牛干扰素-γ基因的克隆、序列分析及表达研究

›› 2014, Vol. 41 ›› Issue (10): 1-6.

• 生物技术 • 下一篇

牛干扰素-γ基因的克隆、序列分析及表达研究

杨洁¹, 张书光¹, 李长青², 刘世高², 柴俊¹, 刘旭川¹, 史宪伟¹, 张以芳¹

1. 云南农业大学动物科学技术学院, 云南昆明 650201;
2. 云南省大理市动物疫病预防控制中心, 云南大理 671003

收稿日期:2014-03-05 出版日期:2014-10-20 发布日期:2014-10-29
通讯作者: 张以芳 E-mail:zyfkm@qq.com
作者简介:杨洁(1991－),女,河南人,硕士,研究方向:动物微生物与免疫学。
基金资助:
国家自然科学基金(31260598);云南省应用基础重点项目(2011FA014)

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

YANG Jie¹, ZHANG Shu-guang¹, LI Chang-qing², LIU Shi-gao², CHAI Jun¹, LIU Xu-chuan¹, SHI Xian-wei¹, ZHANG Yi-fang¹

1. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China;
2. Dali Animal Disease Control and Prevention Center in Yunnan Province, Dali 671003, China

Received:2014-03-05 Online:2014-10-20 Published:2014-10-29

摘要/Abstract

摘要： 为了使干扰素-γ(interferon γ,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23 ku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244 ng/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。

关键词: 牛干扰素-γ; 序列分析; 原核表达; 生物学活性

Abstract: 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-γ gene were identified by sequencing and BLAST. Then,the BoIFN-γ was inserted into the expressing plasmid pET-28a for expression in E. coli BL21 (DE3). The recombinant bacteria were induced with IPTG. Sequence analysis showed the homology of BoIFN-γ gene between Yunnan and Holstein cows was 99.60%. The molecular size of IFN-γ expressed 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-γ expressed was obviously,the content in bacteria was 244 ng/mL. The experiment lays a foundation for the further research and application of bovine IFN-γ.

Key words: bovine interferon-gamma; sequence analysis; prokaryotic expression; biological activity

中图分类号:

杨洁, 张书光, 李长青, 刘世高, 柴俊, 刘旭川, 史宪伟, 张以芳. 牛干扰素-γ基因的克隆、序列分析及表达研究[J]. , 2014, 41(10): 1-6.

YANG Jie, ZHANG Shu-guang, LI Chang-qing, LIU Shi-gao, CHAI Jun, LIU Xu-chuan, SHI Xian-wei, ZHANG Yi-fang. Cloning,Sequence Analysis and Expression of Bovine Interferon-gamma Gene[J]. , 2014, 41(10): 1-6.

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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

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