【作者】 唐小千；

【导师】 战文斌；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2010， 博士

【摘要】 白斑症病毒(WSSV)病是危害我国对虾养殖业健康发展的主要疾病之一,目前仍无切实有效的防控措施。病毒细胞受体是介导病毒的侵入门户,因此WSSV细胞受体的寻找及鉴定是弄清病毒感染机理的关键点之一,也是实现白斑病防控的重要理论基础。目前研究表明WSSV的多个囊膜蛋白含有RGD (Arg-Gly-AsP)模体,且其中多个囊膜蛋白的抗体被证实具有病毒中和活性,说明其与WSSV感染密切相关,由于细胞膜受体蛋白整合素能保守地与RGD模体结合,所以其可能为WSSV潜在受体蛋白。本论文克隆了中国对虾血细胞p-integrin基因cDNA全序列,序列特性分析显示其含有整合素p亚基所有的经典结构域。RT-PCR分析该基因在不同组织内和WSSV感染后的差异表达情况,显示其与WSSV感染密切相关。在序列分析的基础上对其进行分段表达,分别构建β-integrin胞外区和VWA结构域重组表达载体,特性研究显示β-integrin重组蛋白具有的WSSV体外结合活性和体内中和活性,证实了β-integrin为WSSV一个重要的细胞膜受体蛋白。制备了抗β-integrin单克隆抗体,利用其分析β-integrin的组织分布特性,结果显示在WSSV易感组织器官内β-integrin蛋白丰度显著高于其他组织,揭示WSSV组织亲嗜性的分子基础；同时,大量制备抗中国对虾血细胞膜单克隆抗体,建立受体单抗体外筛选模型,筛选到两株具明显阻断效果的受体单抗,并对相应的受体蛋白进行鉴定和功能验证,结果显示中国对虾血蓝蛋白和血细胞beta-actin能与WSSV发生结合,这种结合可以被筛选到的单抗3C4和2F9所抑制,说明建立的WSSV受体单抗筛选体系可以应用于WSSV受体研究。具体研究内容和结果如下：(1)设计简并引物扩增中国对虾血细胞整合素β亚基编码基因的中间保守序列,通过RACE技术获得整合素β亚基cDNA序列,序列全长2853 bp,含一个2349 bp的开放阅读框,编码783氨基酸。同源比对分析显示该基因与整合素β1型亚基相似性最高。同时,利用半定量RT-PCR分析了β-integrin基因在中国对虾不同组织内及其WSSV感染后鳃组织中的转录差异情况,结果显示该基因在血细胞中转录水平最高,心脏和鳃组织次之,在肠道和性腺中也有较高的转录水平,在肌肉和肝胰腺中转录水平最低。中国对虾在感染WSSV后,鳃组织中β-integrin基因转录水平显著上调,在感染后24小时到达最高水平,然后呈下降趋势。从结果中可以发现该基因转录较高的组织为WSSV感染的主要靶器官,且在WSSV感染后出现明显上调,说明该基因与WSSV感染密切相关。(2)构建了β-integrin胞外区和VWA结构域融合表达载体pET32a-ER-βintegrin和pET32a-VWA-βintegrin,转入大肠杆菌后成功表达,分别获得分子量大小为100和50 kDa的重组蛋白。ELISA和dot blotting实验结果显示,β-integrin胞外区和VWA结构域重组表达蛋白均具有WSSV结合活性。通过螯虾体内中和实验发现,β-integrin胞外区重组表达蛋白具有显著的WSSV中和活性,以上结果说明中国对虾血细胞β-integrin为WSSV一个重要的细胞膜受体蛋白,在WSSV感染过程中发挥重要作用。(3)研制出5株特异性稳定分泌抗中国对虾血细胞β-integrin的杂交瘤细胞2C5、2C10、1F4、1C10和1D10。间接免疫荧光结果显示5株单抗均可与中国对虾血细胞膜结合,western blotting结果显示,5株单抗均可以与β-integrinVWA结构域重组蛋白发生特异性结合,同时能特异性识别1条分子量为120 kDa左右中国对虾血细胞蛋白,推测其为中国对虾血细胞β-integrin。以研制的抗中国对虾血细胞β-integrin单克隆抗体,检测β-integrin在不同组织器官内的分布特性,在心脏、鳃和肠道中可以观察到明显的绿色荧光信号,而在肌肉和肝胰腺组织中则没有观察到明显的荧光阳性信号,说明WSSV细胞膜受体蛋白β-integrin在病毒易感组织心脏、鳃和肠道中的丰度显著高于肌肉和肝胰腺组织,这一结果为揭不WSSV组织亲嗜性的分子基础提供了重要支持。(4)大量制备抗中国对虾血细胞单克隆抗体,基于ELISA和dot blotting技术,以中国对虾血细胞膜和地高辛标记的WSSV (WSSV-DIG)建立病毒受体单抗体外筛选模型,从制备的中国对虾血细胞膜单克隆抗体中筛选到具有阻断效果的单抗3C4和2F9。Western blotting结果显示,筛选到的单抗3C4与中国对虾血细胞1条分子量为42 kDa蛋白发生特异性结合,经质谱鉴定显示为beta-actin蛋白；单抗2F9与中国对虾血细胞分子量约为73和75 kDa蛋白发生特异性结合,经质谱鉴定后,显示为血蓝蛋白不同亚基。Dot blotting结果显示,分离纯化的beta-actin和血蓝蛋白亚基都具有WSSV结合活性,说明它们在WSSV感染过程中起着一定的作用。更多还原

