pIRES2-EGFP-BMP-2真核表达质粒的构建及其活性测定

Construction of the pIRES2-EGFP-BMP-2 plasmid and study on its transcriptional activity

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： The purpose of this study was to construct the plRES2-EGFP-BMP-2 plasmid of hu- man and explore the feasibility of introducing bone morphogenetic protein 2（BMP-2）gene into 3T3 cell and the function of introduced gene in 3T3 cell. Methods： pIRES2 EGFP-BMP-2 plasmids were constructed, identified and then introduced into 3T3 cell. RT PCR,IH and WB were done to detect the integration of BMP-2 gene in 3T3 cell. Results： The successful construction of pIRES2-EGFP-BMP2 plasmids were con- firmed by the identification of endonuclease cutting,PCR,EGFP protein,IH and WB. RT PCR demonstrated the specific bands of BMP-2 cDNA in 3T3 cell. WB and IH confirm the expression of BMP-2 protein in cell. Conclusion： The exogenous human BMP-2 gene could be introduced into 3T3 cell. The introduced BMP-2 gene was able to replicate itself and express its protein in 3T3 cell. Our results provided a new pIRES2-EGFP-BMP-2 plasmid and gave us an opportunity to study more.