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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: To synthesize and label human bone morphogenetic protein-2 (hBMP-2) cDNA probe by molecular biology experiments. Methods Total RNA was extracted from the human condylar fracture specimens. The cDNA fragment encoding the coding region of hBMP-2 mature peptide was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into pGEM-T plasmid , And transformed into Escherichia coli; selected positive clones were cultured, extracted recombinant plasmids, and nucleotide sequence analysis; restriction enzyme digestion, and then labeled with digoxin labeling kit random primer method, Synthesis of digoxigenin-labeled BMP-2 cDNA probe; In situ hybridization of BMP-2 mRNA in human maxillofacial osteosarcoma specimens using this probe was performed to identify probe reliability. Results The hBMP-2 cDNA probe was synthesized by experimental method. The in situ hybridization of this probe confirmed BMP-2 mRNA expression in osteosarcoma of the jaw. Conclusion The self-synthesized and labeled human BMP-2 cDNA probe is stable and reliable and can be applied to in situ hybridization of BMP-2 mRNA expression.