[1]李咏梅,陈金龙,杨海文,等.融合蛋白EGF-TCS的纯化及体外靶向杀伤肿瘤细胞的研究[J].第三军医大学学报,2007,29(13):1316-1319.
 LI Yong-mei,CHEN Jin-long,YANG Hai-wen,et al.Purification of EGF-TCS recombinant fusion protein and its targeting action on human tumor cells in vitro[J].J Third Mil Med Univ,2007,29(13):1316-1319.
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融合蛋白EGF-TCS的纯化及体外靶向杀伤肿瘤细胞的研究(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第13期
页码:
1316-1319
栏目:
论著
出版日期:
2007-07-15

文章信息/Info

Title:
Purification of EGF-TCS recombinant fusion protein and its targeting action on human tumor cells in vitro
作者:
李咏梅陈金龙杨海文罗仁
南方医科大学南方医院中医科;军事医学科学院放射医学研究所
Author(s):
LI Yong-mei CHEN Jin-long YANG Hai-wen LUO Ren
Department of Traditional Chinese Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515; Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China
关键词:
表皮生长因子天花粉蛋白纯化靶向作用凋亡
Keywords:
epidermal growth factor Trichosanthin purification target action apoptosis
分类号:
R392-33; R730.51; R967
文献标志码:
A
摘要:
目的    在大肠杆菌中表达并纯化EGF-TCS融合蛋白,观察该融合蛋白对肿瘤细胞的靶向选择性杀伤作用。    方法    将重组表达质粒PQE30/EGF-TCS转化至大肠杆菌M15中,表达该融合蛋白(EGF-TCS)。产物经Ni-NTA Agrose亲和层析柱纯化;用流式细胞仪检测肿瘤细胞株和正常肝LO2细胞株的EGFR表达量;进行细胞杀伤实验,验证EGF-TCS的选择性杀伤能力;用流式细胞仪检测细胞凋亡的方法进一步考察EGF-TCS的靶向选择性杀伤能力,并用电镜观察细胞形态。    结果    重组表达质粒PQE30/EGF-TCS在大肠杆菌M15中获得稳定、高效的融合表达,表达上清柱纯化纯度达95%以上;肝癌BEL-7402细胞表面EGFR表达量最高(72.33%),正常肝LO2细胞表面EGFR表达量最低(5.51%),重组的融合蛋白对肿瘤细胞具有较大的杀伤能力(BEL-7402、MCF-7和BGC-823的IC50分别为11.40、22.47、12.53 μg/ml),对正常细胞的杀伤能力微弱(IC50为53.19 μg/ml)。    结论    应用基因工程技术,成功构建了融合蛋白EGF-TCS,该融合蛋白可明显促进肿瘤细胞的凋亡。
Abstract:
Objective    To express and purify EGF-TCS fusion protein and observe the targeted and selective killing effect on cancer cells of the fusion protein.     Methods    The recombinant expression plasmid PQE30/EGF-TCS was transformed to E. coli. M15 and the fusion protein (EGF-TCS) was expressed. Ni-NTA Agrose affinity chromatography was used to purify the protein, flow cytometry to detect EGFR expression rate in cancer cells (BEL-7402, MCF-7, BGC-823) and normal liver LO2 cells, and the killing test to verify selective killing ability of EGF-TCS; The cell apoptosis detection by flow cytometry and microscopic observation were used to confirm the selective killing ability of EGF-TCS.     Results    Recombinant expression plasmid PQE30/EGF-TCS was expressed in E. coli. M15 stably and effectively. The purity of EGF-TCS was over 95% by chromatography. EGFR expression rate was highest in hepatoma cells BEL-7402 (72.33%) and lowest in normal liver LO2 cells (5.51%). The killing ability of recombinant protein was more effective to cancer cells (IC50 of BEL-7402, MCF-7 and BGC-823 was 11.4, 22.47 and 12.53 μg/ml respectively) and was weak to normal cells (IC50 53.19 μg/ml).     Conclusion    The recombinant protein EGF-TCS that induces apoptosis of cancer cells was successfully constructed by gene engineering technology.

参考文献/References:

李咏梅, 陈金龙, 杨海文, 等. 融合蛋白EGF-TCS的纯化及体外靶向杀伤肿瘤细胞的研究[J]. 第三军医大学学报, 2007, 29(13):1316-1319.

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更新日期/Last Update: 2008-10-15