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。
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. Conclusions： The PcDNA3A-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.
出处
《现代生物医学进展》
CAS
2014年第21期4014-4017,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(81070003
81370173)
国家青年科学基金项目(81200002)
