人腮腺多形性腺瘤中骨形成蛋白-2成熟肽编码区基因克隆及序列分析

Molecular cloning and sequence analysis of BMP-2 gene fragmentencoding mature peptide in human pleomorphic adenoma of parotid gland

目的克隆腮腺多形性腺瘤(pleomorphic adenoma,PA)中骨形成蛋白-2(bone morphogenetic protein,BMP-2)成熟肽编码区基因片段,构建含有该基因片段的重组质粒,并DNA序列分析。方法从5例腮腺PA标本中分别提取总RNA,用特异性引物经逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)扩增BMP-2成熟肽编码区基因片段,连接质粒载体pGEM-T,转化至感受态大肠杆菌JM109,挑选阳性克隆培养,抽提重组质粒DNA,测序鉴定。结果重组质粒DNA测序表明,插入的基因片段中有2个碱基发生改变,但所编码的氨基酸不变。结论从腮腺PA标本中成功克隆BMP-2成熟肽编码区基因片段,并构建重组质粒。

Objective To clone bone morphogenetic protein- 2 （BMP -2） gene fragment encoding mature peptide in human pleomorphic adenoma （PA） of parotid gland, and to construct recombinant plasmid for BMP -2 and to analyze DNA sequence. Methods Total RNA was extracted from 5 specimens with human PA of parotid gland. The BMP - 2 gene fragment encoding mature peptide was amplified by RT-PCR with specific primers. The cDNA fragment was inserted into pGEM-T and then was transformed into sensitive Bacillus Coli JM109. After the bacteria whose clone was positive were cultured, the recombinant plasmid for BMP - 2 was extracted, and identified by DNA sequencing. Results Sequence analysis revealed that there were 2 nucleotides in the inserted cDNA fragment different from those in human BMP - 2 mRNA reported by GenBank , with All7→G, C252→T, respectively. But there was no amino acid change with nucleotide changing. Conclusion Conclusion BMP -2 gene fragment encoding mature peptide in human PA of parotid gland was successfully cloned and the recombinant plasmid for BMP -2 was constructed.