鱼类白细胞介素4的基因克隆和表达分析

【作者】 郦佳慧；

【导师】 邵健忠； 项黎新；

【作者基本信息】 浙江大学 ， 细胞生物学， 2007， 硕士

【摘要】 白细胞介素4(IL-4)是Th2型免疫应答的关键细胞因子，对很多免疫细胞因子具有调节作用，如γ干扰素，白细胞介素1和α肿瘤坏死因子等。目前对人类和一些哺乳动物的IL-4已开展了较多研究，但对鱼类IL-4的研究尚未见报道。本文成功克隆了河豚IL-4 cDNA全序列，并在毕赤酵母系统中诱导表达了重组IL-4。河豚IL-4 cDNA全长834bp，包括39bp的5’非编码区(5’UTR)，375bp的3’非编码区(3’UTR)和420bp的开放阅读框架(ORF)，其翻译得到的多肽含139个氨基酸，预测分子量约16.131kDa。河豚IL-4基因与已知的哺乳动物和鸟类有相同的结构，即由四个外显子和三个内含子组成。它的编码蛋白与其他同源体有11％—16％的同源相似性。用优化条件的RT-PCR半定量估测IL-4在河豚各个组织中的表达水平，结果显示，IL-4在所选组织中均为组成性表达，包括头肾、脾脏、肝脏、大脑、鳃、肌肉和心脏，其中头肾、脾脏和肝脏的表达量较低。IL-4的组成性表达与它对免疫细胞因子调节的潜在作用是相一致的。用2μg ConA、2μg PHA和2μg PMA的混合诱导剂刺激河豚，可以发现IL-4在以上所检测组织中的表达量显著上升，这一结果也预示了有丝分裂原激活IL-4的炎症免疫应答。此外，成功构建了重组表达质粒pPIC9k-IL4，并将其重组整合入GS115毕赤酵母基因组，通过组氨酸缺陷型培养基MD和G418筛选获得含有多个拷贝的毕赤酵母转化子，经甲醇诱导，在毕赤酵母表达系统中检测到约16kDa的条带，与预期的重组蛋白大小相一致。这是关于鱼类IL-4的克隆、鉴定和表达的首例报道。更多还原

【Abstract】 Interleukin-4 (IL-4) is one of the key cytokines in Th2 mediated immune responses, which has been shown to regulate the responses of many immune cytokines, such as interferon-gamma (IFN-γ), interleukin-1 (IL-1) and TNF-a. Much work on 1L-4 has been done in human and several mammal species while little in fish. In this study, we have cloned and characterized the full-length cDNA of IL-4 in Tetraodon, and obtained recombinant IL-4 (rIL-4) expressed in Pichia pastoris. The Tetraodon IL-4 cDNA is 834 bp in length and contains a short 5’UTR of 39 bp, a 3’UTR of 375 bp and an open reading frame of 420 bp translating into a protein of 139 aa with a predicted molecular mass of 16.131 kDa. The Tetraodon IL-4-encoding gene with the same organization as the mammalians and birds consists of four exons and three introns. The encoded protein shows 11-16% identities to other homologues. RT-PCR was optimized to estimate the expression level of IL-4 in Tetraodon. The results showed that IL-4 is constitutively expressed in all selected tissues, including head kidney, spleen, liver, brain, gill, muscle and heart, although low levels were observed in head kidney, spleen, and liver. The ubiquitous expression of IL-4 is consistent with a postulated role in immune cytokines regulation. Stimulating the fish with a mixed stimulant that contained 2μg ConA, 2μg PHA, and 2μg PMA, significantly up-regulated the expression of IL-4 in most tissues examined, which potentially indicated that IL-4 was involved in the immune inflammatory responses triggered by mitogens. Recombinant expression plasmid pPIC9k-IL4 was successfully constructed, and transformed into P. pastoris strain GS115. Transformants were screened with auxotrophic medium MD and G418. Induced with methanol, the predicted expression of rIL-4 was detected. This is the first report of cloning, characterization and expression of IL-4 in fish.更多还原