山东医药2017，Vol.57Issue(6)：5-8,4.DOI:10.3969/j.issn.1002-266X.2017.06.002
稳定表达 Notch1胞内结构域肺腺癌细胞株的构建及鉴定
Construction and identification of lung adenocarcinoma cell line with stable expression of N1ICD
摘要
Abstract
Objective To construct a lung adenocarcinoma cell line A 549 with stable expression of Notch1 intracellu-lar domain (N1ICD).Methods The N1ICD fragment was amplified by PCR, which was used to construct pHBLV-N1ICD-ZsGreen-Puro plasmid (LV-N1ICD).The recombinant plasmid was identified by PCR and gene sequencing .LV-N1ICD was packaged by transfection of 293T cells with recombinant plasmid and auxiliary plasmids which were purified without endotoxin extraction .Lentivirus empty plasmid ( LV-ZsGreen-Puro ) was also constructed .Drug screening method was used to determine the titer of LV-N1ICD and LV-ZsGreen-Puro.A549 cells were divided into A549-N1ICD group and A549-ZsGreen-Puro group.Puromycin was used to screen stable transfection cell line after infection of A 549 cell with LV-N1ICD or LV-ZsGreen-Puro.Transfection efficiency was observed by fluorescence microscope and cells with green fluores -cent were preliminarily identified as stable transfection cell lines .Taking A549 cells as the control group , and N1ICD mR-NA and protein expression was further detected by qPCR and Western blotting , respectively .Results PCR and gene se-quencing showed that pHBLV-N1ICD-ZsGreen-Puro plasmid was successfully constructed .The titer of LV-N1ICD was 1 × 108 TU/mL and the LV-ZsGreen-Puro was 1 ×108 TU/mL.Cells shape had no obvious changes and green fluorescence was detected in about 70%-80%cells of the A549-ZsGreen-Puro and A549-N1ICD group by fluorescence microscope after Pu-romycin screening .The N1ICD mRNA expression of the control group , A549-ZsGreen-Puro group and A549-N1ICD group was 1.59 ±0.11, 1.09 ±0.10 and 70.81 ±6.39, respectively and the N1ICD mRNA expression of the A549-N1ICD group was significant higher than that of the other two groups (all P<0.01).The N1ICD protein expression of the three groups was 1.99 ±0.13, 1.73 ±0.08 and 6.58 ±0.43, respectively and the N1ICD protein expression of the A549-N1ICD group was significant higher than that of the other two groups (all P<0.01).Conclusions A lung adenocarcinoma cell line A549 with stable expression of N1ICD was successfully constructed .关键词
肺腺癌/Notch蛋白/胞内结构域/慢病毒重组质粒Key words
lung adenocarcinoma/Notch protein/intracellular domain/lentivirrus分类
医药卫生引用本文复制引用
邹斌,吴霞,周学亮,詹宇亮,赖松青,刘季春..稳定表达 Notch1胞内结构域肺腺癌细胞株的构建及鉴定[J].山东医药,2017,57(6):5-8,4.基金项目
国家自然科学基金资助项目（81570262）；江西省研究生创新专项资金（YC2015-B009）。 （）
