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(Lc LCK)基因c DNA全长序列,并利用实时荧光定量PCR(q RT-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Lc LCK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Lc LCK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Tyr Kc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Lc LCK与红鳍东方鲀(Takifugu rubripes)LCK聚为一支;Lc LCK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,Lc LCK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p GEX-4T-2-LCK在大肠杆菌中成功表达,其新增Lc LCK重组蛋白条带在SDS-PAGE电泳检测中约为84 k D,与预期表达的分子量相符。大黄鱼Lc LCK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。
To identify the structure and function of the lymphocyte-specific protein tyrosine kinase（LCK）gene（Lc LCK）in largeyellow croaker（Larimichthys crocea）,a full length c DNA of Lc LCK was cloned from large yellow croaker by RT-PCR and RACE. Theexpressions of Lc LCK in various tissues of adult male and female large yellow croaker as well as at different stages of the embryonic developmentwere analyzed and examined via q RT-PCR. The recombinant prokaryotic vector was also constructed and transferred into Escherichia coli forefficient expression. The results were as following ：Its full-length c DNA sequence was 2 334 bp,with a 1 503 bp open reading frame encodinga protein of 501 amino acids. The predicted protein contained the typical domains of SH3,SH2 and Tyr Kc in the Src kinase family of nonreceptor tyrosine. Two motifs of GCXCS and CXXC were found in the N-terminal region of LCK,while the two conserved Tyr sites in accordancewith the Tyr^（394） and Tyr^（505） of LCK from human were present in C-terminal. Phylogenetic analysis showed that the deduced LCK clustered withJapanese pufferfish（Takifugu rubripes）. q RT-PCR revealed that the expressions of Lc LCK were detected in all adult tissues examined,withhigher expression in the main immune tissues such as spleen,head kidney,and gills of both sexes;and the expression level of Lc LCK gene in these tissues from female was higher than that of the male. Early in the embryonic development,the high levels of Lc LCK were observed during the multiple cells,blastula,and gastrula stages with the expression peak in blastula stage. The significant decrease of Lc LCK 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 p GEX-4T-2-LCK was expressed successfully in E. coli. In SDS-PAGE analysis,the new recombinant Lc LCK protein band was about 84 k D that was in accordance with the expected. In conclusion,Lc LCK 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.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第11期180-187,共8页
Biotechnology Bulletin
基金
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)
关键词
大黄鱼
LCK基因
胚胎发育
原核表达
Larimichthys crocea
LCK gene
embryonic development
prokaryotic expression
