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

以大鼠激活素βA基因为探针，筛选了非洲爪哇基因组DNA文库。我们已经分离到5个独立的基因，其中一个基因被发现编码与哺乳动物激活素密切相关的蛋白。有趣的是，该基因的其余部分含有哺乳动物骨形态发生蛋白的两栖对应蛋白，该蛋白是根据它们在体内诱导异位骨形成的能力而提纯的。最终，我们分离了非洲爪哇BMP-2、-4和-7的cDNA。为了获得纯净的BMP蛋白并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，将BMP-4基因导入含有PSD(X)表达载体的CHO细胞中，表达效率较高(20ug/L)。另一方面，我们成功地制备了抗骨形态发生蛋白-2多肽和β-半乳糖苷酶/骨形态发生蛋白融合蛋白的多克隆抗体。前者抗体(Ab383)与BMP-4和BMP-2反应，后者(Ab97)与BMP-4特异。根据其免疫反应性，我们对在CHO细胞中表达的BMP-4进行了部分纯化。最终制备的重组非洲爪哇BMP-2在生化和生物学上与哺乳动物BMP-2几乎没有区别。因此，非洲爪哇BMPs被认为是一种很有前途的分子，用于开发治疗骨折和骨质疏松症的药物。此外，我们的抗体被证明是有用的筛选牛骨提取物中的BMPs。

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.