罗氏沼虾白斑综合征病毒囊膜蛋白VP28基因的克隆及分析

根据已公布的罗氏沼虾白斑综合征病毒(WSSV)囊膜蛋白VP28基因序列设计一对特异性引物,从疑似患白斑病毒病的罗氏沼虾(Macrobrachium rosenbergii)中提取总DNA,并以此为模板,经PCR扩增、克隆并测序后将该片段通过GenBank比对,证实为WSSV的VP28基因;与20个已公布的WSSV VP28进行同源性比较,结果显示:从中国对虾、斑节对虾、南美白对虾、日本对虾、波纹龙虾提取的病毒株聚为一类,印度对虾WSSV VP28为另一类,罗氏沼虾WSSV VP28又单独为一类。根据测序结果推测VP28蛋白的二级结构在氨基酸的7～29区间可能为跨膜螺旋区,且该区域高度保守。     

对虾白斑综合征病毒囊膜蛋白VP28研究进展

对虾白斑综合征病毒(white spot syndrome virus,WSSV)是在水产养殖中引起对虾等甲壳类动物发生严重病害的病原体。VP28是WSSV中最重要的囊膜蛋白之一,在WSSV感染对虾的初期起着至关重要的作用。文章从基因和蛋白质结构、VP28在病毒入侵中的作用及免疫应用等方面概述了VP28的研究进展,可为WSSV的防治研究提供参考。     

对虾白斑综合征病毒囊膜蛋白VP19的单克隆抗体制备及其定位

为了更好地研究对虾自斑综合征病毒(WSSV)蛋白VP19在WSSV感染过程巾的作用,利用VP 19的单克隆抗体直接对VP19进行了定位.从患白斑综合征的中国明对虾(Fenneropenaeus chinensis)鳃丝中提取WSSV,将提纯的WSSV经十二烷基磺酸钠-聚内烯酰胺凝胶电泳(SDS-PAGE)分离,然后洗脱提纯其病毒蛋白VP19并免疫Balb/c小鼠,取免疫小鼠脾细胞和骨髓瘤细胞融合,用间接免疫荧光技术(IFAT)和Western-Blot技术筛选出1株阳性杂交瘤细胞,将检测出的阳性杂交瘤细胞经有限稀释法克隆,研制出抗VP19的单抗,再利用免疫胶体金技术对病毒蛋白VP19进行定位,结果显示,胶体金粒子位于WSSV病毒的囊膜上,说明病毒篮白VP19位于WSSV囊膜上.[中国水产科学,2009,16(1):69-74]     

对虾白斑综合征病毒囊膜蛋白VP28和VP26的毕赤酵母组成型分泌表达

近年来,重组 VP28和 VP26蛋白作为蛋白亚单位疫苗,在增强对虾抗白斑综合征病毒(WSSV)感染的过程中具有重要作用。本研究根据GenBank中WSSV的基因序列设计引物,以WSSV粗提液为模板进行普通PCR扩增,得到VP28和VP26基因,再用引物悬挂法将EcoRⅠ和XbaⅠ酶切位点分别添加到 VP28和 VP26基因的5￠端和3￠端。目的基因经双酶切后插入到表达载体pGAPZαA,转化TOP10大肠杆菌,经博莱霉素(Zeocin)抗性筛选阳性重组酵母表达载体。AvrⅡ酶切线性化之后,电击转化 X-33毕赤酵母感受态细胞,经 Zeocin 抗性筛选得到阳性重组酵母。SDS-PAGE电泳分析重组酵母表达上清液的目的蛋白,没有检测到VP28和VP26重组蛋白。随后,采用蛋白质银染法,结果显示,与空载pGAPZαA组相比,VP28和VP26表达上清液组有明显的条带,证明VP28和VP26在毕赤酵母中成功表达,蛋白分子量大小约为32 kDa。     

