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

骨诱导因子的分子克隆及其临床应用基础研究。

基本信息

批准号：
03670937
负责人：
IWAKI Hiroshi
金额：
$ 1.28万
依托单位：
TOKYO MEDICAL AND DENTAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

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.

为了解骨诱导因子的生物学作用机制，本研究根据人骨形态发生蛋白（BMP）-2/4和小鼠BMP-6（Vgr-1）的核苷酸序列，制备了寡核苷酸引物，并克隆了这些因子的基因。从新生小鼠组织和人骨肉瘤细胞系MG 63中分离信使RNA，并用于用逆转录酶合成互补DNA（cDNA）。利用这些引物和cDNA模板进行PCR（Polymerase Chain Reaction），我们克隆了小鼠BMP-2、小鼠BMP-6和人BMP-4的cDNA。将在人胎盘cDNA文库筛选中检测到的阳性克隆亚克隆到质粒载体中并进行核苷酸测序。结果发现阳性克隆是人BMP-4的全长cDNA。用同样的方法，从人肺小细胞癌（NCL-H69）cDNA文库中克隆了一个几乎全长的人BMP-6 cDNA，并尝试合成人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，并获得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。这些融合蛋白的骨诱导活性正在研究中。