Liu Zhen, Zhen Yan, Yu Xiao-li, Jiang Qing-ping, Long Jie, Fang Wei-yi
Department of Pathology, Guangzhou Medical University, Guangzhou 510182, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2011 Jan;31(1):65-8.
To construct a lentiviral vector carrying human NESG1-EGFP gene and observe its expression in 293FT cells.
The CDS region of NESG1 gene was amplified from a plasmid containing the full-length NESG1 sequence and cloned into the lentiviral vector pGC-FU-EGFP by restriction endonuclease AgeI digestion and T(4) DNA ligase ligation. After transformation into competent E. coli cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the two packaging plasmids were co-transfected into human embryonic kidney cell line 293FT cells by lipofectamine 2000 to produce the lentiviral particles, and the viral titer was determined. The 293FT cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope. Western blotting was used to detect the expression of NESG1 protein in the transfected cells.
The lentiviral vector pGC-FU-NESG1-EGFP for NESG1 gene was constructed successfully. Strong green fluorescence was observed in 293FT cells under fluorescent microscope after co-transfection of the cells with the 3 plasmids of lentiviral vector. The virus in the supernatant reached a titer of 2×10(7) TU/ml. The transfection efficiency of the collected virus exceeded 90% in 293FT cells with a multiplicity of infection of 1. Western blotting identified the presence of NESG1 expression in the transfected 293FT cells.
The lentiviral vector for NESG1 has been successfully constructed with a high yield of lentivirus, which facilitate further investigation of the roles of NESG1 gene in the development and progression of nasopharyngeal carcinoma.
构建携带人NESG1 - EGFP基因的慢病毒载体，并观察其在293FT细胞中的表达。
从含有全长NESG1序列的质粒中扩增NESG1基因的CDS区，经限制性内切酶AgeI酶切和T(4) DNA连接酶连接，克隆至慢病毒载体pGC - FU - EGFP中。转化感受态大肠杆菌细胞后，通过PCR和测序鉴定候选克隆。将重组质粒与两个包装质粒用脂质体2000共转染入人胚肾细胞系293FT细胞以产生慢病毒颗粒，并测定病毒滴度。用获得的慢病毒颗粒感染293FT细胞，在荧光显微镜下评估转染效率。采用蛋白质免疫印迹法检测转染细胞中NESG1蛋白的表达。
成功构建了用于NESG1基因的慢病毒载体pGC - FU - NESG1 - EGFP。将慢病毒载体的3种质粒共转染细胞后，在荧光显微镜下293FT细胞中观察到强绿色荧光。上清液中的病毒滴度达到2×10(7) TU/ml。在感染复数为1的情况下，收集的病毒在293FT细胞中的转染效率超过90%。蛋白质免疫印迹法鉴定了转染的293FT细胞中存在NESG1表达。
已成功构建NESG1慢病毒载体，且慢病毒产量高，这有助于进一步研究NESG1基因在鼻咽癌发生发展中的作用。
