免疫毒素
分子生物学
重组DNA
限制性酶
HEK 293细胞
生物
转染
表达式向量
融合蛋白
质粒
融合基因
穿梭机载体
表情盒
基因
载体(分子生物学)
遗传学
细胞毒性
体外
作者
Changchen Hu,Yiquan Ke,Binquan Wang,Liyuan Zhou,Jun Lu,Fabing Zhang,Jian-kan Lu,Yangyang Cai,Ling-sha Qin
出处
期刊:Cancer Research and Clinic
日期:2009-04-28
卷期号:21 (4): 222-225
标识
DOI:10.3760/cma.j.issn.1006-9801.2009.04.003
摘要
Objective To construct a new recombinant immunotoxin expression vector by using human VEGF165 and a truncated pseudomonas exotoxin A ramification (PE38) gene, and explore the expression of the VEGF165-PE38 fusion protein in HEK293 cells. Methods VEGF165 was cloned by polymerase chain reaction (PCR). PE38 gene was gained from an vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. After the eukaryotic recombinant vector pIRES2-VEGF165-PE38-EGFP was identified by restriction endonuclease digestion and sequence analysis, the vector was transfected into HEK293 cells by liposome protocol. RT-PCR and ELISA method was used to confirm the expression of the fusion gene in the HEK293 cells. Results Restriction endonuclease digestion and sequence analysis revealed the VEGF165-PE38 fusion gene was cloned into the eukaryotic expression plasmid vector pIRES2-EGFP successfully. The pIRES2-VEGF165-PE38-EGFP fusion gene could express in the HEK293 cells. Conclusion The result provide the basis for search of the targeted cytotoxic activity to tumor vascular endothelial cells and may have some potential value in clinical application.
Key words:
Vascular endothelial growth factor; PE38; Recombinant; Immunotoxin; HEK293 cells; Eukaryotic expression
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