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ΔNp63α及其SAM结构域的原核及真核表达

发表时间:2015-06-15  浏览量:1434  下载量:674
全部作者: 陈静,易勇,李成华
作者单位: 四川大学生命科学学院,生长代谢与衰老研究中心
摘 要: 为建立高效的ΔNp63α及其SAM(sterile alpha motif)结构域的真核细胞表达和原核细胞表达及蛋白纯化体系并进一步研究ΔNp63α的功能,研究扩增出人ΔNp63α及其SAM结构域编码序列,闭幕并克隆至慢病毒表达载体pCMV-phage和原核表达载体pGEX-6P-1. 通过将构建的pCMV-phage-ΔNp63α包装至慢病毒颗粒,感染人头颈鳞状细胞癌FaDu细胞后,用Western blotting检测到ΔNp63α能在FaDu细胞中有效地过表达;DNA损伤药物Doxorubicin(Dox)处理能诱导FaDu细胞发生凋亡,而用pCMV-phage-ΔNp63α在FaDu细胞过表达则能有效挽救Dox诱导的细胞凋亡;通过将构建的pGEX-6P-1-ΔNp63α或pGEX-6P-1-SAM转化至BL-21,成功诱导表达并纯化出GST融合的ΔNp63α及其SAM结构域蛋白。研究证实了ΔNp63α在抑制细胞凋亡中具有重要作用,并为进一步研究ΔNp63α及其SAM结构域的功能奠定了基础。
关 键 词: 医学生物化学;ΔNp63α;慢病毒载体;GST融合蛋白纯化
Title: Prokaryotic and eukaryotic expression of ΔNp63α and its SAM domain
Author: CHEN Jing, YI Yong, LI Chenghua
Organization: Center of Growth, Metabolism and Aging, School of Life Sciences, Sichuan University
Abstract: To establish efficient prokaryotic and eukaryotic expression and purification systems of ΔNp63α and its sterile alpha motif (SAM) domain, and for further function research of ΔNp63α, we amplified the coding sequences of human ΔNp63α and its SAM domain, and cloned them into lentiviral expression vector pCMV-phage and prokaryotic expression vector pGEX-6P-1. After packaging the established pCMV- phage-ΔNp63α into lentiviral particles and infecting carcinoma FaDu cells in human head and neck, we find that exogenous ΔNp63α were successfully overexpressed by using Western blotting detection. DNA damage reagent Doxorubicin (Dox) induces apoptosis in FaDu cells, while overexpression of ΔNp63α with pCMV-phage-ΔNp63α efficiently rescued this apoptosis. On the other hand, by the conversion of pGEX- 6P-1-ΔNp63α or pGEX-6P-1-SAM into BL-21, GST-fused ΔNp63α or its SAM domain was successfully expressed and purified. This study demonstrates that ΔNp63α plays a key role in inhibition of cell apoptosis, and lays a foundation for further investigations on function of ΔNp63α and its SAM domain.
Key words: medical biochemistry; ΔNp63α; lentiviral vector; GST fused protein purification
发表期数: 2015年6月第11期
引用格式: 陈静,易勇,李成华. ΔNp63α及其SAM结构域的原核及真核表达[J]. 中国科技论文在线精品论文,2015,8(11):1141-1146.
 
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