鸭子α-干扰素基因表达及多样性分析

Analysis of polymorphism and expression of duck interferon-α gene

目的 :采用分子杂交及PCR方法 ,分析鸭α 干扰素基因的表达及多态性。方法 :从鸭外周血分离出的单核细胞在体外经PHA(5 μg/ml)刺激不同时间后 ,提取总RNA。以RT PCR方法检测鸭α 干扰素 (DuIFN α)mRNA表达状况。引物根据最近公布的DuIFN α基因序列设计。从鸭外周血单核细胞中提取的基因组DNA经限制性内切酶BamHI,HindIII,PstI,XbaI消化后 ,以公布的DuIFN α序列为探针 ,采用Southern杂交分析DuIFN α基因的多样性。结果 :在未经PHA刺激的鸭外周血单核细胞 (PBMCs)中 ,未检测到DuIFN α表达 ;PHA刺激 4h后 ,即可检测到DuIFN α表达 ,一直持续到 2 4h。基因组DNA限制性内切酶多态性分析表明 ,PstI酶切后 ,出现片段大小各异的杂交信号 ,提示DuIFN α存在多样性。结论 :鸟类α 干扰素基因与哺乳动物类似 。

Objective: To analyse polymorphism and expression of duck interferon α (DuIFN α) using nucleic acid hybridisation and PCR amplification Methods: The total RNA was isolated from duck PBMCs stimulated with 5 μg/ml PHA at different timepoint and transcribed to cDNA with oligodT The expression of DuIFN α was detected by RT PCR with primers based on the published sequence of DuIFN α gene To analyse the polymorphism of type I DuIFN gene, the genomic DNA isolated from duck PBMCs was digested with BamHI, HindIII, PstI, XbaI respectively and subjected to southern hybridisation with 32 P labeled DuIFN α probe Results: The data indicated that the DuIFN α transcript was undetectable in PBMCs without PHA stimulation, but detectable after 4 h stimulation with PHA and last until 24 h The RFLP analysis of duck genomic DNA showed that the hybridisation signals with varying sizes were occurred in PstI digested sample, which suggested the presence of other type I DuIFN gene Conclusion: The type I IFN gene from avian species has a similar expression and polymorphism pattern with its mammalian counterpart