Molecular characteristics of three thymosin-repeat proteins from Marsupenaeus japonicus and their responses to WSSV infection
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摘要: 胸腺肽是一类高度保守的多肽,在伤口愈合、血管生成、抗微生物过程和抗病毒免疫等方面起着重要作用。本文利用抑制性差减文库的表达序列标签克隆了日本囊对虾三个新的β胸腺肽多结构域蛋白,根据所包含结构域的数目分别命名为mjthm4、mjthm3和mjthm2。mjthm4、mjthm3和mjthm2的开放阅读框分别编码166、128和90个氨基酸多肽,它们的氨基酸序列分别含有4个、3个和2个β胸腺肽肌动蛋白结合结构域。氨基酸序列比对分析发现,mjthm4、mjthm3和mjthm2与已知的无脊椎动物β胸腺肽多结构域蛋白存在较高的同源性。在被检测的组织中,每个组织都能检测到三个蛋白的转录表达,其中肠道中的转录表达水平最高。实时定量PCR检测了白斑综合症病毒感染日本囊对虾后肝胰脏中三个蛋白的转录表达水平,结果显示mjthm4、mjthm3和mjthm2在注射病毒6小时后转录表达水平显著上调,但mjthm4在抗白斑综合症病毒的日本囊对虾肝胰脏中的转录表达水平没有变化,而mjthm3和mjthm2的转录水平却明显下调。研究结果表明克隆的日本囊对虾mjthm4、mjthm3和mjthm2是新的β胸腺肽多结构域蛋白成员,它们与白斑综合症病毒感染有着密切关系,该研究有助于更好地理解虾对病毒感染的先天性防御和/或与病毒入侵的关系。Abstract: β-thymosins, a family of highly conserved peptides, play a vital role in wound-healing, angiogenesis, antimicrobial process and antiviral immunity. Three novel β-thymosin-repeat proteins, named mjthm4, mjthm3 and mjthm2, were cloned from Marsupenaeus japonicus using expressed sequence tags(EST) from suppression subtractive hybridization. The mjthm4, mjthm3 and mjthm2 cDNAs possessed open reading frames that encoded 166, 128 and 90 amino acid residue polypeptides and contained four, three and two β-thymosin actin binding modules, respectively. Blast analysis demonstrated that mjthm4, mjthm3 and mjthm2 shared high homology with known invertebrate multi-repeat β-thymosins. These proteins are ubiquitously expressed in all of the examined tissues, and the transcriptional levels were highest in the intestine. Further investigation revealed that mjthm4, mjthm3 and mjthm2 were remarkably up-regulated 6 h after WSSV infection. Moreover, while mjthm4 transcriptional levels displayed no changes, mjthm3 and mjthm2 levels decreased in the virus-resistant shrimps. The results indicate that mjthm4, mjthm3 and mjthm2 are novel multi-repeat β-thymosin homologues, have a close relationship with WSSV infection, and might contribute to a better understanding of host defense and/or virus invasion interactions in shrimps.
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