人骨形态发生蛋白-7全长基因cDNA的克隆和序列分析

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

目的 克隆人骨形态发生蛋白-7(BMP-7)全长基因,并进行测序及序列分析,研究其结构和功能。方法 采用异硫氰酸胍一步法从人胎儿肾中提取总RNA,利用逆转录—聚合酶链反应(RT-PCR)的方法,分段扩增出hBMP-7全长基因cDNA,与PGEM-Teasy连接成PGEM-Teasy/hBMP-7重组质粒,采用Sanger双脱氧链终止法测定基因序列。结果以4号特异引物引导的RT-PCR扩增出BMP-7基因3′端540bp片段,以2号特异引物引导的RT-PCR扩增出BMP-7基因5′端750bp片段,重组连接两片段得到BMP-7全长基因cDNA。与Genebank上发表的hBMP-7序列(XM30619)比较有一个碱基不同,第862位核苷酸由T→G,编码的氨基酸由苯丙氨酸变为缬氨酸。结论 首次从中国人胎肾中分段克隆出BMP-7全基因。cDNA,该基因在骨关节系统的损伤与修复中具有重要的临床应用价值。

Objective To clone the full-length human bone morphogenetic protein-7 (BMP- 7) gene and analyses its sequence, to aid in investigation of its function and structure. Methods Total RNA was isolated from Chinese fetal kidney by the acid guanidinium thio-cyanate 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 Sanger, 1977 # 423 was used to sequence the cD-NA. Results 750 bp fragment from 5' end of BMP- 7 gene was prepared by RT- PCR using # 2 primer (PCR by using #2 and # 1), and 540bp fragment from 3'end was generated by RT - PCR using # 4 primer ( PCR using # 3 and # 4). Full-length cDNA encod-, ing BMP- 7 was obtained by religation of two segments. When compared with hBMP - 7 sequence in Genebank (XM30619) , our full-length BMP - 7 cDNA has a G instead of a T at nucleotide 842. 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 plays an important role in healing injuries of the osteo-articular system. This makes BMP - 7 is an attractive target for various clinical applications.