+Gz重复暴露大鼠脑cDNA消减文库的构建  

Construction of a cDNA Subtractive Library of Rat Brain after Repeated +Gz Exposure.

作　　者：蔡庆[1] 刘红巾[1] 林凯[1] 姜建东[1] 朱美财[1] 

机构地区：[1]空军总医院分子生物学研究中心,北京100036

出　　处：《航天医学与医学工程》2002年第6期415-418,共4页Space Medicine & Medical Engineering

基　　金：全军医药卫生科研基金课题 (98D0 3 3 ) ;空军后勤部科研基金课题 (KH99190 76)

摘　　要：目的应用抑制性消减杂交技术构建 +Gz重复暴露大鼠脑和正常大鼠脑差异表达的cDNA消减文库 ,为筛选 +Gz暴露大鼠脑差异表达基因 ,探讨 +Gz引起脑损伤的分子机制奠定基础。方法Wister大鼠随机分成对照组和 +Gz重复暴露组 ,对照组大鼠G值为 + 1Gz,实验组大鼠在动物离心机上经历了 3次+ 1 0Gz/1min(两次间间隔 30min)作用 ,于暴露后 6h处死取脑。分别从 +Gz重复暴露组和对照组大鼠脑中提取poly(A) +RNA ,依次合成单链及双链cDNA ,用RsaⅠ酶切成大小不等的片段。将 +Gz重复暴露组cDNA分为两组 ,分别与两种不同的接头连接 ,再与对照组cDNA进行两次消减杂交及两次抑制性PCR ,将PCR产物与T/A载体连接构建 +Gz暴露大鼠脑cDNA消减文库。结果成功构建具有高消减效率的 +Gz暴露大鼠脑cDNA消减文库 ,非特异性cDNA片段被有效地消减 ,特异表达的cDNA得到富集。结论 +Gz暴露大鼠脑cDNA消减文库的建立为进一步筛选、克隆 +Gz重复暴露大鼠脑差异表达基因奠定了基础。Objective To construct a cDNA subtractive library of rat brain after repeated +Gz exposures with suppression subtractive hybridization (SSH). Method Wister rats were randomly divided into control group and repeated +Gz exposure group. Using an animal centrifuge, control rats were exposed to +1 Gz and exposure rats were exposed to +10 Gz for three times, each for 1 min with 30 min interval in between. Brains were taken 6 h after the last centrifuge run and Poly(A) +RNA were isolated. Moreover, single strand cDNAs and double strand cDNAs were synthesized in turn. After RsaⅠenzyme restriction, +Gz exposure rat brain cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2R, respectively. Then +Gz exposure rat brain cDNAs were hybridized with the control rat brain cDNA twice and underwent nested PCR twice. The PCR product was ligated with T/A plasmid vectors to set up the subtractive library. Result The cDNA subtractive library of rat brain after repeated +Gz exposures with high subtractive efficiency was set up successfully. Conclusion The highly efficient cDNA subtractive library may provide a solid foundation for screening and cloning differentially expressed genes in rat brain after repeated exposures to +Gz.

关 键 词：+GZ 重复暴露 大鼠 脑 CDNA消减文库 抑制性消减杂交 

分 类 号：R854[医药卫生—航空、航天与航海医学]