一种抗对虾白斑综合症病毒(WSSV)线性抗原表位单链抗体的筛选

对虾白斑综合症病毒(WSSV)是全世界对虾养殖业最主要的病原体之一, 虽然对该病毒的研究已较为深入, 但目前仍无有效的防治方法。本研究应用噬菌体展示技术, 构建了抗变性WSSV的单链抗体噬菌体展示文库, 分别以WSSV病毒粒子和原核表达的囊膜蛋白VP28为靶分子对该文库进行淘选。经过数轮淘选后, 得到5个能特异识别WSSV的单链抗体, 且首次获得了能特异识别WSSV线性抗原表位的单链抗体P75E8。并通过免疫胶体金电镜分析, 对5种单链抗体对应在病毒粒子上的表位进行了定位。为获取识别多种WSSV抗原的抗体提供了新的方法路线, 也为获取特异性识别线性表位的单链抗体提供了一种新的淘选技巧。     

抗对虾白斑综合症病毒的单链抗体A1的表达和生物化学特性

用噬菌体展示技术制备了抗对虾白斑综合症病毒(WSSV)的单链抗体A1。该抗体在30℃培养条件下诱导表达20h后,其蛋白表达量可达总菌体蛋白的3.67%。用亲和层析柱和SephadexG-100层析柱可将单链抗体A1纯化为一条单电泳条带,其分子量约为31.5kD。用等电聚焦电泳测定,其等电点为pH5.8。ELISA测定表明冻干的单链抗体A1在室温储藏4年后与WSSV结合仍具有较高的活力。       

一种改良的对虾白斑综合征病毒的提纯技术

对虾白斑综合征病毒(White spot syndrome virus,WSSV)是对虾养殖业的主要病原,自1992年以来一直严重影响对虾的产量和质量,造成巨大的经济损失.     

注意杆状病毒科分类的变化

杆状病毒由于被广泛用作目的基因的表达载体倍受重视.近年来证实某些杆状病毒对虾具有致病性,从而成为研究热点之一.目前公认的最重要的对虾致病病毒是对虾白斑综合征病毒(white spot syndromviruS,WSSV),在我国养殖对虾中广为流行,引致重大经济损失.不少文献将WSSV称作对虾白斑病杆状病毒,其实并不准确.     

抗对虾白斑综合症病毒单链抗体A1基因在酵母中表达及其与抗原结合活性

通过PCR从噬菌粒载体上扩增一种抗对虾白斑综合症病毒 (WSSV)的单链抗体A1(ScFvA1)基因 ,并构建于大肠杆菌 酵母穿梭质粒载体pPIC9K上。经PCR ,酶切 ,测序鉴定重组克隆 ,发现重组成功。将重组质粒pPIC9K ScF vA1转化毕赤酵母 (Pichiapastoris)GS115中 ,利用甲醇诱导 ,将单链抗体A1在酵母中进行了初步表达。经SDS PAGE电泳 ,发现其大小约为 32KD ,通过ELISA实验 ,证明表达上清液中的单链抗体具有很高的WSSV结合活性。     

对虾白斑综合征杆状病毒同源性比较的研究

比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株即唐海分离株（渤海湾）,宁波分离株（东海）,深圳分离株（南海）的同源性。三个WSSV分离株基因组的限制性内切酶(Sac I,Hind III,Pst I)酶切多态（RFLP）以及病毒结构蛋白图谱完全一致,证实造成我国从南至北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒（RV-PJ＝PRDV）,斑节对虾白斑综合征杆状病毒（WSBV＝PmNOBIII）基因组核酸片段特异性引物进行PCR扩增,结果均能从中国对虾白斑杆状病毒（WSSV）基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒（WSSV）与斑节对虾白斑综合征杆状病毒（WSBV＝PmNOBIII）,日本对虾杆状病毒（RV－PJ＝PRDV）同源率分别为100％与97％,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了证据。     

噬菌体展示随机十肽库的构建及抗WSSV单链抗体A1的模拟抗原表位的淘选和鉴定

本实验以pCANTAB5E噬菌粒为载体,成功构建了较高容量的噬菌体展示随机十肽库,并将其应用于抗原模拟表位的淘选和鉴定。将一种特异识别对虾白斑综合症病毒(Whitespotsyndromevirus,WSSV)的单链抗体A1对十肽库和十五肽库分别进行淘选,结果得到一系列能与单链抗体A1特异性结合的阳性克隆。将这些阳性克隆所编码的多肽氨基酸序列与已知的单链抗体A1的抗原WSSV388片段氨基酸序列做比对,发现多数阳性多肽序列都与WSSV388片段序列的C端一处K????R??R?QS的氨基酸片段相似,由此推论单链抗体A1的模拟抗原表位可能是由该不连续氨基酸片段所构成的构象表位,而非线性表位。研究结果表明,噬菌体展示随机肽库技术是一种用于研究抗原表位结构的有效方法,有助于进一步探讨WSSV的结构蛋白的构象及功能,以及相应单链抗体与细胞受体相互作用的机理。     

