在CHO细胞中表达具有高效成骨活性的BMP-2／7异源二聚体  

Expression of BMP-2/7 Heterodimers with Potent Ability to Stimulate Osteogenic Differentiation in CHO Cells

作　　者：潘秋辉[1] 杨松海[2] 孙奋勇[2] 

机构地区：[1]中山大学附属第二医院医学研究中心,广州510120 [2]暨南大学生命科学技术学院生物工程研究所,广州510632

出　　处：《生物工程学报》2008年第3期473-479,共7页Chinese Journal of Biotechnology

基　　金：广州市科技计划项目(No.2006Z1-E0031,No.2006Z3-E0791)~~

摘　　要：PCR扩增BMP-2与BMP-7的编码基因,利用重叠PCR以柔性肽(Gly4Ser)5编码序列将二者串连并克隆到质粒pIRESneo3上,转染CHO-K1细胞得到混合稳定克隆。ELISA检测培养液中BMP-2/7异源二聚体蛋白的表达水平为230.75±13.34ng/mL,以此为条件培养基处理成骨细胞株MC3T3,对照组为分别含有CHO-K1细胞及大肠杆菌表达的BMP-2同源二聚体以及PBS的条件培养基。结果发现碱性磷酸酶染色与茜素红染色差异明显,定量RT-PCR显示分子指标OC、ALP、Runx2与Osx的转录水平明显增高(P<0.05),Luciferase报告基因检测BMP/Smad通路活性较对照组升高明显(P<0.05)。首次设计构建了BMP-2/7异源二聚体蛋白,其成骨活性显著高于BMP-2同源二聚体。Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide （Gly4Ser）5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230,75±13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coll. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased （P〈0.05）, and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers （P〈0.05）. The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.

关 键 词：骨形态发生蛋白 异源二聚体 成骨细胞 分化 

分 类 号：Q78[生物学—分子生物学]