不同宿主及不同组织细胞与白斑综合症病毒（WSSV）的结合研究

【作者】 冯书营；

【导师】 黄倢； 张仕璀；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2004， 硕士

【摘要】 本文利用病毒与其宿主细胞上受体蛋白的特异性结合原理，建立了用于研究WSSV与宿主细胞结合的荧光素标记法，该方法具有简单、快捷、经济性等优点。在该方法的基础上初步查明了在日本囊对虾、凡纳滨对虾和克氏原螯虾等宿主的不同组织细胞与WSSV的结合特异性。日本囊对虾的鳃细胞和血细胞能够与WSSV发生特异性的结合，肝胰腺细胞和肌细胞与WSSV没有该结合关系，该特异性的结合关系反映出组织细胞上存在有病毒特异性的受体蛋白。凡纳滨对虾和克氏原螯虾二者宿主不同组织细胞与WSSV的结合结果与日本囊对虾宿主不同组织细胞的结合结果存在着相似性和一致性，凡纳滨对虾和克氏原螯虾的鳃细胞、血细胞和甲壳下表皮组织与WSSV产生了特异性的结合，肌细胞和肝胰腺细胞没有此结合关系，不仅查明了WSSV在二者宿主上不同组织细胞结合的特异性，而且初步弄清了WSSV在不同宿主相同组织细胞上的结合共性，可以得出病毒侵染组织细胞的共同性，加速病毒受体的确定和分离工作。 为了查明WSSV在不同宿主不同组织细胞上的结合活性高低，又分别提取了各宿主的组织细胞膜与WSSV进行了结合试验，其结果显示：凡纳滨对虾的鳃细胞膜、血细胞膜和甲壳下表皮组织细胞膜与WSSV产生了特异性的结合，并以鳃细胞膜的结合活性最高，甲壳下表皮组织细胞膜次之，血细胞膜的结合活性低于二者；肌细胞膜和肝胰腺细胞膜不与WSSV发生结合；不同组织细胞膜与WSSV的结合结果和在细胞上的结合结果完全一致，进一步确定了WSSV在凡纳滨对虾宿主不同组织细胞上的结合特异性。同时比较凡纳滨对虾不同组织细胞膜之间的相互作用，证明了血细胞膜和甲壳下表皮组织膜对鳃细胞膜与WSSV的结合具有封闭抑制作用，初步查明WSSV在凡纳滨对虾宿主不同组织细胞上的受体蛋白是相同的。在克氏原螯虾宿主中，存在类似的结合结果，鳃细胞膜和血细胞膜与WSSV具有特异性的结合，肌细胞膜和肝胰腺细胞膜不与WSSV发生结合，同时比较了凡纳滨对虾鳃细胞膜对克氏原螯虾鳃膜、血细胞膜与WSSV结合活性的影响作用，结果显示了前者对后者与WSSV之间的结合具有封闭抑制作用，初步证明了在二者宿主上WSSV的受体蛋白相同。凡纳滨对虾和克氏原螯虾不同的组织细胞膜经过SDS-PAGE分析，初步确定了各细胞膜的蛋白条带组成。 日本蟳宿主的不同组织细胞膜与WSSV结合试验中，鳃细胞膜和血细胞膜能与WSSV产生结合，并以鳃膜的结合活性稍高一点；肝胰腺细胞膜与肌细胞膜不与WSSV 不同宿主及不同组织细胞与白斑综合症病毒(WSSv)的结合研究产生结合。同时比较了日本崛鳃细胞膜和血细胞膜对凡纳滨对虾鳃膜与WSsV结合的影响作用，证明了前者组织细胞膜对后者鳃细胞膜与WSSV结合基本上没有影响，初步表明WSSV在日本崛和凡纳滨对虾宿主上的结合位点或受体蛋白不同，从侧面说明WSSV的受体蛋白并非单一性的，有可能是多个受体蛋白位点。经过SDS一PAGE对不同细胞膜进行分析，初步确定了各细胞膜的蛋白条带组成。 查明了挠足类细胞膜、卤虫细胞膜和藤壶细胞膜与WSSV之间存在特异性的结合关系，该结合关系表明了在三者宿主细胞膜上存在有WSSV的受体蛋白，为支持三者宿主属于WSSV的侵染宿主范围提供了重要依据，为查明三者宿主在白斑病的暴发和流行过程中所起的作用有着积极的意义。三者宿主细胞膜经过SDS一PAGE分析，确定了各细胞膜的蛋白条带组成。同时比较了凡纳滨对虾鳃膜对挠足类细胞膜、藤壶细胞膜、卤虫细胞膜与WSSV结合的影响作用，前者对后者的结合有明显的封闭作用，初步说明了WSSV在挠足类、卤虫、藤壶和凡纳滨对虾宿主细胞膜上的受体蛋白相同。表面活性剂Tween 20对挠足类细胞膜与WSSV的结合具有明显的增强作用，而TritonX一100对二者的结合没有明显的影响，初步表明了WSSV的受体蛋白在细胞膜上占有较小的一段跨膜区域。 本文着重查明了以上七种宿主及其不同组织细胞与WSSV结合关系，并进一步比较了提取各种组织细胞膜与WSSV的结合活性，其结果显示了日本蝎、卤虫和藤壶等宿主的结合活性较高，凡纳滨对虾、克氏原鳌虾和挠足类等宿主的结合活性低于前三者，不仅说明了卤虫和藤壶属于WSSV的侵染宿主，而且证明了二者的结合活性明显高于其它宿主。在日本崛、卤虫和藤壶三者宿主中以日本崛结合活性最高，卤虫细胞膜和藤壶细胞膜结合活性次之。在凡纳滨对虾、克氏原鳌虾和挠足类三者宿主中以凡纳滨对虾宿主的结合活性最高，克氏原鳌虾和挠足类二者宿主的结合活性相似，低于凡纳滨对虾宿主。初步查明了WSSV在不同宿主以及不同组织细胞上受体的有无以及不同细胞膜的结合活性高低，加速该病毒受体的确定和进一步分离工作，从而反映出WSSV在白斑病的暴发和流行过程中感染宿主的先后次序以及不同宿主对该病毒病所起的作用，有可能WSsV首先侵染蟹类、藤壶和卤虫等环境宿主，由环境宿主携带和传播WSSV，造成大面积的白斑病的暴发，为控制和监测白斑病的暴发提供了警示线索。最终找到阻断病毒入侵的方法或有效途径，控制对虾白斑病暴发和流行。更多还原

