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目的:构建pIRES2-绿色荧光蛋白(EGFP)-骨形成蛋白2(bone morphogenetic protein 2,BMP-2)真核表达质粒,并检测其表达活性。方法:采用逆转录聚合酶链式反应(RT-PCR)技术从人骨肉瘤组织中扩增出人骨形态发生蛋白2(hBMP-2)的基因片段,构建成pIRES2-EGFP-BMP-2重组质粒,经酶切、测序检测其构建的正确性,通过转染3T3细胞后分别采用RT-PCR、免疫组化及Western免疫印迹(Western blotting,WB)法检测其转录、表达活性。结果:构建的pIRES2-EGFP-BMP-2质粒经酶切、测序、RT-PCR、免疫组化、EGFP表达鉴定、WB等检验表明质粒构建成功并具有转录活性。结论:本实验成功构建了具有表达活性的hBMP-2真核表达质粒,为进一步研究用BMP-2基因转染的方法来促进实验动物骨折的愈合奠定了基础。
OBJECTIVE: To construct the eukaryotic expression plasmid pIRES2-EGFP-BMP2 and to detect its expression activity. Methods: The hBMP-2 gene fragment was amplified from human osteosarcoma tissue by reverse transcription-polymerase chain reaction (RT-PCR) and constructed into recombinant plasmid pIRES2-EGFP-BMP-2. After being transfected into 3T3 cells, the transcription and expression of 3T3 cells were detected by RT-PCR, immunohistochemistry and Western blotting (WB) respectively. Results: The constructed plasmid pIRES2-EGFP-BMP-2 was verified by restriction endonucleases, sequencing, RT-PCR, immunohistochemistry and EGFP. WB and other tests showed that the plasmid was successfully constructed and transcribed. Conclusion: The hBMP-2 eukaryotic expression plasmid was successfully constructed in this experiment, which laid the foundation for the further study of the BMP-2 gene transfection method to promote the healing of experimental animal fracture.