林麝IL-1β基因的克隆、表达及生物学活性检测

doi:10.11843/j.issn.0366-6964.2013.11.006

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (11): 1734-1738.doi: 10.11843/j.issn.0366-6964.2013.11.006

林麝IL-1β基因的克隆、表达及生物学活性检测

邹丹丹，杨东，姜立春，邹方东^*

(四川大学生命科学学院，四川省濒危野生动物保护生物学重点实验室，生物资源与生态环境教育部重点实验室，成都 610064)

收稿日期:2013-05-02 出版日期:2013-11-23 发布日期:2013-11-23
通讯作者: 邹方东，教授，博导，主要从事分子细胞生物学研究，E-mail: fundzou@163.com
作者简介:邹丹丹(1988- )，女，重庆人，硕士，主要从事动物分子生物学研究，E-mail: dandanzou88@163.com

Molecular Cloning, Expression and Biological Activities Detection of Interleukin-1β (IL-1β) Gene from Moschus berezovskii

ZOU Dan-dan, YANG Dong, JIANG Li-chun, ZOU Fang-dong^*

(Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China)

Received:2013-05-02 Online:2013-11-23 Published:2013-11-23

摘要/Abstract

摘要：

旨在克隆和表达林麝IL-1β，为其用于林麝疾病的防治奠定基础。从林麝外周血单核细胞中提取总RNA，RT-PCR扩增IL-1β(Interleukin-1β)基因。将IL-1β成熟肽编码序列连接到pET32a(+)原核表达载体进行表达，纯化目的蛋白并检测其生物学活性。序列分析表明，林麝IL-1β开放阅读框(ORF)长801 bp，编码266个氨基酸,且在反刍动物中高度保守。IPTG诱导林麝IL-1β融合蛋白(MB-rIL-1β)表达，得到约35 ku的目标带，且大部分以可溶形式存在。对上清蛋白分离纯化得到约35 ku的单一条带。细胞增殖试验结果显示，林麝IL-1β融合蛋白能明显促进小鼠成纤维细胞(L929)增殖，表明其具有生物学活性。运用大肠杆菌表达系统可高效可溶性表达具有生物学活性的MB-rIL-1β融合蛋白。

Abstract:

The aim of this study was to clone and expression IL-1β from Moschus berezovskii for further study on the role of IL-1β in infectious diseases. Moschus berezovskii IL-1β (MBIL-1β) was cloned from peripheral blood mononuclear cells using RT-PCR. The sequence encoding the IL-1β mature peptide was ligated with pET32a(+) vector and expressed in Escherichia coli. The fusion protein was purified by His-Bind Column. Results showed that the length of MBIL-1β was 801 bp, encoding 266 amino acids. Sequence analysis showed that MBIL-1β had a high homology with other ruminants species. The M. berezovskii recombinant IL-1β (MB-rIL-1β) protein was expressed with IPTG induction and mainly in soluble form, the molecular weight was 35 ku. After purified, there was an aimed protein obtained. MTT essay confirmed that the MB-rIL-1β protein could enhance mouse fibroblast cell (L929) proliferation obviously and had biological activities. The result provide a feasible and convenient approach to produce soluble protein MB-rIL-1β with biological activity.

中图分类号:

邹丹丹，杨东，姜立春，邹方东. 林麝IL-1β基因的克隆、表达及生物学活性检测[J]. 畜牧兽医学报, 2013, 44(11): 1734-1738.

ZOU Dan-dan, YANG Dong, JIANG Li-chun, ZOU Fang-dong. Molecular Cloning, Expression and Biological Activities Detection of Interleukin-1β (IL-1β) Gene from Moschus berezovskii[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1734-1738.

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林麝IL-1β基因的克隆、表达及生物学活性检测

Molecular Cloning, Expression and Biological Activities Detection of Interleukin-1β (IL-1β) Gene from Moschus berezovskii

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