斜带石斑鱼IL-10基因的克隆及其原核表达

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.018

生物技术通报 ›› 2015, Vol. 31 ›› Issue (9): 131-137.doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.018

斜带石斑鱼IL-10基因的克隆及其原核表达

肖周婷^1，2，3, 黄郁葱^1，2，3, 简纪常^1，2，3, 鲁义善^1，2，3, 吴灶和^2，3，4

1.广东海洋大学水产学院，湛江 524088
2.广东省水产经济动物病原生物学及流行病学重点实验室，湛江524088
3.广东省教育厅水产经济动物病害控制重点实验室，湛江 524088
4.仲恺农业工程学院，广州 510225

收稿日期:2014-12-07 出版日期:2015-09-15 发布日期:2015-09-16
作者简介:肖周婷，女，硕士研究生，研究方向：水产动物病害防治；E-mail：xihu1tingyu@163.com
基金资助:
广东教育厅科技创新重点项目(2012CXZD0026)，十二五国家科技支撑计划(2012BAD17B03)

Cloning and Prokaryotic Expression of IL-10 Gene of Epinephelus coioides

Xiao Zhouting^1，2，3, Huang Yucong^1，2，3, Jian Jichang^1，2，3, Lu Yishan^1，2，3, Wu Zaohe²,^，3，4

1. College of Fisheries，Guangdong Ocean University，Zhanjiang 524088
2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals，Zhanjiang 524088
3. Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions，Zhanjiang 524088
4. Zhongkai University of Agriculture and Engineering，Guangzhou 510225

Received:2014-12-07 Published:2015-09-15 Online:2015-09-16

摘要/Abstract

摘要：

白介素10(Interleukin-10，IL-10)是一个重要的多效细胞因子，主要作用是介导炎症反应和调控一些免疫细胞的分化和增殖。利用同源克隆和RACE-PCR技术获得全长为1 104 bp的斜带石斑鱼(Epinephelus coioides)IL-10基因。该基因ORF为564 bp，编码187个氨基酸，预测蛋白分子量为21.7 kD，等电点为5.74。氨基酸同源性比对及进化分析结果显示，斜带石斑鱼IL-10氨基酸序列和吉富罗非鱼(O. niloticus)同源性最高，达72.25%。利用双酶切及质粒重组技术构建IL-10原核表达载体，并转化大肠杆菌BL21(DE3)中诱导原核表达。SDS-PAGE分析显示，融合蛋白分子量为37.5 kD，与预期相符；在IPTG为0.02 mmol/L，37℃诱导3 h，蛋白包涵体的表达量最大。

关键词: 斜带石斑鱼(Epinephelus coioides), Interleukin-10, 基因克隆, 重组表达

Abstract:

Interleukin 10(IL-10)is the critical multiple-functional cytokine playing the major role of mediating inflammatory responses and regulating the differentiation and proliferation of some immune cells. The full-length cDNA of IL-10 of Epinephelus coioides was cloned using homological cloning and rapid amplification of cDNA ends(RACE-PCR). Results showed the full-length of IL-10's cDNA was 1 104 bp, containing an open reading frame of 564 bp encoding 187 amino acids with an estimated molecular weight of 21.7 kD and an estimated isoelectric point of 5.74, and it had a signal peptide of 22 amino acid residues and no transmembranous region. Amino acid homology analysis showed that the amino acid sequences of IL-10 with Oreochromis niloticus had the highest homology and up to 72.25%. SDS-PAGE indicated that the molecular weight of the fusion protein was 37.5 kD which was in accord with the estimated. The optimal condition of inducible expression for IL-10 protein in Escherichia coli was at 37℃ for 3 h with 0.02 mmol/L of IPTG.

Key words: Epinephelus coioides, Interleukin-10, gene cloning, prokaryotic expression

肖周婷, 黄郁葱, 简纪常, 鲁义善, 吴灶和. 斜带石斑鱼IL-10基因的克隆及其原核表达[J]. 生物技术通报, 2015, 31(9): 131-137.

Xiao Zhouting, Huang Yucong, Jian Jichang, Lu Yishan, Wu Zaohe,. Cloning and Prokaryotic Expression of IL-10 Gene of Epinephelus coioides[J]. Biotechnology Bulletin, 2015, 31(9): 131-137.

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斜带石斑鱼IL-10基因的克隆及其原核表达

Cloning and Prokaryotic Expression of IL-10 Gene of Epinephelus coioides

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