人骨形态发生蛋白12前体蛋白cDNA的克隆及序列分析

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (03): 260-263.

人骨形态发生蛋白12前体蛋白cDNA的克隆及序列分析

路艳蒙¹, 付文玉², 朴英杰¹

1. 第一军医大学中心实验室, 广东, 广州, 510515;
2. 潍坊医学院组织胚胎学教研室, 山东, 潍坊, 261041

出版日期:2004-03-20 发布日期:2004-03-20
基金资助:
收稿日期:2003-8-22。
基金项目:“973”国家重点基础研究发展规划资助项目(G1999054038);国家自然科学基金(30070368);广州市科委科技攻关项目(2000-Z- 017-01-05)
作者简介:路艳蒙(1970- ),女,2002年毕业于第一军医大学,博士,讲师,电话:020-61640114-89057,E-mail:tse@fimmu.com

Cloning and sequence analysis of the preprotein cDNA of human bone morphognetic protein 12

LU Yan-meng¹, FU Wen-yu², PIAO Ying-jie¹

1. 第一军医大学中心实验室, 广东, 广州, 510515;
2. 潍坊医学院组织胚胎学教研室, 山东, 潍坊, 261041

Online:2004-03-20 Published:2004-03-20

摘要/Abstract

摘要： 目的克隆人骨形态发生蛋白12前体蛋白的基因。方法根据Genbank人骨形态发生蛋白12的基因序列合成两条引物,从人胎盘组织中提取总RNA,用反转录聚合酶链反应(RT-PCR)技术扩增出长920 bp编码人骨形态发生蛋白12(BMP12)前体蛋白的基因序列。将所得的基因片段插入载体pT_ARGET^TM质粒中并转化大肠杆菌JM109,提取重组质粒进行鉴定并测序。结果 RT-PCR产物琼脂糖凝胶电泳显示一长约920 bp的条带,阳性克隆质粒经PCR扩增出约920 bp的片段,全自动DNA测序结果表明和Genbank中的序列完全相符。结论通过RT-PCR可从人胎盘组织中成功地克隆出人BMP12前体蛋白基因,基因序列完全正确。

Abstract: Objective To clone the cDNA of the preprotein cDNA of human bone morphogenetic protein 12 (hBMP12). Methods Two primers were designed according to hBMP12 sequence reported in GenBank. The hBMP12 preprotein cDNA was obtained by reverse transcriptional (RT)-PCR from the mRNA extracted from human placenta, followed by cloning into pT_ARGET^TM plasmid and sequence analysis of the plasmid pTARGETTM-BMP12. Results DNA agarose gel electrophoresis showed that the product of RT-PCR was about 920 bp, as was consistent with the result of PCR detection of the recombinant plasmid. The result of sequence analysis was in agreement with the reported hBMP12 sequence in GenBank. Conclusion The preprotein of hBMP12 cDNA has been successfully cloned with correct sequence.

中图分类号:

路艳蒙¹, 付文玉², 朴英杰¹. 人骨形态发生蛋白12前体蛋白cDNA的克隆及序列分析[J]. 南方医科大学学报, 2004, 24(03): 260-263.

LU Yan-meng¹, FU Wen-yu², PIAO Ying-jie¹. Cloning and sequence analysis of the preprotein cDNA of human bone morphognetic protein 12[J]. Journal of Southern Medical University, 2004, 24(03): 260-263.

参考文献

[1] Wolfman NM, Hattersley G, Cox K, et al. Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family [J]. J Clin Invest, 1997, 100(2): 321-30.
[2] Urist MR. Bone: formation by autoinduction[J]. Science, 1965, 150 (698): 893-9.
[3] Urist MR, Chang JJ, Lietze A, et al. Preparation and bioassay of bone morphogenetic protein and polypeptide fragments [J]. Meth ods Enzymol, 1987, 146: 294-312.
[4] Wu ZY, Hu XB. Separation and purification of porcine bone mor phogenetic protein[J]. Clin Orthop, 1988, (230): 229-36.
[5] Hogan BL. Bone morphogenetic proteins: multifunctional regulatorsof vertebrate development[J]. Genes Dev, 1996, 10(13): 1580-94.
[6] Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair [J]. Clin Orthop, 1998, Jan(346): 26-37.
[7] Kaplan FS, Shore EM. Encrypted morphogens ofskeletogenesis: bi ological errors and pharmacologic potentials [J]. Biochem Pharma col, 1998, 55(4): 373-82.
[8] Bessho K, Tagawa T, Murata M. Comparison of bone matrix -de rived bone morphogenetic proteins from various animals [J]. J Oral Maxillofac Surg, 1992, 50(5): 496-501.
[9] Celeste AJ, Ross AJ, Yamaji N, et al. The molecular cloning of hu man morphogenetic protein-10, -11, -12, three new members of the transforming growth factor-β superfamily [J]. J Bone Miner Res, 1995, 10(Suppl): S336.
[10] Fu SC, Wong YP, Chan BP, et al. The roles of bone morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix pro duction of human patellar tendon fibroblasts[J]. Life Sci, 2003, 72 (26): 2965-74.
[11] Lawton LN, Bonaldo MF, Jelene PC, et al. Identification of a novelmember of the TGF -beta superfamily highly expressed in hman placenta[J]. Gene, 1997, 203(1): 17-26.
[12] Furuya K, Nifuji A, Rosen V, et al. Effects of GDF7/BMP12 on pro liferation and alkaline phosphatase expression in rat osteoblastic os teosarcoma ROS 17/2.8 cells [J]. J Cell Biochem, 1999, 72(2): 177-81.
[13] 金丹,曾位森,裴国献,等.经转染人骨形态发生蛋白7基因后骨髓间充质干细胞mRNA的表达[J].第一军医大学学报,2002,22 (3):222-4.Jin D, Zeng ws,Pei GX, et al. Expression of human bone mor phogentic protein 7 mRNA after the gene transfection in rabbit bone marrow-derived mesenchymal stem cells [J]. J First Mil Med Univ/ Di Yi Jun Yi Da Xue Xue Bao, 2002,22(3): 222-4.
[14] 魏宽海,金丹,裴国献,等.逆转录病毒介导人重组骨形态发生蛋白7基因转染骨髓肌卫星细胞mRNA的表达[J].第一军医大学学报,2002,22(4):312-4.Wei KH, Jin D, Pei GX, et al. mRNA Expression of recombinanthuman bone morphogentic protein 7 in skeletal muscle satellite genetic cells with retroviral gene transfection [J]. J First Mil Med U niv/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(4): 312 -4.