红笛鲷p38β MAPK基因的克隆及原核表达

doi:10.13560/j.cnki.biotech.bull.1985.2014.12.030

生物技术通报 ›› 2014, Vol. 0 ›› Issue (12): 177-183.doi: 10.13560/j.cnki.biotech.bull.1985.2014.12.030

红笛鲷p38β MAPK基因的克隆及原核表达

夏洪丽^1,2,3，蔡佳^1,2,3，鲁义善^1,2,3，简纪常^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:2014-03-27 出版日期:2014-12-08 发布日期:2014-12-12
作者简介:夏洪丽，女，硕士，研究方向:水产动物病害防治;E-mail:xiahongli0427@163.com
基金资助:
广东省国际合作项目(2012B050600029)，广东高校国际合作创新平台项目(2013gjhz0008)

Cloning and Prokaryotic Expression of p38β MAPK from Lutjanus sanguineus

^1,2,3Xia Hongli,^1,2,3Cai Jia,^1,2,3Lu Yishan, ^1,2,3Jian Jichang, ^2,3,4Wu Zaohe

(1. Fisheries College, Guangdong Ocean University, Zhanjiang 524088; 2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquantic Economic Animals, Zhanjiang 524088; 3. Key Laboratory of Control for Disease of Aquatic Animals of Guangdong Higher Education Institutes, Zhanjiang 524088; 4. Zhongkai University of Agriculture and Engineering, Guangzhou 510225)

Received:2014-03-27 Published:2014-12-08 Online:2014-12-12

摘要/Abstract

摘要： 鱼类 p38 MAPK 基因在宿主免疫调控及抵御病原侵染的过程中具有重要作用。利用 RACE 技术克隆了红笛鲷 p38β MAPK 基因(Ls-p38β，GenBank 登录号为 KJ502277)。序列分析结果显示，Ls-p38β cDNA 全长 1 628 bp，开放阅读框 1 086 bp，可编码 361 个氨基酸，预测其编码蛋白的分子量为 41.6 kD，理论等电点为 5.74。该基因推导的氨基酸序列含有 p38 MAPK 家族保守的双磷酸化激活环(TGY)，与尼罗罗非鱼的 p38β 有 97% 的相似性。将此基因定向插入原核表达载体构建重组表达质粒 pET32- p38β 后导入 BL21(DE3)菌株，利用 IPTG 进行诱导表达。SDS-PAGE 与 Western blot 分析显示约在 60 kD 出现特异蛋白条带，与预期分子量大小相符，说明 Ls-p38β 融合蛋白表达成功。

关键词: 红笛鲷, p38β, 丝裂原活化蛋白激酶, 原核表达

Abstract: Humphead snapper(Lutjanus sanguineus)p38β MAPK(Ls-p38β)was cloned using RACE method. The GenBank accession number is KJ502277. The full-length cDNA of p38β MAPK was 1 628 bp containing an open reading frame of 1 086 bp, encoding 361 amino acids with an estimated molecular weight of 41.6 kD and a theoretical pI of 5.74. Amino acid alignment indicated that Ls-p38β possessed a Thr-Gly-Tyr(TGY)dual phosphorylation motif of p38 MAPK family and shared 97% similarity with Oreochromis niloticus p38β. The prokaryotic expression vector pET-p38β was constructed and then transformed into BL21(DE3). SDS-PAGE and Western blotting analysis indicated that the recombinant protein of Ls-p38β was successfully expressed and molecular weight of expressed fusion protein was 60 kD.

Key words: Lutjanus sanguineus, p38β, MAPK, Prokaryotic expression

夏洪丽，蔡佳，鲁义善，简纪常，吴灶和. 红笛鲷p38β MAPK基因的克隆及原核表达[J]. 生物技术通报, 2014, 0(12): 177-183.

Xia Hongli,Cai Jia,Lu Yishan, Jian Jichang, Wu Zaohe. Cloning and Prokaryotic Expression of p38β MAPK from Lutjanus sanguineus[J]. Biotechnology Bulletin, 2014, 0(12): 177-183.

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红笛鲷p38β MAPK基因的克隆及原核表达

Cloning and Prokaryotic Expression of p38β MAPK from Lutjanus sanguineus

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