The Cloning And Sequencing Of Partial Human Bone Morphogenetic Protein 3 Gene

Bone morphogenetic proteins (BMPs) were originally discovered based on their ability to induce cartilage and bone formation at ectopic sites in animals. As members of TGF-13 superfamily(excepting BMP-l),we know today that BMPs function not only in directing bone formation, but also in patterning embryonic morphogenesis, in the differentiation of nerve cells, in the development of mesoderm and hematopoietic organs, in spermatogenesis and placentation, and in programmed cell death. Since the BMPs extracted from animal and human bones can not meet the requirements of basic research and clinical use, scientists have expressed BMP genes in E.coli and some eukaryotic expression systems, such as COS, CHO. The expression of BMPs in mammary gland cells has not been reported yet. Most scientists obtained hBMP genes from cDNA libraries established from some human cell lines, however, it's time consuming to establish a cDNA library by ourselves and expensive to buy one. Therefore, we tried to clone the complementary DNA of hBMP-3 gene by RT-PCR, for the purpose of producing human morphogenetic protein in the mammary glands of transgenic animals.With the application of RT-PCR, we amplified, cloned, sequenced and analyzed partial cDNA of hBMP-3 gene from human bone fiber sarcoma tissue. At the outset, we successfully isolated total RNA from human bone fiber sarcoma tissue by using TRIZOL regent. Then the total RNA was transcripted into cDNA, which was used as template in PCR. Utilizing specific primers designed according to published sequence, a band about 0.85kb was obtained after PCR. The PCR was carried out with 5 min initial denaturation(94癈)followed by 35 cycles:30s at 94癈,30s at 52.5癈 and 90s at 72癈,and ended by a 10 min final extension at 72 癈. The target fragment was purified using a UNIQ-IO Column DNA Gel Extraction Kit, then was cloned into a pMD18-T vector. After being transformed into E.coli.DHS a Identified by LacZ blue-white selection, and tested by Hindlll restriction endonuclease digestion, the positive clone was sequenced. The result indicated that the size of the fragment cloned is 845bp,and-IV-its sequence homology with the designed one is exactly 100%. Also, the corresponding 280 amino acid sequence of the fragment was deduced from the sequencing result.Taken together, these data suggest that we have successfully amplified and cloned the major part of the cDNA of human bone morphogenetic protein 3 gene.