【作者】 王帅；

【导师】 王金星；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 对虾的养殖是我国沿海地区重要的水产支柱产业。但是白斑病毒(white spot syndrome virus, WSSV)等病毒使对虾养殖业遭受了极大的损失。因此,研究对虾的抗病毒先天免疫防御机制,对于预防和控制对虾疾病具有重要意义。1.对虾TRBP和eIF6之间存在直接的相互作用,并在对虾抗病毒RNAi途径中起作用HIV转录激活反应RNA结合蛋白(Trans-activation response RNA-binding protein, TRBP)是一种多功能蛋白,在多种生命进程中发挥重要作用。我们从中国明对虾(Fenneropenaeus chinensis)中克隆得到三个TRBP家族成员,分别命名为Fc-TRBP1-3.我们在大肠杆菌Escherichia coli中重组表达并纯化了Fc-TRBP1,随后使用纯化的Fc-TRBP1进行对虾血细胞T7噬菌体展示文库淘选,并得到中国明对虾真核翻译起始因子(Fc-eukaryotic initiation factor 6, Fc-eIF6)的部分序列。我们克隆得到Fc-eIF6的全长cDNA序列,并在大肠杆菌中进行重组表达。利用pull down实验验证了TRBP和eIF6之间存在直接的相互作用。RT-PCR和Northern blot结果表明Fc-TRBP和Fc-eIF6在所有检测的组织中都有表达,对虾受鳗弧菌刺激后在所有组织中表达有上调趋势,在对虾受到病毒刺激时TRBP和eIF6的表达模式在不同组织中有较大差异,在血细胞中呈现下调趋势而在鳃和肠中上调。Western blot结果验证了TRBP在肝胰腺和鳃中的表达模式。注射重组TRBP蛋白可抑制WSSV在对虾体内的复制。通过这些结果可以推测TRBP和eIF6可能在抗病毒免疫中起重要作用。随后我们在日本囊对虾(Marsupenaeus japonicus)中克隆得到TRBP和eIF6的全长序列。我们对日本囊对虾TRBP(Mj-TRBP)的不同结构域进行重组表达。pulldown实验证明TRBP结构域B,C介导与eIF6的结合。Gel-shift结果表明TRBP结构域A对dsRNA具有很高的亲和力,而结构域B亲和力很弱,结构域C不结合dsRNA但可通过介导TRBP全长分子的二聚化提高TRBP分子的亲和力。RNAi结果表明TRBP和eIF6参与dsRNA介导的RNA沉默,沉默TRBP和eIF6均可促进WSSV的增殖。以上结果表明,TRBP和eIF6可能通过抗病毒RNA沉默参与对虾抗病毒免疫。2.组织蛋白酶C参与对虾的抗病毒先天免疫组织蛋白酶C, (Cathepsin C, Cath C)是一种溶酶体半胱氨酸蛋白酶,属于木瓜蛋白酶家族。Cath C能够激活许多糜蛋白酶样的丝氨酸蛋白酶(chymotrypsin-like serine proteases)并在免疫和炎症细胞中参与调控许多丝氨酸蛋白酶的活性。在本研究中,我们克隆了中国明对虾Cath C的cDNA全长序列,命名为Fc-Cath C。Fc-Cath C的cDNA长1445bp,包含1356的bp开放读码框(open reading frame, ORF)编码了451氨基酸长度的蛋白包括17个氨基酸的信号肽。Real time PCR结果表明Fc-Cath C mRNA在肝胰腺中转录,并在受鳗弧菌Vibrioanguillarum和白斑病毒(white spot syndrome virus, WSSV)刺激后上调。通过向对虾体内注射Cath C的抗体后,可促进WSSV的增殖以及提高对虾受病毒感染后的死亡率。我们的结果表明Fc-Cath C可能在对虾的抗病毒先天免疫中起重要的作用。3.翻译控制肿瘤蛋白的基因克隆及功能研究翻译控制肿瘤蛋白(translationally controlled tumor protein, TCTP)是一种多功能的蛋白,在许多生命过程中发挥重要的功能。我们从中国明对虾中克隆得到TCTP的cDNA全长序列命名为Fc-TCTP。Fc-TCTP cDNA长711bp,包含507 bp的开放读码框(open reading frame, ORF)编码了168氨基酸,包含一个TCTP结构域的蛋白。我们Fc-TCTP受到鳗弧菌刺激和WSSV刺激后的表达模式进行分析发现,在受到WSSV刺激后TCTP的mRNA和蛋白水平上的表达量显著上调。结果表明Fc-TCTP可能在对虾的抗病毒先天免疫中起重要的作用。更多还原

【Abstract】 The Chinese white shrimp (Fenneropenaeus chinensis) cultivation is an important economic activity in China. However, the aquaculture of shrimp has suffered vast economic losses because of serious viral diseases such as white spot syndrome virus (WSSV). So investigating the mechanism of antiviral immunity will help us to control those viral diseases.1. Fc-TRBP interacts with Fc-eIF6 directly, and both are involved in the antiviral RNA interferenceThe Trans-activation response RNA-binding protein (TRBP) plays an important role in many biological processes. In these studies, three new cDNAs of the TRBP family were cloned from Fenneropenaeus chinensis, and designated Fc-TRBP 1-3. Recombinant expressed Fc-TRBPl in Escherichia coli was used for panning of a T7 phage display library constructed using Chinese shrimp hepatopancreas. From this panning, eukaryotic initiation factor 6 (eIF6) was isolated and sequenced. After cloning and recombinantly expressing Fenneropenaeus chinensis-derived Fc-eIF6, the interaction between Fc-TRBP and Fc-eIF6 was confirmed using pull-down assays and far western analysis. Expression of Fc-TRBPs was detected in many tissues, with its expression level increasing in