对虾白斑综合征杆状病毒同源性比较的研究

比较我国沿海不同海域对虾白斑综合征杆状病毒三个分离株：即唐海分离株（渤海湾）、宁波分离株（东海）,深圳分离株（南海）的同源性。三个WSSV分离株基因组的限制笥内切酶（Sac Ⅰ,HindⅢ,PstⅠ）酶切多态（RFLP）以及病毒结构蛋白图谱完全一致,证实造成我国从南对北对虾爆发性流行病的对虾白斑杆状病毒为同一种病毒。利用高保真Taq酶,分别以报道的日本对虾杆状病毒（RV－PJ－PRDV）,斑节对虾白斑综合征杆状病毒（WSBV－PmNOBⅢ）基因组核酸片段特异性引物进行PCR扩增,结果均能从中国一杆状病毒（WSSV）基因组中扩增得到相应大小的PCR产物,扩增产物序列分析表明中国对虾白斑杆状病毒（WSSV）与斑节对虾白斑综合征杆状病毒（WSBV－PmNOBⅢ）,日本对虾相状RV-PJ=PRDV)同源率分别为100％与97％,其结果为证实亚洲及太平洋地区对虾白斑综合征杆状病毒为同一种病毒或同一种病毒的不同株系提供了依据。     

噬菌体展示随机十肽库的构建及抗WSSV单链抗体A1的模拟抗原表位的淘选和鉴定

本实验以pCANTAB 5 E噬菌粒为载体,成功构建了较高容量的噬菌体展示随机十肽库,并将其应用于抗原模拟表位的淘选和鉴定.将一种特异识别对虾白斑综合症病毒(White spot syndrome virus, WSSV)的单链抗体A1对十肽库和十五肽库分别进行淘选,结果得到一系列能与单链抗体A1特异性结合的阳性克隆.将这些阳性克隆所编码的多肽氨基酸序列与已知的单链抗体A1的抗原WSSV388片段氨基酸序列做比对,发现多数阳性多肽序列都与WSSV388片段序列的C端一处K····R··R·QS的氨基酸片段相似,由此推论单链抗体A1的模拟抗原表位可能是由该不连续氨基酸片段所构成的构象表位,而非线性表位.研究结果表明,噬菌体展示随机肽库技术是一种用于研究抗原表位结构的有效方法,有助于进一步探讨WSSV的结构蛋白的构象及功能,以及相应单链抗体与细胞受体相互作用的机理.     

白斑综合征病毒囊膜蛋白在对虾免疫保护应用中的研究进展

白斑综合征自上世纪90年代初在水产养殖业中爆发以来,其病原体白斑综合征病毒的研究一直在深入开展,特别是WSSV结构蛋白的功能学研究尤为广泛,其主要方向集中在病毒囊膜蛋白对虾体的免疫保护上,并取得了显著的保护效果。从利用病毒囊膜蛋白作为亚单位疫苗免疫虾体、利用囊膜蛋白对应抗体保护虾体、构建囊膜蛋白基因核酸疫苗和利用RNAi干扰技术保护虾体等四个方面,对当前WSSV囊膜蛋白在对虾免疫保护中的应用进行了概述,并对其应用前景作一展望,旨在为及早开发出有效防治白斑综合征疾病的技术途径提供借鉴参考。     

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.     

抗对虾白斑综合症病毒单链抗体P1D3基因在毕赤酵母中的分泌表达

为了将可中和对虾白斑综合症病毒(WSSV)的单链抗体P1D3在酵母中实现表达,以原核表达载体M13噬菌粒为模板,设计带有SnaBⅠ和EcoRⅠ酶切位点的特异性引物,通过PCR方法扩增P1D3基因。经过酶切、连接反应将该基因连入大肠杆菌-酵母穿梭质粒pPIC9K上。重组质粒pPIC9K-scFvP1D3经BglⅡ线性化后,用电转化的方法转入毕赤酵母(Pichiapastoris)GS115中。通过PCR和DNA测序,挑选和鉴定阳性克隆。经甲醇诱导,P1D3在酵母中获得分泌表达。ELISA实验结果表明,酵母表达上清液中的单链抗体具有较高的WSSV结合活性,而且其活性要高于大肠杆菌所表达抗体的活性。表达条件优化后,单链抗体在酵母中最高表达量可达302mg/L,为开展对虾被动免疫研究提供了新的抗体来源。     

