CAJ | 학술논문

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.
배경:골형태발생단백등인자급이기위중심구건적지가혹재체이경엄범응용우실험연구중,병축점응용우림상,연이,치료효과적시종방법위실험제출료도전.증강형록색형광단백기인적발현화응용해결료저일난제.목적:구건쌍순반자진핵표체재체PIRES2-증강형록색형광단백-인골형태발생단백2,검측기표체정황.방법:이용RT-PCR방법종인골육류조직중제취인골형태발생단백2기인,사지여PMD18-T재체련접,경매절회수후,여목적재체pIRES2-증강형록색형광단백련접.용PCR기술급기인측서감정구건결과.연후장구건호적재체전염인배신293세포,통과형광현미경급Western blot기술관측기표체.결과여결론:실험성공확증료인골형태발생단백2기인병구건료pIRES2-증강형록색형광단백-인골형태발생단백2재체,장pIRES2-EGFP-hBMP-2용지질체전염입인배신293세포후48 h,형광현미경관찰약30%세포발출록색형광,Western blot결과발현재Mr46×106처유일특이성골형태발생단백조대,표명소구건재체중파기인능성공표체.
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 plRES2-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 Mr46×10s side, which indicated that the target gene could be expressed in constructed pIRES2-EGFP-hBMP-2 vector.