视神经慢性损伤相关差异表达cDNA文库的鉴定与分析

Identification and analysis of subtracted cDNA library of differentially expressed genes associated with chronic optic nerve injury

目的：对构建的猫视神经慢性损伤相关差异表达cDNA文库进行初步克隆、鉴定和分析。方法：用氯化钙转化法转化大肠杆菌进行cDNA文库扩增和蓝白斑筛选，每个文库随机挑取300个白色克隆用菌落PCR进行鉴定。对阳性克隆菌落进行DNA测序以获得差异表达基因片段序列。将测序所得的序列通过互联网用Blast程序查找日本国家DNA数据库进行同源性分析。用实时定量PCR验证差异克隆。结果：PCR鉴定获得1000个阳性菌落，测序获得674个EST片段，序列长度在200～800bp之间。同源性分析结果提示4周正向文库得到14个同源基因，4周反向文库得到20个同源基因，8周正向文库得到23个同源基因，8周反向文库得到19个同源基因。这些基因可归类于能量代谢、物质转运、信号转导、基因转录、细胞损伤与修复、MHC分子等。实时定量PCR检测的4个克隆证实是差异表达序列。结论：本研究构建的视神经慢性损伤相关差异表达cDNA文库为进一步鉴定和研究慢性视神经损伤相关基因提供了实验基础。

Objective：To clone,identify and analyze subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve compression in cats. Methods： The constructed cDNA libraries were amplified by E. coli transformation with calcium chloride and were subjected to blue and white screening. Three hundred positive bacterial clones were randomly chosen from each library and were identified by colony PCR. The positive clones were sequenced to screen for the differentially expressed genes. The identified sequences were then analyzed for homology using Blast program against the DNA database bank of Japanese through internet. Real-time PCR was performed to verify the expression of the 4 differential clones. Results： One thousand positive clones were identified by PCR and 674 ESTs were obtained by seqeuncing, with length ranging from 200 to 800bp. Results from Blast analysis revealed 14,20,23 and 19 homolog genes in 4-w forward,4-w reverse, 8-w forward and 8-w reverse subtractive libraries, respectively. These genes fell into several functional groups, such as energy and metabolism, ion transport, gene transcription, signal transduction, cellular injury and repair, and MHC molecules. Result of real-time PCR verified that the 4 clones were differentially expressed. Conclusion： The constructed subtractive cDNA libraries lay an experimental basis for further identification and study of the differentially expressed genes related to chronic optic nerve injury.