大黄鱼酪氨酸激酶基因克隆及原核表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.021

生物技术通报 ›› 2016, Vol. 32 ›› Issue (11): 180-187.doi: 10.13560/j.cnki.biotech.bull.1985.2016.11.021

大黄鱼酪氨酸激酶基因克隆及原核表达分析

张在鹏¹, 林鹏¹, 郭松林¹, 王艺磊¹, 张子平², 冯建军¹

1. 集美大学水产学院鳗鲡现代产业技术教育部工程研究中心农业部东海海水健康养殖重点实验室,厦门 361021;
2. 福建农林大学动物科学学院,福州 350002

收稿日期:2016-03-28 出版日期:2016-11-25 发布日期:2016-11-11
作者简介:张在鹏,男,硕士研究生,研究方向：水产动物免疫学;E-mail：wyzzp4331@163.com
基金资助:
国家自然科学基金项目（31272685）,福建省海洋与渔业厅项目（201212140006）,福建省自然科学基金项目（2016J01164）,集美大学创新团队基金项目（2010A001）,集美大学科研基金项目（C60819）

Cloning and Prokaryotic Expression Analysis of Tyrosine Kinase Gene in Large Yellow Croaker

ZHANG Zai-peng¹, LIN Peng¹, GUO Song-lin¹, WANG Yi-lei¹, ZHANG Zi-ping², FENG Jian-jun¹

1. Fisheries College,Jimei University,Engineer Research Center of Eel Modern Industry Technology,Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture,Xiamen 361021;
2. College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002

Received:2016-03-28 Published:2016-11-25 Online:2016-11-11

摘要/Abstract

摘要： 为了研究大黄鱼（Larimichthys crocea）T淋巴细胞酪氨酸激酶（lymphocyte cell kinase,LCK）的基因结构和功能,通过RT-PCR和RACE技术克隆了大黄鱼LCK（LcLCK）基因cDNA 全长序列,并利用实时荧光定量PCR（qRT-PCR）技术对该基因在大黄鱼各组织器官和胚胎发育各时期的表达情况进行了检测与分析,同时构建了LcLCK基因的原核表达载体,在大肠杆菌中进行了高效表达。结果表明,获得的LcLCK基因全长为2 334 bp,开放阅读框为1 503 bp,编码501个氨基酸。该蛋白具有非受体酪氨酸激酶Src家族典型的SH3、SH2和TyrKc三个结构域,其N端含有GCXCS和CXXC基序,C末端出现2个与人类LCK基因中的Tyr³⁹⁴和Tyr⁵⁰⁵相一致的保守酪氨酸位点。系统发育树表明LcLCK与红鳍东方鲀（Takifugu rubripes）LCK聚为一支;LcLCK基因在雌雄大黄鱼各组织器官中均有表达,其中在主要免疫器官脾脏、头肾和鳃有高丰度表达,雌性个体的表达量要高于雄性个体;胚胎发育过程中,LcLCK基因表达水平在多细胞期、囊胚期和原肠期较高,囊胚期达到峰值,卵黄栓形成期显著降低,眼泡出现期至孵出期持续下降,而初孵仔鱼期则有所回升;构建的原核表达载体pGEX-4T-2-LCK在大肠杆菌中成功表达,其新增LcLCK重组蛋白条带在SDS-PAGE电泳检测中约为84 kD,与预期表达的分子量相符。大黄鱼LcLCK在雌雄成熟个体的细胞免疫应答以及胚胎发育过程中T淋巴细胞免疫器官的形成密切相关。

关键词: 大黄鱼, LCK基因, 胚胎发育, 原核表达

Abstract: To identify the structure and function of the lymphocyte-specific protein tyrosine kinase（LCK）gene（LcLCK）in large yellow croaker（Larimichthys crocea）,a full length cDNA of LcLCK was cloned from large yellow croaker by RT-PCR and RACE. The expressions of LcLCK in various tissues of adult male and female large yellow croaker as well as at different stages of the embryonic development were analyzed and examined via qRT-PCR. The recombinant prokaryotic vector was also constructed and transferred intoEscherichia coli for efficient expression. The results were as following：Its full-length cDNA sequence was 2 334 bp,with a 1 503 bp open reading frame encoding a protein of 501 amino acids. The predicted protein contained the typical domains of SH3,SH2 and TyrKc in the Src kinase family of non-receptor tyrosine. Two motifs of GCXCS and CXXC were found in the N-terminal region of LCK,while the two conserved Tyr sites in accordance with the Tyr³⁹⁴ and Tyr⁵⁰⁵ of LCK from human were present in C-terminal. Phylogenetic analysis showed that the deduced LCK clustered with Japanese pufferfish（Takifugu rubripes）. qRT-PCR revealed that the expressions of LcLCK were detected in all adult tissues examined,with higher expression in the main immune tissues such as spleen,head kidney,and gills of both sexes;and the expression level of LcLCK gene in these tissues from female was higher than that of the male. Early in the embryonic development,the high levels of LcLCK were observed during the multiple cells,blastula,and gastrula stages with the expression peak in blastula stage. The significant decrease of LcLCK expression was found at formation of yolk plug stage,and remained at the low expression level from formation of eye lens stage to pre-hatching stage,whereas an increase of gene expression was observed at alevin stage. The constructed prokaryotic vector pGEX-4T-2-LCK was expressed successfully in E. coli. In SDS-PAGE analysis,the new recombinant LcLCK protein band was about 84 kD that was in accordance with the expected. In conclusion,LcLCK is closely related to the cellular immune responses of adult male and female large yellow croaker as well as the mature of immune organs producing T-cell during embryonic development.

Key words: Larimichthys crocea, LCK gene, embryonic development, prokaryotic expression

张在鹏, 林鹏, 郭松林, 王艺磊, 张子平, 冯建军. 大黄鱼酪氨酸激酶基因克隆及原核表达分析[J]. 生物技术通报, 2016, 32(11): 180-187.

ZHANG Zai-peng, LIN Peng, GUO Song-lin, WANG Yi-lei, ZHANG Zi-ping, FENG Jian-jun. Cloning and Prokaryotic Expression Analysis of Tyrosine Kinase Gene in Large Yellow Croaker[J]. Biotechnology Bulletin, 2016, 32(11): 180-187.

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大黄鱼酪氨酸激酶基因克隆及原核表达分析

Cloning and Prokaryotic Expression Analysis of Tyrosine Kinase Gene in Large Yellow Croaker

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