栉孔扇贝CfLITAF基因的克隆与表达分析

IOCAS-IR > 海洋环流与波动重点实验室

	栉孔扇贝CfLITAF基因的克隆与表达分析
其他题名	Molecular cloning and characterization of a putative LITAF gene homologue from Zhikong scallop Chlamys farreri
	余允东
学位类型	博士
	2007-06-08
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	栉孔扇贝 Lps诱导的tnf-α因子（litaf）脂多糖实时定量pcr
摘要	关于无脊椎动物中是否存在细胞因子一直都存在着异议。从细胞因子本身入手，运用细胞生物学和免疫学手段，已经在软体动物、节肢动物和棘皮动物等无脊椎动物中证实了高等动物细胞因子样活性物质的存在，然而利用分子生物学手段至今没有得到任何核苷酸或蛋白序列信息，而从与细胞因子相关的分子如调控其表达的转录因子入手来研究无脊椎动物中的细胞因子样物质的存在，或许会得到意想不到的惊喜。最近从人的单核细胞系THP-1中分离纯化出一种新型的转录因子，它可以被LPS诱导表达，产生的蛋白在TNF-α的表达过程中起着非常重要的作用，因而命名为LPS-induced TNF-α factor（LITAF）。随后该基因在小鼠和鸡中也被克隆分离出来，其编码的蛋白已经证实在TNF-α的表达过程中起着不可或缺的作用。迄今为止，在无脊椎动物中还没有相关同源基因的报道。本研究采用大规模EST测序方法，结合锚定PCR技术从栉孔扇贝体内克隆到了高等动物LITAF基因的同源基因CfLITAF的全长cDNA序列。该cDNA全长为1240bp，其中5' 非编码区（UTR）为112 bp；开放阅读框（Open Reading Frame, ORF）包括450 bp，编码149个氨基酸残基，该多肽的理论分子量为16.08 kDa，等电点为6.77；3' UTR为678bp，包含一个poly A尾巴。SMART结构域预测发现CfLITAF蛋白含有一个保守的LITAF结构域。实时定量PCR检测CfLITAF基因在栉孔扇贝主要免疫器官中的表达分布情况，发现在所检测的六种组织血细胞、外套膜、肌肉、心、腮、性腺中均能检测到CfLITAF基因转录本的不同丰度的表达。血淋巴和肌肉中的表达量相差不大，而在心、外套膜、腮和性腺中的表达量要高于血淋巴和肌肉中的表达量，分别是血中表达量的2.45、2.82、9.23和24.93倍。为了验证其表达量是否会受到LPS的诱导，利用QRT-PCR（quantitative real time PCR）检测了CfLITAF基因在受到LPS和PGN刺激后在血细胞中的表达规律。结果如下：在LPS刺激后3h，CfLITAF的表达量显著上调，达到了原初水平的1.5倍，随着时间的延长，其表达量逐渐恢复到刺激前水平；在用PGN刺激血细胞后的9个小时内均没有检测到CfLITAF基因mRNA表达量的显著变化。实验中所用血细胞为同一批原代培养的栉孔扇贝血淋巴细胞，细胞活力通过台盼蓝拒染实验测定。 CfLITAF基因的克隆与表达分析，不仅为进一步认识栉孔扇贝的免疫防御机制提供了实验参考，更重要的是为无脊椎动物中细胞因子样物质的研究提供了一个分子水平上的线索，为无脊椎动物中细胞因子样物质的研究奠定了理论基础。
其他摘要	Whether cytokines-like molecules exist in the invertebrates is controversial for a long time. By using cellular biological and immunological approaches, several cytokines-like molecules, such as TNF-α, IL-1α and IL-6, have been verified to exist in mollusk, arthropod and echinoderm. Unfortunately, there are still no direct evidences of nucleotide or protein sequences about the presence of cytokine-like molecules in invertebrates. Transferring eyes on the transcription factor that regulates cytokine expression could be a new method to research the cytokine-like molecules in invertebrate. Recently, a novel transcriptional factor LPS-induced TNF-α factor (LITAF) was cloned and characterized from human monocyte/macrophage cell line THP-1. Subsequently homologues of LITAF were cloned from mouse and chicken. Functional experiments indicates that this factor is necessary for TNF-α expression. However, there were no reports about the homologues of LITAF in invertebrates. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5’ untranslated region (UTR) of 112 bp, a 3’ UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad was measured by quantitative real-time PCR. CfLITAF mRNA transcripts could be detected in all tissues examined. In the tissues of heart, mantle, gill and gonad, there was a significant 2.45-fold, 2.82-fold, 9.23-fold and 24.93-fold relative abundance compared with mRNA abundance in haemocytes, while there was not significant difference of CfLITAF expression between muscle and haemocytes. The temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) was also measured by quantitative real-time PCR. The results indicated that CfLITAF was up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. Molecular cloning, characterization, and the temporal expression profile of CfLITAF in scallop C. farreri not only simply explicated the innate immune response of scallop to LPS stimulation, but importantly provided the clue for the existence of TNF-α or TNF-α-like molecules in invertebrates.
页数	71
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/897
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	余允东. 栉孔扇贝CfLITAF基因的克隆与表达分析[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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