Molecular cloning and basic study of bone inducing factors for their clinical use.

为了了解骨诱导因子的生物学作用机制，我们尝试克隆这些因子的基因。根据人骨形态发生蛋白(BMP)-2/4和小鼠BMP-6（Vgr-1）核苷酸序列制备寡核苷酸引物。从新生小鼠组织和人骨肉瘤细胞系MG63中分离信使rna，并利用逆转录酶合成互补dna （cdna）。利用这些引物和cDNA模板，通过PCR（聚合酶链式反应），我们克隆了小鼠BMP-2、小鼠BMP-6和人类BMP-4的cDNA。利用PCR克隆的cDNA探针筛选不同cDNA文库，分离BMP全长cDNA。将筛选到的人胎盘cDNA文库阳性克隆亚克隆到质粒载体上，进行核苷酸测序。结果发现阳性克隆为人BMP-4全长cDNA。用同样的方法，从人肺小细胞癌（NCL-H69） cDNA文库中分离出一个几乎全长的人BMP-6 cDNA。进一步，我们尝试合成人BMP-4重组蛋白。将人BMP-4 cDNA转入大肠杆菌表达载体的两个衍生物中，获得了β -半乳糖苷酶-人BMP-4融合蛋白和谷胱甘肽- s -转移酶-人BMP-4融合蛋白。这些融合蛋白的成骨诱导活性目前正在研究中。

To understand the mechanism of biological actions of bone inducing factors, we attempted to clone the genes for these factors.According to the human BMP(Bone Morphogenetic Protein)-2/4 and mouse BMP-6(Vgr-1) nucleotide sequences, oligonucleotide primers were prepared. Messenger RNAs were isolated from neonatal mouse tissue and human osteosarcoma cell line MG63, and used for the synthesis of complementary DNAs (cDNAs) with reverse transcriptase. By PCR(Polymerase Chain Reaction) using these primers and cDNA templates, we have cloned cDNAs for mouse BMP-2, mouse BMP-6, and human BMP-4.To isolate full length of BMP cDNAs, various cDNA libraries were screened with the cDNA probes cloned by PCR. A positive clone detected in the screening of a human placenta cDNA library was subcloned into a plasmid vector and subjected to nucleotide sequencing. The result was that the positive clone was found to be a full length cDNA for human BMP-4. Using the same method, an almost full length human BMP-6 cDNA was also isolated from a human lung small cell carcinoma(NCL-H69) cDNA library.Furthermore, we attempted to synthesize the human BMP-4 recombinant protein. The human BMP-4 cDNA was transferred into two derivatives of the E.coli expression vector, and a beta - Galactosidase-human BMP-4 fusion protein and a Glutathione-S- transferase-human BMP-4 fusion protein were obtained. Osteoinductive activities of these fusion proteins are now under investigation.