从人软骨提取总RNA和CDMP-1成熟肽片段的克隆

Isolation of Total RNA from Cartilage and Cloning of Human CDMP-1 Gene

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 and partially sequence of human cartilage-derived protein-1 (CDMP-1) encoding mature protein. Methods In the study, total RNA was extracted respectively by the improved acid guanidinium thiocyanata-phenol-chloroform method from the human cartilage tissue, and chondrocytes from the human cartilage through collagenase digestion. The desired DNA product was obtained from the total RNA by RT-PCR with the primers including Oligo(dt) and two gene specific primers. The segment (about 1.0 kbp) was inserted into the pBluescript vector and the interesting plasmid was transformed into the E. coli host strain DH5α. The double-stranded DNA of the positive clone was analyzed by restriction endonuclease mapping and DNA sequencing. Results The restriction endonuclease map and sequence of human CDMP-1 encoding mature protein were consistent with those of the references published. Conclusion The CDMP-1 cDNA encoding mature protein may be obtained for further research.