双顺反子真核表达载体pIRES2-EGFP-hBMP-2的构建及鉴定

背景：骨形态发生蛋白等因子及以其为中心构建的支架或载体已经广泛应用于实验研究中，并逐渐应用于临床，然而，治疗效果的示踪方法为实验提出了挑战。增强型绿色荧光蛋白基因的发现和应用解决了这一难题。 目的：构建双顺反子真核表达载体PIRES2-增强型绿色荧光蛋白-人骨形态发生蛋白2，检测其表达情况。 方法：利用RT-PCR方法从人骨肉瘤组织中提取人骨形态发生蛋白2基因，使之与PMD18-T载体连接，经酶切回收后，与目的载体pIRES2-增强型绿色荧光蛋白连接。用PCR技术及基因测序鉴定构建结果。然后将构建好的载体转染人胚肾293细胞，通过荧光显微镜及Western blot技术观测其表达。 结果与结论：实验成功扩增了人骨形态发生蛋白2基因并构建了pIRES2-增强型绿色荧光蛋白-人骨形态发生蛋白2载体，将pIRES2-EGFP-hBMP-2用脂质体转染入人胚肾293细胞后48 h，荧光显微镜观察约30%细胞发出绿色荧光，Western blot结果发现在Mr46×106处有一特异性骨形态发生蛋白条带，表明所构建载体中靶基因能成功表达。

Construction and identification of pIRES2-EGFP-hBMP-2 bicistronic eukaryotic expression vector

BACKGROUND: Bone morphogenetic protein (BMP) and its derived scaffold or vector here been widely used in experiment and gradually applied in the clinic. However, the tracing method challenges the therapeutic effect. The discovery and application of enhanced green fluorescent protein (EGFP) can solve this problem. OBJECTIVE: To construct the bicistronic eukaryotic vector pIRES2-EGFP-hBMP-2 and to observe its expression in human embryo kidney (HEK) 293 cells. METHODS: hMBP-2 gene was extracted from human osteosarcoma by RT-PCR and inserted into PMD18-T vector. Following the DNA sequence verification, it was then sub-cloned into the eukaryotic vector pIRES2-EGFP. After restriction enzyme analysis, the pIRES2-EGFP-BMP-2 was transfected into HEK 293 cells. Then its expression was observed using fluorescence microscopy and Western blot. RESULTS AND CONCLUSION: hBMP-2 gene was amplified and the eukaryotic vector pIRES2-EGFP-hBMP-2 was constructed successfully. At 48 hours after tranfection of pIRES2-EGFP-hBMP-2 into HEK 239 cells, approximately 30% of cells emitted green fluorescence under a fluorescence microscope. Western blot results demonstrated that there was a specific BMP strap at Mr46×106 side, which indicated that the target gene could be expressed in constructed pIRES2-EGFP-hBMP-2 vector.