【作者】 刁菁；

【导师】 战文斌；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2008， 博士

【摘要】 鱼类淋巴囊肿病(Lymphocystis disease,LCD)是一种世界范围内流行的病毒病,宿主遍及42科125种以上的海水、淡水和半咸水鱼类。研究黏附蛋白与细胞受体以及两者之间的相互作用是探讨淋巴囊肿病毒(Lymphocystis disease virus,LCDV)感染机制和综合防治淋巴囊肿病的一个有效途径。本文利用牙鲆LCDV敏感细胞系—牙鲆鳃细胞系(Flounder gill cell line,FG),建立了牙鲆LCDV中和抗体的筛选体系,从制备的抗牙鲆LCDV单克隆抗体中,筛选出2株牙鲆LCDV中和单抗,并对病毒的黏附蛋白进行初步确认;利用病毒铺覆蛋白印迹技术(VOPBA)鉴定出牙鲆LCDV靶器官及FG细胞的LCDV受体蛋白,研究分析了FG细胞的LCDV受体蛋白特性及其在病毒感染中的作用。具体结果如下: FG细胞的培养是本文研究的基础,在本文所选择的培养条件下(含10%胎牛血清的Eagle’s MEM培养基培养,pH7.4,温度20℃),FG细胞生长迅速,状态良好,细胞生长曲线显示,细胞3-4d即可达到增殖顶峰,7d之内均保持良好状态。将粗提的牙鲆LCDV接种于FG细胞,1-2d后细胞即出现明显的病变,半数细胞培养物感染量(TCID50)为22.88/40μL。细胞病变有两种形式,一是细胞崩解、脱落形成明显的空斑,二是细胞融合形成大量的合胞体。利用制备的抗牙鲆LCDV单克隆抗体,通过免疫荧光技术和免疫细胞化学方法,均可检测到FG细胞内增殖的牙鲆LCDV,进一步证实FG细胞是牙鲆LCDV的敏感细胞。另外免疫荧光检测病毒对FG细胞的吸附发现,牙鲆LCDV可与FG细胞表面结合,说明FG细胞表面具有牙鲆LCDV的受体,为受体蛋白的研究提供理论依据。本文利用FG细胞,以患LCD的牙鲆血清和通过免疫获得的兔抗牙鲆LCDV血清为研究对象,通过微量细胞病变中和实验,建立了检测牙鲆LCDV中和抗体的方法,且对中和实验的主要参数包括病毒与血清的孵育条件、病毒与血清的浓度设定以及判定标准进行探讨。结果显示患病牙鲆血清以及兔抗血清对牙鲆LCDV感染都具有明显的中和作用,具有中和效果的血清最高稀释度分别为1:64和1:160。本文采用差速离心和密度梯度法分离提纯了牙鲆LCDV,以此作为抗原,运用单克隆抗体技术,通过免疫荧光抗体技术和斑点免疫印迹技术筛选,有限稀释法克隆出9株分泌抗牙鲆LCDV单克隆抗体的杂交瘤细胞。经微量细胞病变中和实验筛选,得到2株具有中和性的牙鲆LCDV单克隆抗体(3G3和1B2)。Western blotting结果显示,中和单抗3G3和1B2能特异性结合的病毒多肽分子量分别为38.2 kDa和32.9 kDa。本文运用免疫荧光技术,检测到牙鲆的鳃、表皮、肾、胃、肠是牙鲆LCDV的靶器官。提取了各靶器官蛋白,通过病毒铺覆蛋白印迹技术(VOPBA)进行受体蛋白的鉴定,结果如下:鳃组织蛋白中鉴定出1条分子量为37.6 kDa的受体蛋白带;表皮组织蛋白中鉴定出1条分子量为56 kDa的受体蛋白带;胃、肠和肾组织蛋白中均鉴定出1条分子量为27.8 kDa的受体蛋白带。本文利用NP-40裂解和差速离心法,提取了FG细胞的膜蛋白,利用VOPBA鉴定出FG细胞膜上的1个牙鲆LCDV受体蛋白,其分子量大小为37.6 kDa。采用电洗脱的方法提纯了该受体蛋白,双向电泳结果显示此蛋白为单一多肽,其等电点在6.0左右。将该受体蛋白经胰蛋白酶和高碘酸钠作用后,发现病毒与受体蛋白的结合变弱,推测此受体蛋白可能为糖基化的蛋白受体。用该受体蛋白免疫Balb/c小鼠,制备其多抗,免疫荧光结果显示,受体蛋白抗体能够与FG细胞表面结合。通过VOPBA阻断实验发现,稀释50倍和100倍的受体蛋白抗体能够阻断病毒与受体蛋白的结合,稀释200倍的受体蛋白抗体阻断效果不明显。病毒体外感染阻断实验发现,稀释50倍、100倍和200倍的受体蛋白抗体能够在不同程度上阻断病毒的感染。由此推测37.6 kDa受体蛋白可能是介导牙鲆LCDV感染FG细胞的一个功能蛋白。更多还原

