and Child Health Hospital Affiliated to Nanjing Medical University,Nanjing,Jiangsu 210004,China

Received:2013-01-05 Online:2013-11-06 Published:2013-11-06
Contact: ZHANG Chun-mei,E-mail:zhangcm79@163.com

摘要/Abstract

摘要： 目的构建人miR-146b慢病毒表达载体,包被病毒并检测其在人脂肪细胞中过表达效果。方法以人基因组DNA为模板,PCR高保真扩增包含miR-146b区域上下游各100bp左右的基因组DNA,亚克隆入慢病毒表达载体,与包装质粒共转染HEK-293T细胞,包装病毒,收取病毒上清,纯化后感染人脂肪前体细胞,36 h开始观察荧光标记,72 h收取细胞,抽提RNA,Realtime PCR检测慢病毒感染下miR-146b的相应表达量。结果成功构建人miR-146b慢病毒表达载体,包装获得的病毒感染人脂肪细胞的效率可达到85%以上,miR-146b过表达水平可接近4倍。结论本研究成功构建了人miR-146b慢病毒表达载体,包被的慢病毒可以在人脂肪细胞中实现过表达效果,为后续功能研究奠定了基础。

关键词: miR-146b, 慢病毒, 人脂肪前体细胞

Abstract: Objective To construct a recombinant lentiviral vector carrying miR-146b and validate its infection efficiency in human pre-adipocytes. Methods The minigene fragment of miR-146b was amplified from human genomic DNA by PCR,then cloned into a lentivirus expression vector.After sequencing validated exactly,the expression plasmid and packaging plasmids Pcgvp,Rev,Vsvg were co-transfected into HEK-293T cells to produce lentivirus; the supernatant containing lentiviral particles was used to infect human preadipocytes,and its infection efficiency and miR-146b overexpression level were respectively tested by fluorescent observation and real-time quantitative PCR. Results MiR-46b lentiviral vector was constructed successfully,and infection efficiency in human pre-adipocytes reached over 85%,the expression level increased nearly four folds. Conclusion We have successfully constructed the lentiviral vector containing miR-146b with high overexpression in infected human pre-adipocytes,which will be useful to the future studying the function of miR-146b in human pre-adipocytes.

Key words: miR-146b, lentivirus, human preadipocytes

中图分类号:

R394

陈玲,史春梅,杨蕾,朱璐,徐广峰,季晨博,郭锡熔,张春梅. 人miR-146b慢病毒载体构建及人脂肪细胞中表达验证[J]. 中国儿童保健杂志, 2013, 21(11): 1155-1157.

CHEN Ling,SHI Chun-mei,YANG Lei,ZHU Lu,XU Guang-feng,JI Chen-bo,GUO Xi-rong,ZHANG Chun-mei. Construction of a recombinant lentiviral vector carrying miR-146b and validation of its transduction efficiency in human pre-adipocytes[J]. journal1, 2013, 21(11): 1155-1157.