【Abstract】 White spot syndrome virus (WSSV) is the most important viral pathogen that causes considerable economic loss to shrimp farming industry, and it has been extensively studied since its emerging. As the receptors of WSSV target cell mediate virus attachment and entry, so receptors finding and identification are the key points to figure out the WSSV infection mechanism, which is also the theoretical foundation for WSSV disease control. Till now, several WSSV envelop proteins with RGD (Arg-Gly-AsP) motif has been found, and the antibodies against some of these envelop proteins were proved to be having neutralization ability to WSSV infection. In this paper, full length cDNA sequence ofβ-integrin was cloned from the haemocytes of F. chinensis, which was expressed in E.coli by fragments after sequence analysis.β-integrin was proved to be an important membrane receptor of WSSV by binding test in vitro and neutralization test in vivo. The monoclonal antibodies (MAbs) againstβ-integrin were also produced and used to analyze the distribution ofβ-integrin in different tissues. Moreover, for further research on other WSSV receptors, a large number of MAbs against haemocyte membrane of Fenneropenaeus chinensis were produced and a model in vitro was developed to screen the MAbs agaist the receptors of WSSV. Two strains of MAb were obtained with obvious blocking effect on the binding of WSSV to haemocyte membrane, and the receptors were identified respectively. The followings are the details:(1) A full length cDNA sequence ofβ-integrin with 2853 bp was cloned from the haemocytes of F. chinensis, containing an open read frame (ORF) of 2349 bp encoding 783 amino acids. Result of homologous alignment showed cloned sequence was highly similar toβ1-integrin. Amino acid sequence of clonedβ-integrin was identified with all the classical domains ofβ-integrin by SMART. To investigate the expression levels ofβ-integrin gene in different tissues and post WSSV infection, the semi-quantitative RT-PCR was employed. The results showed that the expression level ofβ-integrin was highest in haemocytes, then was in heart, gill, gut and gonad tissues, however, which was very low in muscle and hepatopancreas tissues. After WSSV infection, the expression level in gill was up-regulated and reached to a peak at 24 h post infection, then gradually decreased. These results showed that the tissues with high expression level ofβ-integrin were susceptible to WSSV, and the level ofβ-integrin showed up-regulation after WSSV infection, which suggested thatβ-integrin was closely related to WSSV infection.(2) The cDNA fragments encoding extracellular region or VWA domain ofβ-integrin were amplified and inserted into pET32a plasmid, then transformed into E. coli BL21. The recombinant proteins were successfully expressed in positive clones with molecular masses of 100 kDa (extracellular region ofβ-integrin) and 50 kDa (VWA domain ofβ-integrin), which both showed WSSV binding ability in vitro by ELISA and dot blotting. In vivo neutralization assays of WSSV infection by these recombinant proteins were exerted, the result of which showed the recombinant proteins of extracellular region ofβ-integrin could significantly decrease the mortality of crayfish, showing partial neutralization ability to WSSV infection. The above results proved thatβ-integrin was an important membrane receptor of WSSV, which might play an important role in the process of WSSV infection.(3) Five MAbs (2C5、2C10、1F4、1C10和1D10) againstβ-integrin were produced by immunizing mice with recombinant protein of VWA domain ofβ-integrin. Indirect immunofluensce assay showed that all of these five MAbs could react with haemocyte membrane of F. chinensis. The result of western blotting showed that all of these MAbs could react with the recombinant protein of VWA domain ofβ-integrin, and specifically recognized a protein band with a molecular mass of about 120 kDa in haemocytes proteins, which suggested that 120 kDa protein was the nativeβ-integrin of haemocyte. Tissues of healthy F. chinensis were detected by IFAT using mabs againstβ-integrin. The result showed theβ-integrin in gut, gill and heart, to which WSSV was susceptible, were much more abundant than in muscle and hepatopancreas tissues, which provided important information on the mechanism of WSSV tissue tropism. (4) A large number of MAbs against haemocyte membrane were produced by immunizing BALB/c mice with haemocyte membrane of F. chinensis prepared by differential centrifugation. Based on ELISA and dot blotting techniques, a model for screening mAbs against receptors of WSSV was established with Digoxingenin labeled WSSV (WSSV-DIG) and prepared haemocyte membrane. Two MAbs,3C4 and 2F9, were screened using this model, which showed obvious blocking effects on the binding of WSSV to the haemocyte membrane. The result of western blotting showed that mAb 3C4 could react with a protein band with a molecular mass of 42 kDa, which was identified as a beta-actin by MALDI-TOF-MS peptide mapping, and mAb 2F9 could react with two protein bands with molecular masses of 73 and 75 kDa, which were identified as the subunits of hemocyanin by MALDI-TOF-MS peptide mapping. The results of dot blotting showed that the purified beta-actin and hemocyanin subunits had WSSV binding ability, which suggested that these proteins played an important role in the process of WSSV infection.更多还原