抗WSSV囊膜蛋白（VP28和VP19）单抗的体内中和效果研究

通过酶联免疫吸附法（ELISA）测定不同稀释度对虾白斑综合征病毒（WSSV）与已制备的WSSV囊膜蛋白单克隆抗体结合的OD值。利用克氏原螯虾Cambarus proclarkii动物模型，将不同稀释度病毒与单抗1：1混合孵育2h后，肌肉注射克氏原螯虾（50μl／只），观察记录螯虾的死亡情况。ELISA结果显示，在1×10^-3病毒稀释度下两种单抗均足量。在螯虾体内中和实验中，当病毒浓度为1×10^-3、1×10^-4、1×10^-5和1×10^-6稀释度时，MAb1D6（VP28）螯虾组最终死亡率分别为100％、90％、16．7％和6．7％，而MAb2E9（VP19）螯虾组最终死亡率分别为100％、100％、100％和93．3％。这表明随病毒浓度的降低，MAb1D6（VP28）的中和效果越明显。而MAb2E9（VP19）并无明显的中和效果。     

对虾白斑综合征病毒免疫防治研究进展

对虾白斑综合征病毒(white spot syndrome virus,WSSV)是一种引起养殖对虾爆发性死亡的病毒,由于它宿主范围广、蔓延速度快以及致死率高,已经成为对虾养殖中的第一杀手,因此有效防治WSSV爆发在水产养殖中具有重要意义并极具挑战性。本文主要介绍WSSV基因组以及结构蛋白、对虾WSSV免疫机制、对虾WSSV疫苗研究进展,以及我国对虾养殖中WSSV防治措施,认为对虾WSSV疫苗将是今后对虾大规模养殖中病害防治的一种重要手段。     

乳糖诱导对虾白斑病毒囊膜蛋白VP28基因在重组大肠杆菌中的表达

以含对虾白斑综合症病毒（white spot syndrome virus，WSSV）囊膜蛋白VP28编码基因质粒的重组大肠杆菌Escherichia coliB121作为研究对象，研究了乳糖或乳清粉代替IPTG作为诱导剂诱导重组囊膜蛋白VP28的表达。结果表明，乳糖不仅能够作为诱导剂诱导重组大肠杆菌进行外源蛋白的表达，而且能作为碳源促进菌体的生长。通过对诱导条件的优化，乳糖在发酵培养基的添加量为8g·L^-1，发酵时间为12h时可以获得最高的目的蛋白表达量，为97．36mg·L^-1。试验亦使用乳清粉作为发酵培养基的碳源和诱导工程菌表达的诱导剂。结果表明，在发酵培养基中添加乳清粉作为碳源和诱导剂，使其乳糖终浓度为10g·L^-1，发酵时问为13h时可以获得最高的目的蛋白表达量，为86．24mg·L^-1。     

WSSV-VP37小肽在大肠杆菌中的偏爱型表达

选用对虾白斑综合征病毒(White Spot Syndrome Virus,WSSV)囊膜蛋白VP37的功能区段,利用大肠杆菌密码子偏爱性,在氨基酸不变的情况下将VP37中的密码子通过人工合成改为大肠杆菌偏爱型密码子,并克隆至表达载体pBAD/gIIIA中,构成重组载体pBAD/gIIIA-VP37p′.将重组载体转化入大肠杆菌Top10中,在相同条件下用L-阿拉伯糖与未优化的重组菌株Top10-pBAD/gIIIA-VP37p一同诱导.结果显示,与野生型基因VP37p相比,经密码子优化的VP37p′基因表达目的蛋白量占总蛋白的40.5%,明显高于野生型6.5%的目的蛋白表达量.     

