Expression of BMP family proteins in mammalian cells and production of monoclonal antibodies

以大鼠激活素β - aa基因为探针，筛选了非洲爪蟾基因组DNA文库。我们已经分离出5个独立的基因，其中一个基因被发现编码一种与哺乳动物激活蛋白密切相关的蛋白质。有趣的是，该基因的其余部分包含一种两栖动物的哺乳动物骨形态发生蛋白，这种蛋白是基于它们在体内诱导异位骨形成的能力而纯化的。最终，我们分离出爪蟾BMP-2、-4和-7的cdna。为了获得纯化的BMP蛋白，为实验和临床研究提供依据，我们尝试在哺乳动物细胞中表达BMP蛋白。我们发现用pSD(X)表达载体将BMP-4 cDNA导入CHO细胞后，表达率较高（20 ug/L）。另一方面，我们成功地构建了针对BMP-2肽和β -半乳糖苷酶/BMP-4融合蛋白的多克隆抗体。前者抗体（Ab383）能与BMP-4和BMP-2发生反应，后者抗体（Ab97）对BMP-4具有特异性。我们根据其免疫反应性部分纯化了CHO细胞中表达的BMP-4。最终制备的重组爪蟾bmp -爪蟾BMP-2已被证明与哺乳动物BMP-2在生物化学和生物学上没有区别。因此，爪蟾bmp被认为是开发骨折和骨质疏松药物的一个有前景的分子。此外，我们的抗体被证明可用于筛选牛骨提取物中的bmp。

We have screened a genomic DNA library of Xenopus laevis with rat activin betaA gene as a probe. We have isolated 5 independent genes and one of the genes was found to be encoding a protein closely related to mammalian activin. Interestingly, the rest of the gene contained an amphibian counterpart of mammalian bone morphogenetic proteins, which was purified based on their ability to induce ectopic bone formation in vivo. Eventually, we isolated cDNAs of Xenopus BMP-2,-4 and -7. In order to obtain pure BMP proteins and supply them for experimental and clinical studies, we tried to express BMP proteins in mamalian cells. We have found that expression rate is relatively high(20 ug/L) when BMP-4 cDNA is introduced into CHO cells with pSD(X) expression vector. On the other hand, we successfully raised polyclonal antibodies against BMP-2 peptide and beta-galactosidase/BMP-4 fusion protein. The former antibody (Ab383) was found to react with BMP-4 as well as BMP-2 and the latter (Ab97) was specific to BMP-4. We partially purified the BMP-4 expressed in CHO cells based on its immunoreactivity. The final preparation of the recombinant Xenopus BMP-Xenopus BMP-2 has been shown be indistinguishable from mammalian BMP-2 biochemically ann biologically. Therefore, Xenopus BMPs are thought to be a promising molecule to develop drugs for bone fracture and osteoporosis. In addition, our antibodies are demonstrated to be useful to screen BMPs in the extract of bovine bone.