摘要
为筛选牦牛外周血单核细胞(PBMC)差异性基因,以刀豆素A(ConA)和脂多糖(LPS)联合刺激的PBMC cDNA为实验组,未经诱导刺激的PBMC cDNA为驱动组,利用抑制性消减杂交技术(SSH)构建了丝裂原诱导刺激PBMC的消减文库并对其部分阳性克隆进行了EST序列分析。从消减文库中随机挑取16个阳性克隆,进行PCR鉴定,显示克隆的重组率大于93%,插入片段大小大部分集中在200bp～1000bp之间。随机挑取100个克隆进行测序及同源性分析,初步获得27条差异表达基因片段,其中24个为已知基因,3个为新ESTs序列;随机选择非重复的6个差异表达的序列设计引物,以半定量PCR方法验证其消减效率。结果显示,均从构建的消减文库中扩增到目的片段,其中5个为诱导性差异表达分子,1个为诱导特异性表达分子,说明该文库有较高的质量。本研究应用抑制消减杂交技术构建了牦牛PBMC的差异表达cDNA文库,并高通量克隆鉴定了相关功能基因片段,表明该技术手段有助于快速发现牦牛新功能基因。
SA subtractive cDNA library was constructed by suppression subtractive hybridization （SSH） using cDNA from Yak peripheral blood mononuclear cells （PBMC） treated with lipopolysaccharide （LPS） and concanavalinA （ConA）. The library contained 93 % recombinants with insert size ranged from 200 bp to 1 000 bp. One hundred positive clones were randomly sequenced and analyzed, and 24 differentially expressed genes and 3 novel ESTs were identified. The subtraction efficiency of the SSH cDNA library was confirmed by randomly testing 6 non-repeat sequences by semi-quantitative RT-PCR, of which 5 differential expression molecules were detected. These results suggest that SSH is an effective technique to study gene differential expression gene.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第8期631-636,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家863项目(2006AA10A203)
甘肃自然基金(325061-A25-081)