White spot syndrome virus open reading frame 222 encodes a viral E3 ligase and mediates degradation of a host tumor suppressor via ubiquitination

We have characterized a white spot syndrome virus (WSSV) RING-H2-type protein, WSSV222, which is involved in ubiquitination. WSSV222 exhibits RING-H2-dependent E3 ligase activity in vitro in the presence of the specific conjugating enzyme UbcH6. Mutations in the RING-H2 domain abolished WSSV222-dependent ubiquitination, revealing the importance of this domain in WSSV222 function. Yeast two-hybrid and pull-down analyses revealed that WSSV222 interacts with a shrimp tumor suppressor-like protein (TSL) sharing 60% identity with human OVCA1. To better characterize the interaction of WSSV222 and TSL in vivo, we established a stable TSL-expressing cell line derived from the human ovarian cancer cell line A2780, where we observed a TSL-dependent prolonged G1 phase. Furthermore, we detected WSSV222-mediated ubiquitination and MG132-sensitive degradation of TSL both in shrimp primary cell culture and in the TSL-expressing cell line. Transient expression of TSL in BHK cells leads to apoptosis, which was rescued by WSSV222. Taken together, our data suggest that WSSV222 acts as an antiapoptosis protein by ubiquitin-mediated proteolysis of TSL to ensure successful WSSV replication in shrimp.     

Four viral proteins of white spot syndrome virus (WSSV) that attach to shrimp cell membranes

White spot syndrome virus (WSSV) is a major shrimp pathogen that has a widespread negative affect on shrimp production in Asia and the Americas. It is known that WSSV infects shrimp cells through viral attachment proteins (VAP) that bind with shrimp cell receptors. However, the identity of both WSSV VAP and shrimp cell receptors remains unclear. We used digoxigenin (DIG)-labeled shrimp hemocyte and gill cell membranes to bind to WSSV proteins immobilized on nitrocellulose membranes, and 4 putative WSSV VAP (37 kDa, 39 kDa and 2 above 97 kDa) were identified. Mass spectrometric analysis identified the 37 kDa putative VAP as the product of WSSV gene VP281.     

VP37 of white spot syndrome virus interact with shrimp cells

Aims:  To investigate VP37 [WSV 254 of White spot syndrome virus (WSSV) genome] interacting with shrimp cells and protecting shrimp against WSSV infection.
Methods and Results:  VP37 was expressed in Escherichia coli and was confirmed by Western blotting. Virus overlay protein binding assay (VOPBA) technique was used to analyse the rVP37 interaction with shrimp and the results showed that rVP37 interacted with shrimp cell membrane. Binding assay of recombinant VP37 with shrimp cell membrane by ELISA confirmed that purified rVP37 had a high-binding activity with shrimp cell membrane. Binding of rVP37 to shrimp cell membrane was a dose-dependent. Competition ELISA result showed that the envelope protein VP37 could compete with WSSV to bind to shrimp cells. In vivo inhibition experiment showed that rVP37 provided 40% protection. Inhibition of virus infection by rVP37 in primary cell culture revealed that rVP37 counterparted virus infection within the experiment period.
Conclusions:  VP37 has been successfully expressed in E . coli . VP37 interacted with shrimp cells.
Significance and Impact of the Study:  The results suggest that rVP37 has a potential application in prevention of virus infection.     

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.     

Enzyme E2 from Chinese white shrimp inhibits replication of white spot syndrome virus and ubiquitinates its RING domain proteins

Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.     

RING-H2 protein WSSV249 from white spot syndrome virus sequesters a shrimp ubiquitin-conjugating enzyme, PvUbc, for viral pathogenesis

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.     

对虾白斑综合症病毒与虾鳃细胞膜特异性结合关系的确立

对虾白斑综合症病毒(White spot syndrome virus,WSSV)是养殖对虾的一个主要病原,也是目前发现的基因组最大的动物病毒(基因组约290kDa,双链环状)。WSSV病毒粒子为卵形杆状,外被囊膜,囊膜在尾部延伸成一长尾。它不仅能感染对虾,还能感染其它淡水及海水甲壳类。养殖对虾被感染后,3—10d内累积死亡率可达100％,给对虾养