天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (7): 717-720.doi: 10.11958/j.issn.0253-9896.2015.07.004

• 细胞与分子生物学 • 上一篇    下一篇

miR-223 敲减慢病毒的构建

张欣，关晓慧，王宝利   

  1. 天津医科大学代谢病医院内分泌研究所、 卫生部激素与发育重点实验室(邮编 300070)
  • 收稿日期:2014-11-05 修回日期:2015-02-04 出版日期:2015-07-15 发布日期:2015-07-15
  • 通讯作者: 王宝利 E-mail： bliwang72@163.com E-mail:zhangxinhb@126.com
  • 作者简介: 张欣(1985)， 女， 助理实验师， 硕士， 主要从事生物化学与分子生物学研究
  • 基金资助:
    国家自然科学基金资助项目 （81271977， 81472040）

Establishment of miR-223 knockdown lentivirus

ZHANG Xin, GUAN Xiaohui, WANG Baoli   

  1. Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China
  • Received:2014-11-05 Revised:2015-02-04 Published:2015-07-15 Online:2015-07-15
  • Contact: WANG Baoli E-mail： bliwang72@163.com E-mail:zhangxinhb@126.com

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摘要： 目的 构建 miR-223 敲减慢病毒， 为进一步研究 miR-223 的功能提供工具。方法 采用 Invitrogen 公司miR-RNAi 在线设计工具， 根据 miR-223 成熟体序列设计了一对互补寡核苷酸片段,退火产物连接至 pcDNA6.2-GW/EmGFP-miR 载体， 经酶切连入 pCDH-CMV-MCS-EF1-copGFP 载体， 构建了 miR-223 敲减慢病毒质粒， 利用293T 细胞将其包装成病毒， 并感染 ST2 细胞， 观察感染效率并用 qRT-PCR 检测 miR-223 成熟体表达。结果 酶切鉴定及测序结果显示成功构建了 miR-223 敲减慢病毒载体， miR-223 敲减慢病毒包装并感染靶细胞， 表达绿色 GFP荧光蛋白的细胞约占细胞总数的 80%~90%， 病毒滴度为 1×109 PFU/mL， 感染效率达 90%。感染 miR-223 敲减慢病毒的细胞 miR-223 表达量（0.31±0.03）低于阴性对照（1.00±0.09）， 为阴性对照的 31%， 差异有统计学意义（n=3， t=15.091， P＜0.05）。结论 成功构建了 miR-223 敲减慢病毒，为进一步研究 miR-223 的功能准备了条件。

关键词: 微 RNAs, 慢病毒属, RNA 干扰, 慢病毒源性载体, miR-223

Abstract: Objective To construct miR-223 knockdown lentivirus vector and provide a tool for further study of the function of miR-223. Methods According to the Invitrogen miR-RNAi online design tool, a pair of complementary oligonucleotides encoding miR-223 mature sequence was designed, annealed and ligated with pcDNA6.2-GW/EmGFP-miR vector. Then miR-RNAi expression cassette was cut and subcloned into lentiviral pCDH-CMV-MCS-EF1-copGFP vector. The lentiviruses were packaged and titered, and then ST2 cells were infected with viruses. The efficiency of infection was calculated, and the knockdown of endogenous miR-223 was detected by using real-time RT-PCR. Results Restriction enzyme digestion and sequencing results showed that miR- 223 lentivirus construct was successfully made. Lentivirus that knockdown miR-223 expression packaged and infected of target cells. The expression of GFP green fluorescent protein accounted for 80%-90% and the virus titer was 1×109 PFU/mL. The infection efficiency reached 90%. Compared with negative control virus, miR-223 knockdown lentivirus significantly down-regulated the expression of miR-223 in ST2, and was 31% (n=3, t=15.091, P＜0.05). Conclusion miR-223 knockdown lentivirus is successfully made. It provides a tool for further studying the function of miR-223.

Key words: microRNAs, lentivirus, RNA interference, lentivirus based vector, miR-223