重组WSSV囊膜蛋白VP281和VP31的抗菌功能初步分析

为了解白斑综合征病毒(white spot syndrome virus,WSSV)两种囊膜蛋白VP281和VP31的功能,本实验对二者进行了原核重组表达.利用一种组成型分泌原核表达质粒pBTA1为表达载体,构建了组成型分泌WSSV囊膜蛋白rVP281、rVP28以及rVP28与增强型绿色荧光蛋白rEGFP融合蛋白的重组大肠杆菌菌株DH5α,分别命名为DhpVP281、DhpVP28和DhpVP28-EGFP.3种重组菌在LB固体培养基上生长12 h的菌落直径分别为(164.84±28.44)、(560.47±46.04)和(548.21±58.54)μm,生长19 h的菌落直径分别为(436.31±47.56)、(1 136.90±110.88)和(1 083.33±109.83) μm,生长24 h的菌落直径分别为(594.19±57.17)、(1251.19±188.86)和(1 264.29±172.78) μm;显示在所有培养时间,DhpVP281的菌落均显著小于DhpVP28或DhpVP28-EGFP的菌落(P＜0.05),推测rVP281的表达可能抑制大肠杆菌的生长.以pBTA1为表达载体,未能成功构建组成型分泌rVP31的重组DH5α.为了解其原因,使用pET-30a(+)为表达载体,构建了表达rVP31包涵体的重组菌株BL21(DE3) pLysS.将rVP31蛋白纯化并复性后,采用牛津杯法,检测到rVP31蛋白对溶壁微球菌具有抗菌作用.在动物病毒中发现具有抗菌作用的囊膜蛋白,可以增加有关WSSV的病毒学方面的知识.     

“对虾白斑综合征病毒VP292多肽与应用”获国家发明专利授权

<正>日前,由中国水产科学研究院黄海水产研究所海水养殖生物疾病控制与分子病理学实验室刘庆慧研究员等发明的"对虾白斑综合征病毒VP292多肽与应用"获得国家发明专利授权。对虾白斑综合征病毒是双链DNA病毒,基因组全长305 Kb,是迄今为止发现的最大的动物病毒。近年来,该病毒严重危害我国及亚洲太平洋地区人工养殖的对虾,此外还可侵染淡水及海洋生态系统中的多种蟹类、龙虾类、端足类、水蝇类等甲壳纲动物,具有较广泛的宿主范围,对海洋渔业及海洋生态造成了严重影响。目前还难以对WSSV进行预防和控制,一方面由于WSSV能在自然环境中存活较长时间,另一方面对此病毒分子水平的了解还尚少。因此,研究针对WSSV     

Protection of crayfish, Cambarus clarkii, from white spot syndrome virus by polyclonal antibodies against a viral envelope fusion protein

White spot syndrome virus (WSSV) is a large double-stranded DNA virus, causing considerable mortality in penaeid shrimp and other crustaceans. WSSV produces five major structural proteins, including two major envelope proteins, VP28 and VP19. To produce VP28 and VP19 as a single protein for antibody production, DNA sequences encoding both open reading frames were fused together and cloned into pET-22b(+) expression vector. The fusion protein, VP(19+28), was expressed in Escherichia coli, purified using Ni2+ His affinity chromatography and injected into a rabbit. Antiserum collected from the immunized rabbit was tested in vivo for ability to protect crayfish, Cambarus clarkii, from disease caused by WSSV. Fifteen days after challenge with WSSV, treatment with VP(19+28) antiserum gave 100% protection against disease in the ambient temperature range of 15-22 degrees C and 65% protection at a constant temperature of 26 degrees C. These results demonstrated VP(19+28) antiserum is effective in protection of crayfish from WSSV and confirmed that VP19 and VP28 play an important role in WSSV host infection. Targeting both VP19 and VP28 may be effective for the design of both immunotherapeutic medicines and reagents to detect WSSV.     

