| 先俊 邱娴 张安兵 李理 黄文杰.真核表达质粒pcDNA3.1-CSRP2-HA 的构建及鉴定[J].,2014,14(21):4014-4017 |
| 真核表达质粒pcDNA3.1-CSRP2-HA 的构建及鉴定 |
| Construction and Identification of Eukaryotic Expression Plasmid PcDNA3.1-CSRP2-HA |
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| DOI： |
| 中文关键词: CSRP2 PcDNA3.1 真核表达质粒 |
| 英文关键词: CSRP2 PcDNA3.1 Eukaryotic expression plasmid |
| 基金项目:国家自然科学基金项目（81070003、81370173）；国家青年科学基金项目（81200002） |
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| 中文摘要: |
| 目的：构建并鉴定真核表达质粒PcDNA3.1-CSRP2-HA。方法：据GeneBank 中人CSRP2 CDS序列设计并合成引物，提取
A549 细胞总RNA，并将其逆转录成cDNA作为模板,进行PCR 扩增，获得CSRP2 目的基因后,再与PcDNA3.1-HA载体进行连接
重组，构建PcDNA3.1-CSRP2-HA 真核表达质粒，经限制性内切酶消化、PCR 及DNA 序列测序分析等方法鉴定后, 瞬时转染入
A549细胞，Western blot 法检验CRP2 蛋白表达。结果：成功构建PcDNA3.1-CSRP2-HA 真核表达质粒，Western-blot 结果显示
PcDNA3.1-CSRP2-HA 能够在A549 细胞中表达。结论：PcDNA3.1-CSRP2-HA真核表达质粒构建并鉴定成功，为后续研究CRP2
在炎症诱发氧化损伤机制中的转录调控作用奠定了基础。 |
| 英文摘要: |
| Objective:To construct and identify eukaryotic expression plasmid PcDNA3.1-CSRP2-HA.Methods:According to the
sequence of CSRP2 CDS in GeneBank, a Pair of Primers were respectively designed and Synthesized, and the total RNA was isolated
from A549 cells. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the product was cloned into
PcDNA3.1-HA vector, and they were identified by PCR and double restrictive edonuclease digestion and sequence analysis. Then the
recombinant expression plasmid was transferred into A549 cells, and the CRP2 protein expression was identified by Western-blot.Results:The PcDNA3.1-CSRP2-HA eukaryotic expression plasmid was successfully established, and the expression of PcDNA3.
1-CSRP2-HA could be detected in A549 by Western-blot.Conclusion:The PcDNA3.1-CSRP2-HA eukaryotic expression plasmid was
constructed successfully, which lays the foundation for the research of CRP2 in transcription regulation mechanism of oxidative damage
induced by inflammation. |
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