骨质疏松症骨组织cDNA差减文库的构建

Construction on subtractive cDNA library of osteporosis mice

目的构建人抑制差减文库,为筛选骨质疏松骨相关基因奠定基础。方法分别从正常骨和骨质疏松症骨组织分离mRNA并反转录成cDNA,酶切后骨质疏松cDNA分成两组,分别与不同的接头连接。经过两轮差减杂交和两次抑制性PCR后得到两者之间差异表达的cDNA挑取克隆进行PCR扩增鉴定。结果成功地构建了骨质疏松相关的的差减cDNA文库,获得的正、反向差减文库分别含652、345个重组子;插入片段的平均大小为526bp。结论该差减cDNA文库的建立为进一步在分子水平上阐明骨质疏松症的分子机制奠定了基础。

Objective To construct a subtracted eDNA libraries of osteporosis mice by suppression subtraetive hybridiztion （SSH） ,so as to screen for the differentially expressed genes. Methods The mRNAs were extracted from osteporosis mice and control mice,then converted into double-strand cDNAs, forward and reverse hybridization was performed between mRNAs of osteporosis mice and control mice. Two-directional subtracted eDNA fragments were cloned into Pinpoint plasmid vectors, and the vectors were transformed into E.coli JM109. The clones-selecting were amplified by PCR and identified. Results The subtractive eDNA libraries were obtained including 576 and 322 clones respectively. By PCR detection, the length of the inserted fragments was 530 bp on average. Conclusion A high quality eDNA library from mice bone tissue of osteporosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of osteporosis.