半滑舌鳎AKT-ineracting protein基因的克隆及免疫应答表达分析

研究克隆了半滑舌鳎Cynoglossus semilaevis膜蛋白AKT-interacting protein (AKTIP)基因, 研究了其在健康组织中的表达模式、鳗弧菌(Vibrio anguillarum)感染后免疫组织和不同病原物刺激外周血淋巴细胞中的表达特征。通过常规克隆和RACE技术, 获得的半滑舌鳎AKTIP基因全长cDNA序列为1224 bp, 其中包括5'-UTR为116 bp, 3'-UTR为117 bp和完整的ORF序列891 bp, 编码296个氨基酸, 预测蛋白质的等电点(PI)为9.12,分子量是33.74 kD; 同源比对发现半滑舌鳎AKTIP的氨基酸序列在不同物种之间具有较高的保守性; 荧光实时定量PCR (qRT-PCR)检测到半滑舌鳎各个组织中均有AKTIP基因的表达, 在卵巢中表达量最高; 鳗弧菌感染半滑舌鳎后, AKTIP基因在肝、鳃、血液、肠、头肾和脾中均上调表达; 病原模拟物PGN、LPS、poly I:C和WGP刺激半滑舌鳎外周血淋巴细胞均诱导AKTIP基因下调表达。研究表明半滑舌鳎AKTIP基因参与了机体免疫反应为给半滑舌鳎免疫防御技术的研究提供理论依据。

AKT-INTERACTING PROTEIN GENE CLONING AND ITS EXPRESSION PROFILE IN RESPONSE TO PATHOGEN INFECTION IN HALF SMOOTH TONGUE SOLE (CYNOGLOSSUS SEMILAEVIS)

AKT-interacting protien (AKTIP) is a kind of membrane protein, involvimg in the regulation of PI3K/PDK1/Akt pathway. AKTIP gene is still unknown in fish. In the present study, AKTIP gene of Cynoglossus semilaevis was first cloned and full length of cDNA was 1224 bp in size, including 5'-untranslated region (UTR) of 116 bp, 3'-UTR of 117 bp and a complete open reading frame (ORF) of 891 bp, encoding 296 amino acids. Theoretical isoelectric point (PI) of predicted protein was 9.12 and molecular weight was 33.74 kD. Homologous comparison showed that the amino acids sequence of AKTIP in C. semilaevis had a high identity with those of other species. The AKTIP gene was expressed in all tested tissues in the health C. semilaevis and the highest expression was in the ovary. In order to investigate the expression patterns of the AKTIP gene in immune response, the specific expression of AKTIP was performed after Vibrio anguillarum infection and in peripheral blood lymphocytes with different pathogens stimulated. The results showed that the expression of AKTIP gene was up-regulated in the liver, gill, blood, intestinal, head kidney and spleen after V. anguillarum infection. While the AKTIP gene expression in peripheral blood lymphocytes was down-regulated after PGN, LPS, WGP and poly I: C stimulation. The research revealed that AKTIP gene involved in C. semilaevis immune response. These results provide the evidence to explore AKTIP defense mechanism and to control C. semilaevis disease in the further study.