【作者】 韦秀梅；

【导师】 战文斌；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2010， 博士

【摘要】 对虾白斑症病毒(WSSV)病是危害养殖对虾最严重的病害之一,自该病1993年暴发以来,在对其病原、病理、检测及诊断技术等方面的研究上取得了诸多成果。目前,研究WSSV感染的分子机理,寻求防控WSSV感染的方法已成为热点。已有研究表明WSSV-VP28在WSSV的感染入侵过程中起着非常重要的作用。论文纯化了前期制备的具有中和WSSV特性的抗VP28单克隆抗体(单抗)1D6(Abl),并以之为抗原免疫小鼠,研制抗VP28独特型单抗(Ab2)杂交瘤细胞,利用间接酶联免疫吸附实验(ELISA)和竞争ELISA,从中筛选到一株单抗A1A5,该单抗能与WSSV竞争结合抗VP28抗体；以抗-抗独特型抗体(Ab3)进一步分析单抗A1A5的特性,证实了单抗A1A5是能模拟VP28的抗VP28独特型单抗；利用研制的抗VP28独特型单抗研究了VP28与宿主细胞间的相互作用,检测了VP28与中国对虾组织的结合,鉴定了VP28在中国对虾血细胞上的结合蛋白,为进一步确认VP28在WSSV感染中所起的作用,深入研究WSSV感染机理,切断WSSV感染途径提供新的依据。具体研究内容和结果如下：(1)抗VP28独特型单抗的研制。以前期已制备的具有中和WSSV特性的抗VP28单抗1D6杂交瘤细胞生产腹水,腹水依次经辛酸-硫酸铵法和Protein G亲和层析纯化,获得了纯度高、有活性的抗VP28中和单抗并用以免疫BALB/c小鼠,利用细胞融合技术,研制抗VP28独特型单抗杂交瘤细胞,采用间接ELISA和竞争ELISA方法从杂交瘤细胞中筛选抗VP28独特型单抗,克隆得到1株抗VP28独特型单抗A1A5能识别抗VP28抗体,并能与WSSV竞争结合抗VP28抗体,初步证实单抗A1A5能模拟VP28的抗原构象。(2)以抗-抗独特型抗体分析抗VP28独特型单抗的特性。为了进一步确认单抗A1A5的特性,以A1A5杂交瘤细胞生产腹水,腹水经辛酸-硫酸铵法和ProteinG亲和层析纯化,获得纯化抗体用以免疫BALB/c小鼠制备抗血清,得到Ab3；采用间接免疫荧光分析(IIFA)、金标免疫电镜法(IEM)、竞争ELISA和螯虾体内中和实验多种方法分析了Ab3特性。IIFA证实Ab3可与感染WSSV的虾鳃反应；IEM结果显示Ab3可与WSSV结合,其结合位点位于WSSV囊膜上；竞争ELISA表明Ab3能与抗VP28抗体竞争结合WSSV上结合位点；以螯虾体内中和实验分析Ab3中和性,注射与Ab3孵育后WSSV的螯虾与仅注射WSSV的螯虾相比,其死亡时间明显延迟,证实Ab3能中和WSSV感染。以上结果说明了由Ab2免疫产生的抗体Ab3具有与类似Ab1特性。因此,根据抗独特型抗体理论推断,单抗A1A5是能模拟VP28的抗VP28独特型单抗。(3)抗VP28独特型单抗的应用。论文在成功研制抗VP28独特型单抗的基础上,利用抗VP28独特型单抗探讨了wSSv囊膜蛋白VP28与宿主细胞之间的相互作用关系。中国对虾(Fenneropenaeus chinensis)血细胞与单抗A1A5孵育,再以荧光二抗染色,在中国对虾血细胞膜上可见黄绿色荧光,结果表明单抗A1A5能与中国对虾血细胞膜结合,中国对虾血细胞膜上存在VP28的受体蛋白。以辣根过氧化物酶(HRP)标记的抗WSSV混合单抗,建立了基于斑点免疫印迹(Dot-blot)和ELISA的WSSV与血细胞膜体外结合实验体系,提纯的血细胞膜固定于NC膜或酶标板上,将单抗A1A5或抗中国对虾血细胞颗粒单抗1G8与中国对虾血细胞膜作用后,再与WSSV孵育,结果显示A1A5组的WSSV结合量明显低于1G8组,单抗A1A5与中国对虾血细胞膜的结合能部分阻断WSSV与中国对虾血细胞膜的结合,这为证明VP28是WSSV感染相关的重要蛋白提供了新的依据。以IIFA和免疫组织化学方法(IHC)检测单抗A1A5与中国对虾各组织的结合,发现单抗A1A5可以与中国对虾鳃和肠组织结合,而不与肌肉、性腺、心脏、肝胰腺等组织结合,推断中国对虾鳃和肠组织中存在VP28的受体蛋白。以免疫共沉淀鉴定VP28在中国对虾血细胞上的结合蛋白,单抗A1A5可与中国对虾血细胞膜上分子量为47 kDa的蛋白结合,该蛋白可能是VP28在中国对虾血细胞膜上的受体蛋白。本论文为WSSV感染相关蛋白、分子机理以及预防控制研究提供了新的研究工具和研究方法。更多还原

