大鼠脊髓损伤修复差异表达cDNA文库的构建

Construction of a subtractive library of genes differentially expressed during spinal cord injury and regeneration

目的 运用改良消减杂交技术构建大鼠脊髓损伤修复过程中出现的差异表达基因文库 ,为寻找在脊髓损伤后的修复过程中起关键作用的分子提供帮助。方法 在经典消减杂交的基础上引进SMART反转录、长距离PCR、磁性分离系统、离心柱色谱等方法建立改良消减杂交技术 ,通过两轮消减杂交构建差异表达基因的cDNA消减文库 ,并对文库中的差异基因进行批量克隆与生物信息学分析。结果 将两轮消减杂交后的差异表达基因转化大肠杆菌JM10 9以建立差异表达基因文库 ,库容量大小为 2× 10 3。从文库中随机挑取 90个克隆进行酶切鉴定与测序分析 ,得到 4 0个差异基因序列 ,其中 32个已知序列 ,8个新序列。 结论 通过改良消减杂交成功建立了脊髓损伤修复过程中出现的差异表达基因文库 ,为筛选在大鼠脊髓损伤修复过程中起关键作用的基因 。

Objective To build a library of genes differentially expressed in injured spinal cord and further to understand the molecular mechanisms of CNS regeneration.Methods In our study, SMART reverse transcription method, long-distance PCR, streptavidin magnetic bead-mediated subtraction and spin column chromatography were adopted on the basis of classical subtractive hybridization. After two rounds of subtraction, a subtractive cDNA library was constructed.Then clones were randomly picked out for further identification. Results Differentially expressed genes of the subtractive library were ligated with pGEM-T easy vector and then transformed into JM109 competent cells. The capacity of this library was about 2×10^(3). Among 90 clones randomly picked out for restrictive enzyme digestion and sequencing analysis, 40 differentially expressed genes were obtained,including 32 known sequences and 8 novel sequences. Conclusion Improved subtractive hybridization can be successfully used to construct a subtractive cDNA library of genes differentially expressed in injured spinal cord. Separation and characterization of these genes will provide useful information for understanding the molecular mechanisms of repair and regeneration of injured spinal cord.