Nucleotide sequence variations of the major structural proteins (VP15, VP19, VP26 and VP28) of white spot syndrome virus (WSSV), a pathogen of cultured Litopenaeus vannamei in Mexico

White spot syndrome virus (WSSV) was first reported in farmed Litopenaeus vannamei stocks in Sinaloa and Sonora, Mexico during 1999 and continues to cause severe shrimp losses. WSSV genes encoding nucleocapsid (VP26 and VP15) and envelope proteins (VP19 and VP28) of a Mexican isolate were cloned in the pMosBlue vector. The nucleotide sequences of these genes were compared with WSSV isolates in GenBank. VP15 is highly conserved, and VP26 showed 99% homology to a Chinese isolate. The VP28 fragment demonstrated 100% homology to the majority of the isolates analysed (UniProt accession no. Q91CB7), differing from two Indian WSSV and one Chinese WSSV isolates by two non-conserved and one conserved replacements, respectively. Because of their highly conserved nature, these three structural proteins are good candidates for the development of antibody-based WSSV diagnostic tools or for the production of recombinant protein vaccines to stimulate the quasi-immune response of shrimp. In contrast, VP19 of the Mexican isolate was distinguishable from almost all isolates tested, including an American strain of WSSV (US98/South Carolina, GenBank accession no. AAP14086). Although homology was found with isolates from Taiwan (GenBank accession no. AAL89341) and India (GenBank accession no. AAW67477), VP19 may have application as a genetic marker.     

Increased resistance to white spot syndrome virus in Procambarus clarkii by injection of envelope protein VP28 expressed using recombinant baculovirus

In order to investigate whether protein structure has an effect on protective effect of envelope protein of WSSV, VP28 protein was expressed both in Escherichia coli (pET-VP28) and insect (BmN) cells (BmNPV-VP28). The baculovirus (BmNPV) expression system was used to obtain correctly folded VP28 protein. Procambarus clarkii crayfish were intramuscularly injected with lysates of cells infected with recombinant pET-VP28 and BmNPV-VP28, respectively, and then challenged by intramuscular injection of WSSV to assess the duration of protection. The crayfish injected with BmNPV-VP28 showed generally lower mortality rates when compared to crayfish injected with pET-VP28, resulting in relative percent survivals of 92% and 39%, respectively, when compared to the control groups injected with empty vectors BmNPV and pET-30a. These results showed that VP28 protein produced in BmN cells gave much better protection than VP28 protein produced in E. coli.     

对虾白斑综合征病毒囊膜蛋白VP110在中国明对虾鳃细胞中结合蛋白的鉴定与特性

为了鉴定对虾白斑病综合征病毒(WSSV)囊膜蛋白VP110在中国明对虾(Fenneropenaeus chinensis)鳃细胞中的结合蛋白, 运用pET-32(a)+载体构建了1段含RGD模体的截短VP110原核重组表达质粒, 转化大肠杆菌诱导表达后获得分子量为41 kD的截短重组VP110蛋白(rVP110)。以rVP110作为诱饵蛋白, 运用pull-down实验结合蛋白质谱分析鉴定rVP110结合蛋白, 结果显示, 中国明对虾鳃细胞中的肌动蛋白和精氨酸激酶(arginine kinase,AK)与rVP110具有结合作用。利用PCR扩增中国明对虾AK编码基因, 将其与表达载体pGEX-4T-1连接后转化大肠杆菌诱导表达获得重组AK蛋白(rAK), 通过pull-down实验进一步证实rAK可与rVP110发生结合。克氏原螯虾(Procambarus clarkia)体内中和实验结果显示, rAK对WSSV感染克氏原螯虾具有一定的中和作用, 能延缓螯虾的死亡进程。另外, 中国明对虾在人工感染WSSV后, 荧光定量PCR检测结果显示, AK基因表达水平显著上调, 18 h时达到峰值, 然后下降至正常水平; 酶底物法检测结果同样显示, 鳃细胞中AK酶活性在感染WSSV后发生显著上调。本研究旨在为深入了解WSSV囊膜蛋白VP110在WSSV感染宿主过程中的作用提供基础依据。

     

