hVEGF_(165)和嵌合水蛭肽融合基因的构建、表达和活性检测

Construction,Expression and Characterization of a Fusion Gene of hVEGF_(165) and Fused Hirudin Peptide

根据抗PTCA或支架后再狭窄的基因治疗需要多基因治疗的特点,用基因重组技术构建了hVEGF165和嵌合水蛭肽(fusedhirudin,FH)融合基因,并克隆到真核表达载体pcDNA3.0中,通过脂质体介导将pcDNA3.0/hVEGF165-FH转染到人内皮细胞株(ECV304)中,RT-PCR及蛋白质印迹证明融合基因hVEGF165-FH在ECV304细胞中得到表达(分子质量为24ku左右).通过体外活性检测——MTT法检测hVEGF165-FH对ECV304细胞增殖的影响,通过体外血管生成分析hVEGF165-FH对内皮细胞株ECV304增殖的影响.通过体外抗栓活性检测,表明表达产物具有促进内皮细胞株增殖及加快血管生成的作用,同时显著抑制了ADP诱导的血小板聚集率(P<0.05)并显著延长APTT和TT(P<0.05).实验结果表明,融合基因在内皮细胞株中得到表达,表达的融合蛋白具有hVEGF165和嵌合水蛭肽(FH)的双重活性,这为以后的融合基因治疗再狭窄的动物实验打下了良好基础.

In order to construct a fusion gene encoding hVEGF165 and fused hirudin（FH）, which can not only accelerate endothelial cell proliferation but also inhibit thrombosis, the eukaryotic expression vector hVEGF165-FH/pcDNA3.0 was constructed which contained the hVEGF165 and fused hirudin （FH）. Then, the constructed vector was transfected into endothelial cell strain（ECV304）. The hVEGF165-FH fusion gene transcription and protein expression were examined by RT-PCR and Western blot. The activities of fusion hVEGF165-FH were identified by serials of endothelial cell proliferation tests and antithrombotic tests in vitro. The results showed that the fusion protein was successfully expressed and it had biological activities of both hVEGF165 and fused hirudin. This new fusion gene with multi-function might be a new weapon to prevent restenosis after percutaneous coronary intervention（PCI）.