Construction and identification of mouse killer gene siRNA recombinant adnovirnovius

Yang Gen-ling; et al

doi:10.13406/j.cnki.cyxb.2010.02.006

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Construction and identification of mouse killer gene siRNA recombinant adnovirnovius

更新时间：2024-01-17

- Construction and identification of mouse killer gene siRNA recombinant adnovirnovius
- Journal of Chongqing Medical University Vol. 35, Issue 2, Pages: 165-168(2010)
- 作者机构：
  
  重庆医科大学实验动物中心
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.02.006
  CLC： R341
- Published：2010，
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[1]杨根岭,张晋平,徐倩,谭冬梅,赖国旗,谭毅.小鼠MK基因siRNA重组腺病毒载体的构建及鉴定[J].重庆医科大学学报,2010,35(02):165-168.

Yang Gen-ling, et al. Construction and identification of mouse killer gene siRNA recombinant adnovirnovius. [J]. Journal of Chongqing Medical University 35(2):165-168(2010)
[1]杨根岭,张晋平,徐倩,谭冬梅,赖国旗,谭毅.小鼠MK基因siRNA重组腺病毒载体的构建及鉴定[J].重庆医科大学学报,2010,35(02):165-168. DOI： 10.13406/j.cnki.cyxb.2010.02.006.

Yang Gen-ling, et al. Construction and identification of mouse killer gene siRNA recombinant adnovirnovius. [J]. Journal of Chongqing Medical University 35(2):165-168(2010) DOI： 10.13406/j.cnki.cyxb.2010.02.006.

摘要

目的:构建小鼠肿瘤坏死因子相关凋亡诱导配体的死亡受体（Mouse killer

MK）基因干扰RNA（Small interfering RNA

siRNA）重组腺病毒并在小鼠子宫基质细胞中鉴定其干扰作用。方法:人工合成靶向MK的siRNA干扰序列

将其克隆到穿梭载体pSES-HUS上

并与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组

得到pAdeasy-SES-HUS-MKsiRNA

在HEK293细胞中包装并扩增腺病毒颗粒。用纯化后的腺病毒混合液感染原代培养的小鼠子宫内膜基质细胞

RT-PCR及Western Blot检测基质细胞中MK的mRNA及蛋白表达水平。结果:Adeasy-SES-HUS-MKsiRNA混合液均效价为6.8×10

pfu/ml

感染原代培养的小鼠子宫内膜基质细胞48 h后

MK的mRNA及蛋白水平均显著下调。结论:构建的MK干扰表达腺病毒能有效地抑制MK基因的表达

为进一步研究MK在小鼠子宫基质细胞的功能提供了有效的工具。

Abstract

Objective:To construct the recombinant adenovirus carrying a short interfering RNA（siRNA）targeting MK（mouse killer） gene and identify its inhibitive effect in mouse uterine stromal cells.Methods:The siRNA sequence targeting MK gene was synthesized and cloned into the shuttle plasmid pSES-HUS to generate the vector pSES-HUS-MKsiRNA

which was later homogenously recombinanted with the adenovirus backbone plasmid pAdeasy-1 in Ecoli BJ5183.Then the recombinant adenovirus was transfected into HEK293 cells to be packaged and amplified.The primarily cultured mouse stromal cells were infected with this purified adenovirus and the expression levels of MK mRNA and protein were detected by RT-PCR and Western Blot.Results:The recombinant adenoviral vector containing MK siRNA was correctly constructed with an average titer of 6.8×10

pfu/mL.Both the MK mRNA and protein levels were signifcantly decreased in the uterine stromal cells after 48 hours of infection with MK siRNA recombinant adenovirus.Conclusion:The generated recombinant adenovirus Adeasy-MK-siRNA was demonstrated to significantly abrogate the expression of MK at mRNA and protein level in mouse uterine stromal cells.

关键词

Mouse killer基因RNA干扰重组腺病毒

Keywords

Mouse killer geneRNA interferencerecombinant adenovirus

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Related Institution

Department of Radiology,The First Affiliated Hospital of Chongqing Medical University

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重庆医科大学公共卫生学院生殖医学教研室

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