人骨形态发生蛋白4(2b)成熟区cDNA的克隆和序列分析  

Cloning and sequencing of mature fragment of BMP-4 gene

作　　者：王欣璐[1] 李华[1] 杨广夫[2] 王全颍 杨广笑 

机构地区：[1]西安医科大学第一附属医院骨科,710061 [2]西安医科大学第一附属医院影像学中心 [3]西安医科大学解剖教研室

出　　处：《中华骨科杂志》2000年第9期529-532,共4页Chinese Journal of Orthopaedics

摘　　要：目的 研究人骨形态发生蛋白 4(2b)(BMP-4)成熟区 cDNA的克隆方法 ,并进行测序及序列分析。方法 采用异硫氰酸胍一步法从人骨肉瘤细胞株 U-2OS中提取总 RNA，通过逆转录-聚合酶链反应 (RT-PCR)扩增为 BMP-4成熟区 cDNA基因片段。将此基因片段经适当酶切后克隆入载体 pUC19中，获得重组质粒 pUC19/BMP-4。采用 Sanger双脱氧链终止法双向测定核苷酸序列。结果 通过 RT-PCR方法获得了 BMP-4成熟区的 cDNA，并经核苷酸测序证实。计算机检索 Genebank,选择其中已发表的 BMP-4 DNA序列 (D30751)作为参考，经 DNA SIS软件分析显示， rhBMP-4成熟区 cDNA和 Genebank中 D30751 BMP-4成熟区（ 963～ 1 310 bp）的核苷酸和氨基酸序列同源性均为 99％。 rhBMP-4成熟区 cDNA中有 2个碱基发生突变，一个位于第 1 154(201)位的 G C，但它不引起缬氨酸（ Val）密码子改变，故为无意义突变；另一突变位于 1 222(269)位的 C T，其密码子亦随之发生改变，即由丙氨酸 (Ala)变为缬氨酸。结论 获得克隆的 BMP-4基因成熟区，该基因在骨关节系统的损伤与修复及疾病治疗中具有重要的临床应用价值。Objective To analyse the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was extract from the human osteosarcoma cells line U-2OS by the single-step isolation method with isothiocyanic acid guanidine, the cDNA coding for the mature fragment of BMP-4 was amplified by the reverse transcription-polymerase chain reaction(RT-PCR). The mature fragment of BMP-4 was cloned into the vector pUC19, and sequenced by Sanger dideoxy-mediated chain termination method. Results The mature fragment of BMP-4 cDNA was obtained by RT-PCR and identified by sequencing. The computer search was done on Genebank, and the published DNA sequence of BMP-4 from Genebank (D30751) was chosen for a reference. Analysis showed that the homology and similarity of nucleotides and amino acids between cDNA of rhBMP-4 mature fragment and the published sequence of BMP-4 were both 99％ . Sequence analysis revealed that there were two bases mutations, one was at base 1 154(201) G C, this had no influence on the corresponding amino acids(Val), another was at base 1 222(269) C T, the mutation at the base 1 222 had turned the Ala into Val. Conclusion The mature fragment of BMP-4 gene has been cloned. The gene is of great significance in treatment of skeletal injuries and diseases.

关 键 词：骨形态发生蛋白4(2b) 克隆 序列分析 CDNA 

分 类 号：R341[医药卫生—基础医学]