heart, gill and intestine in the early stages of infection by the white spot syndrome virus (WSSV), and increasing in most tissues following a challenge with Vibrio anguillarum. Western blot studies confirmed the increased expression of Fc-TRBP in gill after WSSV infection. The expression pattern of eIF6 was also analyzed. Infection analysis show the replication of WSSV was reduced after injection with Fc-TRBP.Then we show that the double strand RNA bind domain (dsRBD) B and C of TRBP homologue from Marsupenaeus japonicus (Mj-TRBP) mediated the interaction of TRBP and eIF6. Gel-shift assays reveal the N-terminal dsRBD of Mj-TRBP binds to double strand RNA (dsRNA) with a high affinity, while TRBP might be homo-dimerized by the C-terminal region and increase the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA induced sequence-specific RNAi pathway and facilitates the proliferation of WSSV. These results indicate that TRBP and eIF6 may be components of the RNA-induced silencing complex (RISC), and thereby play a crucial role in the anti-viral defense response of shrimp.2. Cathepsin C is involved in the innate antiviral immunity.Cathepsin C (Cath C) is a lysosomal cysteine protease that belongs to the papain superfamily. Cath C is capable of activating many chymotrypsin-like serine proteases and is reported to be a central coordinator for the activation of many serine proteinases in immune and inflammatory cells. In this study, Cath C cDNA was cloned from Fenneropenaeus chinensis (Fc). The complete cDNA of Fc-Cath C in Chinese white shrimp was found to be 1445-base pairs (bp) long. It contained an open reading frame (ORF) 1356-bp long and encoded a 451-amino acid residue protein, including a 17-amino acid residue signal peptide. Real-time PCR analysis results indicated that Fc-Cath C mRNA was transcribed in the hepatopancreas and upregulated after stimulation by the Vibrio anguillarum and the white spot syndrome viruses (WSSVs). Replication of the WSSV increased after the injection of Fc-Cath C antiserum. These results imply that Fc-Cath C might play an important role in the antiviral immune response of shrimp.3. Molecular cloning and function analysis of TCTPThe translationally controlled tumor protein (TCTP) is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. In this study, a new TCTP cDNA was cloned from Fenneropenaeus chinensis and hence was designated as Fc-TCTP. Its length is 711 bp, and it is characterized by 507-bp open reading frame (ORF) that encodes a deduced 168-amino acid protein, including a TCTP domain. Moreover, this study analyzed the expression patterns of this gene when it responds to infection specifically with Vibrio anguillarum and the white spot syndrome virus (WSSV). Based on the results, Fc-TCTP was present in all the analyzed tissues. Additionally, Fc-TCTP’s expression level decreased after having been infected by bacteria, but was upregulated in the hepatopancreas after having been exposed to WSSV. Likewise, the Fc-TCTP protein was upregulated during its exposure to the virus. These results suggest that Fc-TCTP could well be involved in the antiviral response in F. chinensis.更多还原