【Abstract】 White spot disease (WSD) which is caused by white spot syndrome virus (WSSV) is one of the most important lethal diseases that affect current shrimp aquaculture. During the past more than ten years since WSD outbreak in 1993, many papers on researches of pathogen, pathology, detecting and diagnostic methods of the disease have been published. Recent works focus mainly on figuring out the infection mechanism and searching the strategies of prophylaxis and control of WSSV infection. WSSV-VP28 has been proved to play a very important role in WSSV infection process.In this thesis, anti-VP28 monoclonal antibody (Mab) 1D6 (Ab1), which was proved to specifically recognize VP28 and be able to neutralize WSSV infection in vivo and vitro previously, was purified and used as antigen immunized to BALB/c mice, in order to generate monoclonal anti-idiotypic antibodies (Ab2). After cell fusion, monoclonal antibody A1A5 was obtained after screening by indirect enzyme-linked immunosorbent assay (ELISA) and competitive ELISA. Mab A1A5 was confirmed to be able to inhibit the binding between rabbit anti-VP28 antibodies and WSSV significantly. Anti-A1A5 antibodis (Ab3) were generated and used to further analyze the characterization of Mab A1A5, and Mab A1A5 was proved to represent the internal image of VP28 exactly. Mab A1A5 was used to study the interaction between VP28 and host cells, detect the combination of VP28 with Chinese shrimp (Fenneropenaeus chinensis) tissues, identify the VP28 binding protein on haemocyte membrane of Chinese shrimp. This study shed new light on investigating the role of VP28 in WSSV infection process, provided new information for in-depth study of WSSV infection mechanism and searching the strategies of prophylaxis and control of WSSV infection. The details are as follows:(1) Production of monoclonal anti-idiotypic antibody of VP28. Anti-VP28 Mab 1D6, which was proved to specifically recognize VP28 and be able to neutralize WSSV infection in vivo and vitro previously, was produced in ascites, purified by caprylic acid-ammonium sulfate (CA-AS) and Protein G agrose. Purified Ab1 was immunized to BALB/c mice as antigen, in order to generate monoclonal anti-idiotypic antibodies. After cell fusion, Mab A1A5 which was confirmed to be able to inhibit the binding between rabbit anti-VP28 antibody and WSSV significantly was identified by indirect ELISA and competitive ELISA.(2) Using anti-AlA5 antibodies to further analyze the characterization of Mab A1A5. Mab A1A5 was purified and immunized to BALB/c mice as antigen to generate anti-A1A5 antibody (Ab3), in order to verify whether Mab A1A5 could represent the internal image of VP28 exactly. Competitive ELISA, indirect immunofluorescence assay (IIFA), immunogold electron microscopy (IEM) and in vivo neutralizing assay were developed to analyze Ab3. Competitive ELISA showed that Ab3 could dose-dependently prevent the binding between WSSV and rabbit anti-VP28 antibody. Furthermore, IIFA, IEM and in vivo neutralizing assay showed that both Abl and Ab3 which was induced by Mab A1A5 could combine with WSSV envelope and reduce WSSV infection, Ab3 recognized the same antigen epitope with Ab1. According to the theory of anti-idiotypic antibodies, all the results indicated that the monoclonal anti-idiotypic antibody of VP28 which represented an internal image of VP28 was successfully developed.(3) Application of monoclonal anti-idiotypic antibody of VP28. The monoclonal anti-idiotypic antibody of VP28 was used to study the interaction between VP28 and host cells. IIFA confirmed that Mab A1A5 could combine with the haemocyte membrane of Chinese shrimp, it indicated that there was VP28 receptor protein on the haemocyte membrane of Chinese shrimp. Furthermore, anti-WSSV Mabs were labeled with horse radish peroxidase (HRP) and used in vitro WSSV blocking experiments of Dot-blot and ELISA. Haemocyte membrane of Chinese shrimp was dotted on NC membrane or coated on ELISA plate, after incubated with Mab A1A5 or Mab 1G8 (anti-haemocyte granules Mab), then incubated with WSSV. The binding of WSSV was detected with anti-WSSV-HRP. The blocking experiments showed that the Mab A1A5 could block the binding of WSSV with haemocyte membrane. It provided a new basis to prove that VP28 played a key role in WSSV infection process. IIFA and immunohischemical (IHC) were developed to indentify whether Mab A1A5 could combine with gill, intestine, hepatopancreas, muscle, heart and gonad of Chinese shrimp, both of the results showed that Mab A1A5 could react with gill and intestine, but did not react with hepatopancreas, muscle, heart and gonad. It demonstrated that there was VP28 receptor protein in gill and intestine of Chinese shrimp. Coimmunoprecipitation was developed to identify the VP28 binding protein on haemocyte membrane of Chinese shrimp. A 47 kDa protein which was specially recognized by Mab A1A5 was identified by coimmunoprecipitation, and it was considered to be VP28 receptor protein on Chinese shrimp haemocyte membrane.更多还原