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目的:构建携带Myc标签的人细胞外信号调节激酶2(ERK2)的真核表达载体,表达Myc-ERK2融合蛋白,检测其对人视网膜母细胞瘤细胞生长的影响。方法:采用PCR技术从乳腺文库中扩增人ERK2基因序列,将其插入pXJ-40载体;将重组质粒与空载体转染人视网膜母细胞瘤WERI-Rb-1细胞系后,Western印迹检测表达情况,并进行生长曲线实验。结果:双酶切和测序鉴定表明Myc-ERK2真核表达质粒构建成功;转染WERI-Rb-1细胞后融合蛋白获得表达,生长曲线实验结果表明ERK2可促进肿瘤细胞的生长。结论:携带Myc标签的人ERK2基因的真核表达载体能在人视网膜母细胞瘤WERI-Rb-1细胞中表达,且能促进该细胞的生长。本实验为进一步研究ERK2在肿瘤尤其是眼恶性肿瘤中的功能奠定了基础。
OBJECTIVE: To construct Myc-ERK2 eukaryotic expression vector carrying Myc-tagged human extracellular signal-regulated kinase 2 (ERK2) and detect its effect on the growth of human retinoblastoma cells. Methods: The human ERK2 gene sequence was amplified from the mammary gland by polymerase chain reaction (PCR) and inserted into pXJ-40 vector. The recombinant plasmid and empty vector were transfected into human retinoblastoma cell line WERI-Rb-1 and then detected by Western blot Situation, and the growth curve experiment. Results: Double enzyme digestion and sequencing showed that Myc-ERK2 eukaryotic expression plasmid was successfully constructed. The expression of fusion protein was obtained after transfection of WERI-Rb-1 cells. The results of growth curve showed that ERK2 could promote the growth of tumor cells. CONCLUSION: The eukaryotic expression vector of human ERK2 gene carrying Myc tag can express in human retinoblastoma WERI-Rb-1 cells and promote the growth of the cells. This experiment lays the foundation for the further study of the function of ERK2 in tumor, especially in ocular malignant tumors.