利用抑制性消减杂交技术筛选大鼠哮喘相关基因  

Identification of differentially expressed genes in rat asthma model by suppression subtractive hybridization technology

作　　者：钟波[1] 王慧莲[1] Muhammad Shahzad 宁启兰[1] 韩燕[1] 杨旭东[1] 张富军[1] 吕社民[1] 

机构地区：[1]西安交通大学医学院遗传学与分子生物学系,陕西西安710061

出　　处：《浙江大学学报（医学版）》2009年第4期362-369,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(30630058,30801027,30571725)

摘　　要：目的:利用哮喘大鼠模型筛选与哮喘发病相关的差异性表达基因。方法:采用抑制性消减杂交技术,以哮喘大鼠肺组织总RNA为检验子,对照大鼠肺组织总RNA为驱赶子,将消减杂交获得的DNA片断与pGEM-T Easy Vector连接,构建cDNA文库后,转化大肠杆菌DH5α,筛选阳性克隆。将获得的阳性克隆插入片段进行测序分析,测序结果与GenBank数据库中cDNA和EST序列进行相似性比对,获得差异性表达基因的信息。结果:扩增消减cDNA文库获得300余个白色克隆,随即挑选36个测序,除去重复序列,结果中含有已知的基因4个,ESTs 2个及与任何序列无同源性的新基因片段3个。结论:成功构建了哮喘大鼠肺脏差异性表达基因的正向消减cDNA文库,筛选出数个与哮喘相关的差异表达基因。Objective： To identify differentially expressed genes related to asthma by using a rat model. Methods： Total RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization （SSH） was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E. coli transformation and positive clones of the transformants were screened. The white clones in selective medium from eDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank （NCBI）. Results ： There were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search （http ：//www. ncbi. hlm. nih. gov/BLAST） revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments. Conclusion： The forward- subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.

关 键 词：哮喘/免疫学 哮喘/病理生理学 抑制性消减杂交 差异性表达基因 CDNA文库 

分 类 号：R562.25[医药卫生—呼吸系统]