| 杨乐 郭晓波 陈洪志 成健 董济民 丁海霞 王冠杰.人snail真核表达载体构建与鉴定[J].,2017,17(3):410-413 |
| 人snail真核表达载体构建与鉴定 |
| Construction and Identification of Human Snail Gene Eukaryotic Vector |
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| DOI： |
| 中文关键词: Snail 重组质粒 载体构建 |
| 英文关键词: Snail Recombinant plasmid Vector construction |
| 基金项目:陕西省自然科学基础研究计划项目（2014JM2-8182） |
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| 中文摘要: |
| 目的：构建人snail 基因真核表达载体并鉴定。方法：使用RT-PCR法获取人snail基因全长cDNA，经BanHI、EcoRI双酶
切、连接，插入pcDNA3.1(+)真核表达载体，转化TOP10 感受态细胞，用含氨苄青霉素的LB培养基筛选阳性克隆，提取质粒双酶
切电泳及测序鉴定，瞬时转染siha 细胞Western-blot从蛋白水平鉴定重组质粒在真核细胞内的表达。结果：pcDNA3.1-snail 重组
质粒经酶切电泳符合预期片段，测序鉴定插入片段与NCBI GenBank 文库中人snail 序列一致，重组质粒瞬时转染后snail蛋白表
达量明显增高。结论：成功构建pcDNA3.1-snail 重组质粒载体，为进一步探讨snail 基因生物学功能奠定了基础。 |
| 英文摘要: |
| Objective:To establish pcDNA3.1-snail eukaryotic recombinant expression plasmid.Methods:Human Snail gene cDNA
was amplified by reverse transcription polymerase chain reaction (RT-PCR). After digested by BamH I, EcoRI and ligation, Snail was
inserted into pcDNA3. 1 (+)eukaryotic expression vector. The positive colonies were screened and identified by single, double enzyme
digest, agarose gel electrophoresis and DNA sequence analysis. pcDNA3. 1-snail plasmid was then transfected into siha cell line with
TurboFect Transfection Reagent. Western-blot was used to detect the protein expression of snail.Results:The pcDNA3.1-snail recombinant
plasmid was verified by enzyme digestion electrophoresis showed the expected fragment. The inserted sequence in pcDNA3.1(+)
-snail was the same as the sequence of snail cDNA published in NCBI GenBank. Western blot results validated the recombinant plasmid
expressed in siha cell line efficiently.Conclusion:The eukaryotic recombinant expression plasmid of pcDNA3.1 (+)-snail was successfully
constructed. |
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