| 李俊堂1 王立锋2 王芳1 杨韬1 许彦鸣2△ 杨安钢1.突变型人TRBP 基因的构建及表达[J].,2012,12(16):3033-3036 |
| 突变型人TRBP 基因的构建及表达 |
| Construction and Expression of Mutant TRBP Gene |
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| DOI： |
| 中文关键词: TRBP 基因 突变 pFLAG-CMV4 真核表达 |
| 英文关键词: TRBP Mutation pFLAG-CMV4 Eukaryotic expression |
| 基金项目: |
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| 中文摘要: |
| 目的：探讨RNAi 过程中TRBP 传递双链RNA 的机制，构建RNA 结合结构域突变的人TRBP 基因真核表达载体。方法：设
计突变引物，通过拼接PCR 将突变型TRBP 基因克隆入pFLAG-CMV4 真核表达载体，经双酶切和测序鉴定正确后，命名为
pFLAG-CMV4-TRBPm。瞬时转染HEK-293 细胞，western-blot 检测目的蛋白表达。结果：成功扩增了突变序列，构建了突变型
TRBP 基因的真核表达载体，转染HEK-293 细胞后检测到带标签的目的蛋白表达。结论：突变型TRBP 基因真核表达载体的成功
构建，为进一步研究TRBP 的生物学效应奠定了基础。 |
| 英文摘要: |
| Objective: To investigate the mechanism that TRBP deliver dsRNA in the process of RNAi, and to construct the eukaryotic
expression vector carrying mutant TRBP. Methods: Primers for dsRBD domain mutation were designed, and gene splicing by
overlapping extension PCR was used to obtain mutant TRBP. Then it was cloned into the Flag-labeled eukaryotic expression vector
pFLAG-CMV4, and named pFLAG-CMV4-TRBPm after verified by double-enzyme and sequence detection. After transient transfect into
HEK-293 cells, the expression of target gene was detected by Western Blot. Results: The mutant TRBP cDNA was amplified and the
Flag-labeled eukaryotic expression vector pFLAG-CMV4-TRBPm which encoding mutant TRBP was constructed. Expression of mutant
TRBP was successfully detected by western blot using anti-FLAG monoclonal antibody and anti-TRBP polyclonal antibody respectively.
Conclusion: Flag-labeled pFLAG-CMV4-TRBPm vector was successfully constructed, which is of significance for further research on
biological function of TRBP. |
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