【Abstract】 This paper established a FLUOS labeled method that can be used in the binding assay between WSSV and host cells with the advantages of more convenience, quickness and less expense on the principle of specific binding between virus and receptor on the cells of host. To some extent, it proved the specific binding of WSSV and Marsupenaeus japonicus, Procamburas clarkii and Litopenaeus vannamei etc hosts by right of this method. Gill cells and hemocytes of M. japonicus have a specific binding to WSSV, but cells of muscle and hepatopancreate have not this binding. This binding reflected that receptor of WSSV on the cells of host. The binding results of different tissue cells of hosts P. clarkii and L. vannamei comparable and consistent with the binding results of M. japonicus host. Their gill cells, hemocytes and cephalothorax cuticle can binding to WSSV, but cells of muscle and hepatopancreate can not. It not only proved that the binding result of different cells of these hosts to WSSV, but also testified the binding intercommunity of WSSV and different hosts on which can find out the binding intercommunity of tissue cells to WSSV, accelerated the work for confirm and separate the receptor of WSSV.This paper extracted each host cell membrane binding to the WSSV for testified the binding activity between WSSV and different hosts and different tissue cell. The binding show that gill cell membrane, hemocyte cell membrane and cephalothorax cuticle of L. vannamei can specific binding to WSSV and cell membrane of muscle and hepatopancreate can not. In which gill cell membrane have a highest binding activity, cephalothorax cuticle membrane have a higher binding activity to WSSV and the binding activity of hemocyte cell membrane is lower. This binding result consistent with the binding result of different cells to WSSV, much more confirmed that the binding between WSSV and different tissue cells of L. vannamei. It testified that hemocyte cell membrane and cephalothorax cuticle have a restrained rolling to the binding of WSSV and gill cell membrane of L. vannamei through comparing the mutual influence of each membrane, proved that different tissue cells of L. vannamei have a same receptor to WSSV to a certain extent. P. clarkii host has a similar result that gill cell membrane and hemocyte cell membrane can binding to the WSSV, but cell membrane of muscle and hepatopancreate can not. At the same time, it analysed the influence of gill cell membrane of L. vannamei to the binding between gill cell membrane and hemocyte cell membrane of P. clarkii and WSSV, result show that the former has a inhibitionto later binding to WSSV and proved the host P. clarkii and L. vannamei have a same receptor of WSSV. To some extent, it confirmed their different tissue membrane protein consists through the SDS-PAGE.In the binding assay of different tissue membrane of Charybdis japonica to WSSV, gill cell membrane and hemocyte membrane have a specific binding to WSSV and in which the binding activity of gill cell membrane higher than hemocyte membrane, cell membrane of muscle and hepatopancreate have not this binding. Through comparing the influence of gill cell membrane and hemocyte membrane of C. japonica to binding between WSSV and gill cell membrane of L. vannamei, the result proved that the former hardly any effect to binding of later and WSSV and elementary indicated that host C. japonica and L. vannamei have a same receptor of WSSV. It just as say receptor of WSSV are not singleness, but are multi-binding location. It elementary confirmed the consist of each tissue membrane of this host by SDS-PAGE.It find out the specific binding between WSSV and cell membrane of copepod, barnacle and artemia, this binding indicated the cell membrane of three hosts have a receptor of WSSV. It as a proof for the point that copepod, barnacle and artemia belong to host range of WSSV infection and which have a positive role in illuminating the function of three host in the prevalence and break out of this disease. It confirmed the protein consists of更多还原