摘要
目的构建人c-jun氨基末端激酶3（c-jun N—terminal kinase 3，JNK3）真核表达载体，并在人神经母细胞瘤细胞（SHSY5Y）中稳定表达以探讨JNK3对细胞凋亡的影响。方法以pDBleu—JNK3为模板，PCR扩增人JNK3基因全长，目的片段亚克隆至真核表达载体pEGFP—C3，酶切及测序鉴定。Lipofeetamine^TM 2000转染SHSY5Y细胞，并通过G418选择性培养建立稳定表达JNK3的SHSY5Y细胞系，荧光垃微镜下观察细胞中JNK3表达，RT—PCR、Western印迹检测JNK3表达。结果成功构建了pEGFP—C3-JNK3真核表达载体，并建立了稳定表达人JNK3的SHSY5Y细胞系，与对照组相比，其人JNK3基因表达明显升高。结论稳定表达人JNK3的SHSY5Y细胞系的建立，为进一步研究人JNK3的功能及其与细胞凋亡的关系提供了重要的细胞模型和实验基础。
Objective To construct eukaryotic expressing vector of pEGFP-C3-JNK3, establish neuroblastoma SHSYSY cell line stably expressing JNK3, and study the effect of JNK3 on cell apoptosis. Methods The full-length JNK3 cDNA fragment was amplified by PCR from eukaryotic expressing plasmid pDBleu-JNK3 and subcloned into pEGFP-C3 vector. The recombinant pEGFP-C3- JNK3 vector was transfected into SHSYSY cells by lipofectamineTM 2000. After selected by G418, stahly-transfectcd SHSYSY cell line was establish. Expression of JNK3 was identified by RT-PCR and Western Blot. Results The pEGFP-C3-JNK3 expression vector was successfully constructed. As compared with the control group, JNK3 gene expression was increased significantly in stablytransfected SHSY5Y cell line. Conclusion JNK3 stably expressing SHSY5Y cell line may provide a cell model for further research on JNK3 function and its relationship with cell apoptosis.
出处
《医学分子生物学杂志》
CAS
CSCD
2009年第1期26-29,共4页
Journal of Medical Molecular Biology
基金
国家自然科学基金（No30671008,30801356,30872758）
