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目的克隆人软骨来源形态发生蛋白 - 1(CDMP - 1)成熟肽编码区的基因片段。 方法用异硫氰酸胍一步法从人软骨组织和酶消化法原代培养的软骨细胞中分别抽提总RNA ,利用RT -PCR方法扩增出人CDMP -1成熟区的基因片段 (约 1.0kbp) ,将所得基因片段插入 pBluescript质粒载体 ,重组质粒DNA ,通过酶切分析和核苷酸序列分析鉴定阳性克隆。 结果从软骨组织直接抽取到总RNA ,其质量与从等重量软骨组织中的原代软骨细胞所抽取的基本一致 ,人CDMP - 1酶切图谱正确 ,测序得到的部分序列与基因库公布序列一致。 结论从软骨组织可以直接抽取总RNA ,克隆到人CDMP - 1成熟肽编码区的基因片段。
Objective To clone the gene fragment encoding mature peptide of human cartilage derived morphogenetic protein - 1 (CDMP - 1). Methods Total RNA was extracted from human chondrocytes and chondrocytes cultured by enzymatic digestion by one-step guanidinium thiocyanate method. The gene fragment of human CDMP -1 mature region was amplified by RT-PCR (about 1.0 kbp). The obtained gene fragment was inserted into pBluescript plasmid vector and recombinant plasmid DNA was identified. Positive clones were identified by restriction analysis and nucleotide sequence analysis. Results The total RNA was directly extracted from cartilage tissue and its quality was basically the same as that drawn from the primary chondrocytes in equal weight cartilage tissue. The human CDMP - 1 digestion map was correct, and the partial sequences sequenced were consistent with those published by the gene bank . Conclusion The total RNA can be directly extracted from the cartilage tissue and cloned into the gene fragment of human CDMP - 1 mature peptide coding region.