BmNPV和AcNPV扩大寄主域杂交重组病毒表达载体的构建和改进

苜蓿银纹夜蛾核型多角体病毒(Autographa californica nuclear polyhedrosis virus, 简称AcNPV)和家蚕核型多角体病毒(Bombyx mori nuclear polyhedrosis virus, 简称BmNPV)表达系统是目前最主要的两类昆虫杆状病毒表达系统. 两者各具特点, 前者寄主昆虫个体较小, 但寄主范围广、适合较大规模的细胞悬浮培养生产体系; 而后者虽然寄主专一, 但以个体较大、易饲养的家蚕为寄主, 具有表达量高、 特别适合产业化水平开发的要求. 为了利用AcNPV和BmNPV各自的优势, 扩大昆虫杆状病毒的寄主范围, 以BmNPV为基础, 利用决定病毒寄主范围的DNA解旋酶基因及同源重组原理, 构建了介于BmNPV和AcNPV间的杂交重组病毒(hybrid baculovirus of AcNPV and BmNPV, 简称HyNPV). 这样构建的杂交重组病毒具有AcNPV和BmNPV的双重优点, 既能感染所有AcNPV的寄主, 又可以在家蚕中表达. 以人碱性成纤维细胞成长因子基因作为应用实例, 克隆构建了这样的一种重组杂交病毒, 可在Sf21, Tn-5, BmN培养细胞及家蚕个体中穿梭表达. 为了减少表达后重组蛋白的降解, 利用“基因敲除”法删除了杆状病毒本身的一种主要蛋白酶-半胱氨酸蛋白酶基因, 提高了重组蛋白的稳定性和表达效率.     

WSSV囊膜蛋白VP37和VP39基因酵母双杂交诱饵载体的构建及其激活作用检测

对虾暴发性流行病是近十年来危害对虾养殖业发展的重要病害之一,其主要病原为对虾白斑综合症病毒（WSSV）^[1]。近年来对WSSV的研究主要集中在其囊膜蛋白、黏附蛋白等结构蛋白方面^[2]。本实验室经病毒结合分析^[3]和病毒铺覆蛋白印迹技术（Virus overlay protein blot assay,VOPBA）初步研究,已证实WSSV存在4种病毒黏附蛋白（VAP）,其中VAP1已确定为WSSV囊膜蛋白VP37^[4],该蛋白存在有特征性的细胞结合域（RGD）。编码的蛋白包含281个碱基,与Huang C,et al.^[5]报道的VP37一致,     

对虾白斑综合症病毒结构蛋白VP28的原核表达和性质研究

VP2 8是对虾白斑综合症病毒 (Whitespotsyndromevirus ,WSSV)的一个重要的囊膜蛋白。为了便于研究VP2 8在和宿主细胞相互作用过程中扮演的角色 ,将VP2 8基因克隆到一个原核表达系统 ,对原核表达的VP2 8的特性进行了研究 ,并制备了抗VP2 8的多克隆抗体和单链抗体 ,对原核表达的和天然病毒的VP2 8蛋白免疫原性进行了比较。结果表示 ,原核表达的VP2 8与天然蛋白具有相似的免疫原性。     

从噬菌体展示抗体文库筛选对虾白斑综合征病毒的单链抗体

对虾白斑综合征是一种严重危害对虾养殖业的病毒性疾病.由于目前对其病原体对虾白斑综合征病毒（WSSV）的研究不够深入,所以对WSSV的有效防治仍然是一大难题.为此,用完整的对虾白斑综合征病毒粒子作为靶抗原固相包被,淘选噬菌体展示单链抗体文库,得到两个能够与WSSV结合的单链抗体：E2和H4.单链抗体H4能够结合病毒并抑制病毒对原代培养的对虾淋巴细胞的感染,这些结果表明此单链抗体具有开发为诊断试剂盒和抗病毒药物的潜力.     

对虾白斑综合症病毒结构蛋白60B的原核表达和其基本氨基酸序列的初步分析

VP60B是对虾白斑综合症病毒(WSSV)中含量很少的一个结构蛋白。VP60B的一段序列与腺病毒纤维蛋白(Adenovirus type 5 fiber protein)的knob domain的一段序列具有同源性。本试验将VP60B基因克隆到原核表达系统中,在低温条件下,诱导了VP60B蛋白的表达。结果显示VP60B在该系统主要是以包含体的形式存在。原核表达的VP60B不能被鼠抗WSSV多克隆血清所识别,预示该蛋白的免疫原性较弱。通过对VP60B氨基酸序列的分析,发现有一个跨膜区,这预示着该蛋白可能位于WSSV病毒的囊膜上。     

对虾白斑综合症病毒一个基因的序列分析

对虾白斑综合症病毒)White Spot Syndrome Virus,WSSV)是一种无包含体、具囊膜、杆状的双链DNA病毒 ,其全基因组序列为 300kb左右 ,是目前已知的基因组最大的动物病毒之一1,2.该病毒的基因组结构非常特殊 ,与已知的病毒的基因组相差很远 ,拥有许多该病毒特有的基因 ,很可能为一种新的病毒科成员 ,其分类地位待于进一步研究。从已经登记到GenBank1的对虾白斑综合症病毒全序列知道 ,该病毒存在着至少两种不同的分离株 ,但基因组序列非常保守。本文报道该病毒的一个基因序列的分析结果以及三个不同的白斑综合症病毒分离株的同源基因片段序列的比较结果。       