对虾白斑综合征病毒VP28酵母表面展示

以pYD1为载体,制备以酒精酵母为载体的基因工程免疫制剂。参照GenBank中对虾白斑综合征病毒VP28基因序列设计引物,PCR扩增得到预期长度的产物,双酶切插入用于酒精酵母表面展示的穿梭质粒载体pYD1,转化大肠杆菌TOP10,提取阳性质粒转化酒精酵母菌株EBY100,诱导表达后,用免疫荧光检测外源蛋白的表达。结果获得VP28酵母表面展示菌,测得最佳诱导时间为36～48h。以此展示酵母活细胞分设两个浓度组,腹腔注射螯虾,检测其毒性。结果表明,此重组酵母对螯虾是安全的,该研究为下一步对虾用活载体免疫制剂效果的研究奠定了基础。     

WSSV envelope protein VP51B links structural protein complexes and may mediate virus infection

White spot syndrome virus (WSSV), an enveloped double‐stranded DNA virus, is the causative agent of a disease that has led to severe mortalities of cultured shrimps in Taiwan and many other countries. In the previous study, Penaeus monodon chitin‐binding protein (CBP) and glucose transporter 1 (Glut1), two cell membrane proteins, were found to at least interact with other 10 WSSV envelope proteins including VP51B. These envelope proteins might form a protein complex. According to the known information, VP51B was used to identify its role in the protein complex. Western blotting of the intact viral particles and fractionation of the viral components confirmed that VP51B is one of WSSV envelope proteins. In this study, the protein–protein interaction between VP51B and other WSSV envelope proteins was identified by far‐western blot experiment and VP51B was found to interact with VP24, VP31, VP32, VP39B and VP41A. Furthermore, the in vivo neutralization experiment using recombinant VP51B plus with VP39B showed the best inhibition. These data indicate that VP51B participates in the WSSV protein complex and plays an important role in WSSV infection.     

Protection of Procambarus clarkii against white spot syndrome virus using recombinant subunit injection vaccine expressed in Pichia pastoris

ABSTRACT:   The potentiality of injection vaccine against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. WSSV envelope proteins VP19 and VP28 were expressed in yeast Pichia pastoris GS115. The purified recombinant proteins (2 µg/g of crayfish) were injected intramuscularly, and the same dose injected as a booster shot on fifth day after vaccination. The vaccinated crayfish were divided into two even groups and later challenged orally by WSSV-infected dead crayfish muscle (2 g/individual) on the third and 21st days after the booster shot. The relative percent survival (RPS) in the third-day group was the highest in VP28 (91%), followed by VP19 + VP28 (84%), and VP19 (45%). The RPS for the 21st-day group was the highest in VP28 (78%), followed by VP19 + VP28 (76%), and VP19 (17%). Development of vaccine by using recombinant proteins VP19 and VP28 expressed in yeast is feasible.     

Effect of VP28 DNA vaccine on white spot syndrome virus in <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. Considering the global environmental, the economic and sociological importance of shrimp farming, and the constraints of high intensity cultivation, development of novel control measures against the outbreak of WSSV become inevitable. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the recombinant plasmid in immunized Litopenaeus vannamei. The VP28 gene was cloned in the eukaryotic expression vector pVAX1, and the construct vector was named as lpv28. The protective effect of lpv28 against WSSV was evaluated in L. vannamei by injecting lpv28 construct and later challenging with WSSV. Expression of these proteins from the recombinant plasmids was confirmed in vitro by RT-PCR and Western blot analysis. The result of vaccination trials showed that a survival rate in shrimp vaccinated with lpv28 was 52.5% at most compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups showed that the vaccinated groups owned a high level of lysozyme, alkaline phosphatase, and total superoxide dismutase when compared to the control group. Furthermore, protein expression analysis indicated that VP28 can be detected in gill, muscle and head soft tissue of the shrimps in the immunized group after 14th day injection. Thus, the result indicated that DNA vaccination strategy has a potential utility against WSSV.