Molecular Cloning and Sequence Analysis of Full-Length cDNA Encoding Human Bone Morphogenet-ic Protein-7

摘要

Objective: To clone the full-length human bone morphogenetic protein-7 (BMP-7) gene and analyse its sequence, to aid in investigation of its function and structure. Methods: Total RNA was isolated from Chinese fetal kidney by the acid guanidinium thiocyanate -phenol- chloroform method. Two overlapping segments of human BMP-7 cDNA were obtained by reverse transcription (RT ) - PCR. Following application, the two segments were ligated to each other and subcloned into PGEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results: There was 750 bp fragment obtained RT-PCR using ~#2 primer from 5′ end of BMP-7 gene (PCR by using ~#2 and ~#1), and 540 bp fragment from 3′ end was generated by RT-PCR using ~#4 primer (PCR using ~#3 and ~#4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619), our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion: This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo- articular system. This makes BMP-7 is an at-tractive target for various clinical applications.