人骨形成蛋白-2的cDNA探针的实验合成  

Synthesis and Evaluation 0f Human Bone Morphogenetic Protein 2 cDNA Probe.

作　　者：黄宏杰[1] 平飞云[2] 

机构地区：[1]浙江省杭州市第二人民医院,杭州310008 [2]浙江大学医学院附属第二医院

出　　处：《浙江创伤外科》2004年第1期9-11,共3页Zhejiang Journal of Traumatic Surgery

基　　金：浙江省自然科学基金资助项目(301576)

摘　　要：目的利用分子生物学实验技术合成并标记人骨形成蛋白-2(hBMP-2)的cDNA探针。方法从人髁状突骨折标本中提取总RNA,用逆转录聚合酶链式反应(RT-PCR)方法,扩增出hBMP-2成熟肽编码区基因的cDNA片段,将其插入pGEM-T质粒中,并转化到大肠杆菌;挑选阳性克隆培养,提取重组质粒,并作核苷酸序列分析;用限制性内切酶单酶切后,再经地高辛标记检测试剂盒随机引物法标记,合成地高辛标记的BMP-2cDNA探针;利用这一探针作人颌骨成骨性骨肉瘤标本中BMP-2mRNA表达的原位杂交研究,以鉴定探针的可靠性。结果通过实验方法合成了hBMP-2cDNA探针,利用这一探针的原位杂交研究证实颌骨成骨性骨肉瘤中有BMP-2mRNA表达。结论自行合成并标记的人BMP-2cDNA探针,性状稳定、可靠,能够应用于BMP-2mRNA表达的原位杂交研究。Objective The purpose of this study is to synthesize DNA probe of human bone morphogenetic protein-2(hBMP-2).Methods Extracting total RNA from human condyle sample. hBMP-2 cDNA flanking hBMP-2 mature region was amplified by RT-PCR.The hBMP-2 cDNA was then inserted into cloning vector pGEM-T,Screening by white and blue clone after transforming the recombinant plasmid into E.coli.strain JMl09.Getting the recombinant plasmid pGEMT-T-hBMP-2 which was confirmed by restriction enzyme and sequencing analysis.Cutting pGEMT-T-hBMP-2 by restriction enzymes Nde Ⅰand Sac Ⅱ.Labelling and synthesizing hBMP-2 cDNA probe with Digoxin labeling-detecting Kit.Using this probe to research expression of BMP-2mRNA in human osteosarcoma specimens by in situ hybridization.Results We synthesized hBMP-2cDNA probe with which we demonstrated the positive expression of BMP-2mRNA in osteosarcoma specimens by in situ hybridization. Conclusion The probe we synthesized and labelled was steady and reliable.It could be used in gene expression studies of hBMP-2 by in situ hybridization.

关 键 词：人骨形成蛋白-2 CDNA探针 实验合成 基因克隆 原位杂交 骨肉瘤 

分 类 号：R738.1[医药卫生—肿瘤]