重组血管内皮生长因子165-PE38融合基因的构建及其对人脐静脉内皮细胞的抑制作用

Recombinant immunotoxin VEGF165-PE38 in antiangiogenic therapy for cancer

目的构建血管内皮生长因子（VEGF）基因和铜绿假单胞菌外毒素（PE38）基因的真核融合表达载体，观察其表达产物对人脐静脉内皮细胞（HUVECs）的影响。方法通过聚合酶链反应（PCR）获得VEGF165基因片段，通过HindⅢ和EcoRⅠ双酶切pRB391质粒获得PE38基因，构建两融合基因的真核表达载体pIRES2-VEGF165-PE38-EGFP，转染293细胞后用逆转录（RT）-PCR及ELISA检测VEGF165-PE38融合蛋白在293细胞的表达，并检测转染细胞的培养上清对HUVECs的选择性细胞毒作用。结果成功构建VEGF165-PE38融合基因真核表达载体，其可在293细胞表达，ELISA检测表明空质粒转染组、无转染组和重组质粒转染组VEGF浓度分别为（269．0±23．6）、（306．0±29．3）和（1390．0±136．6）ng／L。CCK-8和TUNEL检测表明重组质粒细胞转染上清对HUVECs具有较强的细胞抑制效应，凋亡细胞率分别为8．34％、7．69％和39．88％，差异有统计学意义（P〈0．05）。结论VEGF165-PE38融合基因构建，表达及其功能的初步研究，为肿瘤血管内皮细胞的靶向治疗及临床应用奠定了基础。

Objective To construct a new recombinant immunotoxin expression vector by fusing human VEGF165 and a truncated pseudomonas exotoxin A ramification （PE38） gene ,and explore the influence of the VEGF165-PE38 fusion protein on human umbilical vein endothelial cells （HUVECs）. Methods VEGF165 was cloned by polymerase chain reaction （PCR）. PE38 gene was gained from a vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. The vector was transfected into 293 cells. RT-PCR and ELISA were used to confirm the expression of the fusion gene in the 293 cells. The selectively killing activities of the immunotoxin in culture supernatant were detected in vitro. Results The fusion gene eukaryotic expression plasmid was constructed successfully. The fusion gene could be expressed in the 293 cells. The VEGF levels of （ 269.0 ± 23.6 ）, （ 306.0 ± 29.3 ） and （ 1390.0± 136.6 ） ng/L were secreted into the culture medium by no transfected cells, pIRES2-EGFP-transfected cells and VEGF165PE38 transfected cells respectively. VEGF165-PE38-containing supernatant was specific to VEGFR-positive HUVECs, and the apoptosis rate was 8. 34%, 7. 69%, and 39. 88% in no transfected group, pIRES2-EGFP-transfected group and VEGF165PE38 transfected group respectively. Conclusion The results provide the basis for research of the targeted eytotoxic activity to tumor vascular endothelial cells, and may have some potential values in clinical application.