对虾白斑综合症病毒极早期基因的研究进展

对虾白斑综合症病毒(white spot syndrome virus,WSSV)是危害对虾养殖业的主要病原之一。WSSV在侵染宿主细胞的过程中,极早期基因(immediate-early gene)对病毒复制增殖起着非常重要的作用。在这个过程中,一方面极早期基因编码的蛋白通过与细胞调控因子相互作用,调节细胞信号通路,为病毒的增殖提供更合适的环境;另一方面,极早期蛋白直接调控病毒基因的转录和表达。综述列举了WSSV的21个极早期基因,并将重点介绍其中5个研究比较深入的基因:ie1、wsv051、wsv083、wsv249和wsv403。     

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     

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。     

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。     

Shrimp vaccination trials with the VP292 protein of white spot syndrome virus

AIMS: Construction of a recombinant vector that expresses VP292 protein of white spot syndrome virus (WSSV) and to exploit the possibility of obtaining the vaccine conferring protection against WSSV infection in shrimps. METHODS AND RESULTS: VP292 protein of WSSV was amplified from WSSV genomic DNA by PCR. The target 814 bp amplified product specific for VP292 protein was inserted in to pQE30 expression vector. The recombinant plasmid of VP292 protein was transformed and expressed in Escherichia coli under induction of isopropyl-1-1-thio-beta-D-galactoside (IPTG) and the immunoreactivity of the fusion protein was detected by Western blot. Shrimp were vaccinated by intramuscular injection of the purified protein VP292 of WSSV and challenged for 0-30 days. Vaccination trial experiments show that two injections with recombinant VP292 (rVP292) protein induced a higher resistance, with 52% relative percentage survival value, in the shrimp at the 30th day postvaccination. CONCLUSIONS: The expression system of protein VP292 of WSSV with a high efficiency has been successfully constructed. Vaccination trials show significant resistance in the shrimp vaccinated twice with recombinant VP292. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study prosper the development of WSSV protein vaccine against WSSV infection in shrimps.     

Conservation of the DNA sequences encoding the major structural viral proteins of WSSV

A cDNA library was constructed from white spot syndrome virus (WSSV)-infected penaeid shrimp tissue. cDNA clones with WSSV inserts were isolated and sequenced. By comparison with DNA sequences in GenBank, cDNA clones containing sequence identical to those of the WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites in the mRNAs of VP28 and VP15 were identified. Genes encoding the major viral structural proteins VP28, VP26, VP24, VP19 and VP15 of 5 WSSV isolates collected from different shrimp species and/or geographical areas were sequenced and compared with those of 4 other WSSV isolate sequences in GenBank. For each of the viral structural protein genes compared, the nucleotide sequences were 100 to 99% identical among the 9 isolates. Gene probes or PCR primers based on the gene sequences of the WSSV structural proteins can be used for diagnoses and/or detection of WSSV infection.     

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.     

Vaccination of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV)

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.     

Passive protection of shrimp against white spot syndrome virus (WSSV) using specific antibody from egg yolk of chickens immunized with inactivated virus or a WSSV-DNA vaccine

White spot syndrome virus (WSSV) causes high mortality and large economic losses in cultured shrimp. The VP28, VP19 and VP15 genes encode viral structural proteins of WSSV. In this study, hens were immunized with recombinant plasmid (pCI-VP28/VP19/VP15) with linkers or with inactivated WSSV, which used CpG oligodeoxynucleotides (CpG ODNs) and Freund's adjuvant as adjuvant, respectively. Egg yolk immunoglobulin (IgY) from hens immunized with inactivated vaccine and DNA vaccine was obtained, purified and used for protection of Metapenaeus ensis shrimp against WSSV. The data showed that the antibody response of the hens immunized with the DNA vaccine was improved by CpG ODNs as adjuvant, but was still inferior to inactivated WSSV in both sera and egg yolks. Using specific IgY from hens immunized with inactivated WSSV and DNA vaccine to neutralize WSSV, the challenged shrimp showed 73.3% and 33.3% survival, respectively. Thus, the results suggest that passive immunization strategy with IgY will be a valuable method against WSSV infection in shrimp.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression

The four main gene expression systems currently used to produce recombinant proteins are the prokary-otic, yeast, insect cell, and mammalian cell expression systems. The baculovirus expression vector system (BEVS) is a protein production system which uses a recombinant baculovirus harboring a foreign gene of interest to produce recombinant protein in an insect or its cultured cells. BEVS has many advantages: (i) BEVS requires less time to establish the production system than is needed in a…     

Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells

White spot disease (WSD) is caused by the white spot syndrome virus (WSSV), which results in devastating losses to the shrimp farming industry around the world. However, the mechanism of virus entry and spread into the shrimp cells is unknown. A binding assay in vitro demonstrated VP28-EGFP (envelope protein VP28 fused with enhanced green fluorescence protein) binding to shrimp cells. This provides direct evidence that VP28-EGFP can bind to shrimp cells at pH 6.0 within 0.5 h. However, the protein was observed to enter the cytoplasm 3 h post-adsorption. Meanwhile, the plaque inhibition test showed that the polyclonal antibody against VP28 (a major envelope protein of WSSV) could neutralize the WSSV and block an infection with the virus. The result of competition ELISA further confirmed that the envelope protein VP28 could compete with WSSV to bind to shrimp cells. Overall, VP28 of the WSSV can bind to shrimp cells as an attachment protein, and can help the virus enter the cytoplasm.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

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.     

Multiple proteins of White spot syndrome virus involved in recognition of β-integrin

The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that β-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of β-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with β-integrin. The β-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of β-integrin. Identification of proteins in WSSV that are associated with β-integrin will assist development of new agents for effective control of the white spot syndrome.