生物学杂志

生物学杂志

• 研究报告 •    下一篇

恒河猴miR-125a-3p慢病毒载体的构建、鉴定以及功能研究

  

  1. 中国医学科学院 北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室， 昆明 650118
  • 出版日期:2017-06-18 发布日期:2017-06-18
  • 通讯作者: 李鸿钧，研究员，从事病毒疫苗研发、病毒与宿主相互关系及分子生物学相关研究，E-mail: lihj6912@163.com
  • 作者简介:刘雅琳，硕士，研究方向为生物化学与分子生物学相关研究，E-mail： 104421990@qq.com
  • 基金资助:
    十二五重大新药创制（2014ZX09102041004）；中国医学科学院医学与健康科技创新工程项目（2016-I2M）；云南省应用基础研究计划项目（2016FB034）；云南省科技计划项目 （2014BC008）

Construction,identification and function of recombinant lentivirus vector for rhesus miR-125a-3p

  1. Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Yunnan
    Key Laboratory of Vaccine Research & Development on Severe Infectious Disease, Kunming 650118， China
  • Online:2017-06-18 Published:2017-06-18

PDF (PC)

128

摘要： 构建携带miR-125a-3p过表达与抑制表达shRNA的慢病毒，研究miR-125a-3p对轮状病毒感染乳鼠导致的腹泻的影响，为进一步研究miR-125a-3p对轮状病毒增殖过程中的影响及探究其机制提供前期研究基础。设计miR-125a-3p前体序列及其反向互补序列，与慢病毒骨架质pLVshRNA-EGFP(2A)Puro载体进行酶切连接，构建过表达与抑制表达miR-125a-3p的慢病毒表达载体。利用HEK293Ta细胞包装慢病毒颗粒，测定病毒滴度后，将获得的miR-125a-3p过表达慢病毒颗粒感染MA104细胞和灌胃感染乳鼠，检测miR-125a-3p的表达。同时对乳鼠进行轮状病毒攻毒试验，观察腹泻情况。研究成功构建了过表达及抑制miR-125a-3p表达的慢病毒载体,获得高效的过表达及抑制miR-125a-3p的慢病毒颗粒，成功感染MA104细胞，以及在乳鼠小肠组织有表达。同时发现miR-125a-3p过表达慢病毒感染乳鼠后对轮状病毒引起的腹泻有明显的抑制作用。

关键词: miR-125a-3p, 慢病毒, 过表达载体, 抑制表达载体, 乳鼠

Abstract: To construct miR-125a-3p overexpression and suppression lentivirus shRNA vectors for studying its effect to diarrhea of miR-125a-3p in mice,which laid a foundation of study miR-125a-3p to provide preliminary preparation for the impact of rotavirus proliferation process and to explore the molecular mechanism, the miR-125a-3p precursor sequence and miR-125a-3p reverse complement sequence were designed, and pLVshRNA-EGFP(2A)Puro vector was used to construct recombination lentivirus plasmid. The recombinant plasmid can generate the miR-125a-3p overexpression and suppression lentivirus vectors. Packaged lentivirus particle contained miR-125a-3p overexpression and suppression lentivirus by HEK293Ta. Virus titer was detected after infection of HEK293Ta. Finally we evaluated miR-125a-3p expression after that the virus infected MA104 cell and injected mice, and diarrhea of mice that was infected by rotavirus.It is successfully to construct the miRNA-125-3p overexpression and suppression lentivirus vectors. The recombinant virus successfully infected MA104 cells and mice small intestine. miR-125a-3p overexpression and suppression lentivirus vector were constructed successfully，which provide a stable cell transfection vector for further study of the impact and mechanism of miR-125a-3p to rotavirus and its host.We also found that the miR-125a-3p overexpression lentivirus vector can inhibitor the diarrhea,which was infected by rotavirus.

Key words: miR-125a-3p； lentivirus vector； overexpression vector； suppression vector, suckling mice