【Abstract】 Lymphocystis disease (LCD) is a chronic worldwide fish disease, characterized by papilloma-like lesions typically on the skin, fins and tail. It has more than 100 hosts including marine and freshwater species. Virus attachment to a cellular receptor through virion attachment proteins is the first step in viral infection. Understanding of how viral proteins and host cell receptors mediate this initial interaction will contribute to understanding the virus infection mechanism and provide a new method for virus disease prevention. In this paper, flounder gill (FG) cell line was used for study of virion attachment proteins and cellular receptors of lymphocystis disease virus (LCDV) isolate from Japanese flounder. An efficient method to detect neutralizing antibodies against LCDV was established using FG cells, through which we screened and obtained two strains of neutralizing monoclonal antibodies against LCDV; Virus overlay protein binding assays (VOPBA) were carried out to find out the virus binding proteins of the LCDV target organs and FG cells. The characteristics and function of the virus binding protein of FG cells were also studied.FG cell culture was the foundation of this study. FG cells were derived from flounder gill tissue and they grew fast and well at 20℃in Eagle’s MEM supplemented with 10% of fetal calf serum. According to the growth curve, the density of cells could reach the peak 3-4 days after inoculation.Obvious cytopathic effect (CPE) could be found on FG cells 1-2 days after inoculation with LCDV purified from lymphocystis cells of Japanese flounder, and the half tissue culture infection dosage (TCID50) was 22.88 per 40μL. Two kinds of CPE were observed including plaques and syncytia. The results of detecting LCDV in FG cells using immunofluoresence assay test (IFAT) and immunocytochemistry (ICC) by monoclonal antibodies against LCDV show that virus could propagated in FG cells. The location of the virions was detected in virus-adsorbed cells by IFAT, suggesting the specific LCDV receptors on the surface of FG cells.Micro-cytopathic effect neutralization test (MCPENT) demonstrated that infectivity to FG cells was reduced by pretreatment of virus with serum obtained from LCD-diseased Japanese flounder and rabbit anti-LCDV serum, the neutralization titers were respectively 1:64 and 1:160. As a sensitive cell line for LCDV, FG cells could be used in the detection of neutralizing antibodies to LCDV.Monoclonal antibodies against LCDV were raised using virus purified by differential centrifugation and sucrose density gradient centrifugation as antigen. By IFAT and dot blotting assay, 9 positive hybridomas were found and then cloned.MCPENT demonstrated that Mab 3G3 and 1B2 were able to neutralize LCDV infection to FG cells. Western blotting analysis showed that Mab 3G3 reacted with a polypeptide with molecular weight of of 38.2 kDa, while Mab 1B2 reacted with a polypeptide with molecular weight of 32.9 kDa.Tissues of LCDV-diseased Japanense flounder were detected by IFAT using Mabs against LCDV. The result showed the gill, stomach, intestine, kidney and epidermis were the target organs of LCDV. Proteins of target organs were extracted and then subjected to VOPBA for LCDV binding protein identification. A gill tissue protein of 37.6 kDa could bind LCDV. A kidney tissue protein of 56 kDa could bind LCDV. The stomach, intestine and epidermis tissue shared the same binding protein of 27.8 kDa. FG cell membrane proteins was extracted by NP-40 lysis buffer and separatedfrom other cellular components by differential centrifugation. The cell membrane proteins were subjected to VOPBA. A protein of 37.6 kDa located on the surface of FG cells bound LCDV. This virus binding protein was purified by electroelution and then analysed by 2D gel electrophoresis. The profile of 2D gel electrophoresis showed it was a single polypeptide with pI of 6.0. After treated by Trypsin and sodium periodate, the ability of virus binding with 37.6 kDa protein was weakened, which suggested this protein was a glycosylated binding protein. Balb\c mice were immunized by 37.6 kDa protein to product polyclonal antibodies. IFAT found the polyclonal antibodies could react with the surface of the FG cells. Virus binding and infection inhibition test demonstrated that the polyclonal antibodies against the 37.6 kDa binding protein could block the virus binding and infection to FG cells, so that the 37.6 kDa binding protein might be a putative receptor of LCDV to FG cells.更多还原