牛干扰素-γ基因的克隆、序列分析及表达研究

Cloning,Sequence Analysis and Expression of Bovine Interferon-gamma Gene

为了使干扰素-γ(interferonγ,IFN-γ)在牛结核等疾病的诊断和防制方面取得更有效的应用,试验采用RT-PCR法从云南本地黄牛外周血淋巴细胞总RNA中扩增牛IFN-γ(BoIFN-γ)基因,测序并进行序列分析;然后亚克隆至pET-28a,转化大肠杆菌BL21(DE3),用IPTG诱导表达。序列分析结果表明,克隆的IFN-γ基因与GenBank中荷斯坦牛IFN-γ序列同源性达99.60%。表达产物经SDS-PAGE分析,在大小约23ku处可见目的蛋白表达条带,与预期结果一致;用ELISA检测,表达的蛋白具有明显的生物学活性,在菌体中的含量为244ng/mL。本试验结果为牛IFN-γ蛋白的进一步研究和应用奠定了基础。

In order to build more effective use of interferon-gamma（IFN-γ）on the diagnosis and control of diseases like bovine tuberculosis,the cDNA of bovine interferon-γ（BoIFN-γ）gene was prepared from PHA-stimulated Yunnan cattle periphetal blood lymphocyte cell by RT-PCR.The BoIFN-γgene were identified by sequencing and BLAST.Then,the BoIFN-γwas inserted into the expressing plasmid pET-28 afor expression in E.coli BL21（DE3）.The recombinant bacteria were induced with IPTG.Sequence analysis showed the homology of BoIFN-γgene between Yunnan and Holstein cows was 99.60%.The molecular size of IFN-γexpressed was 23 ku with SDS-PAGE electrophoresis detection,which was consistent with the forecast.The analysis results of ELISA Kits showed the biological activity of IFN-γexpressed was obviously,the content in bacteria was 244ng/mL.The experiment lays a foundation for the further research and application of bovine IFN-γ.