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。     

Characterization and application of monoclonal antibodies against white spot syndrome virus

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG₁ class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.     

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.     

白斑综合征病毒囊膜蛋白VP19及VP28的研究进展

自二十世纪90年代,白斑综合征病毒(WSSV)就因其暴发范围广、致死率高得到了广泛的关注。研究主要集中在确定该病毒蛋白的结构及功能,以及利用其囊膜蛋白制备亚单位疫苗、研发DNA疫苗等来提高对虾抵抗白斑综合征病毒的能力,尽管免疫防治目前在实验室阶段已取得了显著的保护效果,但因其给药方式局限以及成本较高等因素一直没有应用于实际生产中。VP19和VP28是白斑综合征病毒主要的囊膜蛋白,在WSSV感染对虾的过程中起着非常重要的作用。本文从WSSV的基因组学、VP19和VP28的蛋白质结构及其在免疫防治中的应用等方面概述了VP19和VP28的研究进展,包括蛋白亚单位疫苗、DNA疫苗、RNA疫苗以及相关抗体的研究。在总结了不同类型疫苗的保护效果后发现,VP19和VP28的双价疫苗的保护率较高,为今后制定有效的WSSV控制方法提供了参考。     

Enhancement and confirmation of white spot syndrome virus detection using monoclonal antibody specific to VP26

A portion of the VP26 gene (VP26F109) encoding a structural protein of white spot syndrome virus was expressed, purified by SDS‐PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three groups of MAbs specific to different epitopes on VP26 were selected; these MAbs can be used to detect natural WSSV infection in Penaeus vannamei using dot blotting, Western blotting or immunohistochemistry without cross‐reaction with other shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was ranged 7–14 fmole per spot of the rVP26F109 as determined using dot blotting. A combination of three MAbs specific to VP26 with MAbs specific to VP28, VP19 and ICP11 increased the detection sensitivity of WSSV during early infection. Therefore, the MAbs specific to VP26 could be used to confirm and to enhance the detection sensitivity for WSSV infection in shrimp with various types of antibody‐based assays.     

自然养殖水体投喂CpG ODN和表面展示VP28的解脂耶罗维亚酵母对凡纳滨对虾(Litopenaeus vannamei)抗WSSV的免疫保护作用

用添加CpG寡聚核苷酸(CpG ODN)和表面展示VP28的解脂耶罗维亚酵母(VP28-yl)的饵料投喂凡纳滨对虾,进行田间中试实验。投喂30 d后进行WSSV感染实验,评估其对凡纳滨对虾的免疫保护作用。投喂实验结束后,CpG ODN投喂组对虾的相对增重率达到(65.8±7.8)% (P<0.05),这暗示CpG ODN可能具有促生长作用。WSSV攻毒后,CpG ODN和VP28-yl投喂组对虾中WSSV拷贝数与对照组相比均显著降低(P<0.05),相对免疫保护率分别可达到26.7%和36.7%。在投喂结束和WSSV刺激后,CpG ODN组对虾中的呼吸爆发水平均显著升高(P<0.05)。而在VP28-yl投喂组,WSSV引起的细胞凋亡则显著受到抑制(P<0.05)。此外,WSSV刺激后,STAT基因在CpG ODN组和VP28-yl组对虾中的表达水平均显著上调(P<0.05),分别在第5天和第3天达到最大值,而对照组中则显著下调。研究结果表明,CpG ODN和VP28-yl增强了凡纳滨对虾抗病毒免疫力,对养殖对虾病毒性疫病的防控具有显著作用,可以作为免疫增强剂添加在饵料中,具有在养殖生产中推广使用的前景。     

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

Immune responses of whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.     

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。     

Molecular docking and simulation studies of 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 against VP26 and VP28 proteins of white spot syndrome virus

White spot syndrome virus (WSSV), an aquatic virus infecting shrimps and other crustaceans, is widely distributed in Asian subcontinents including India. The infection has led to a serious economic loss in shrimp farming. The WSSV genome is approximately 300 kb and codes for several proteins mediating the infection. The envelope proteins VP26 and VP28 play a major role in infection process and also in the interaction with the host cells. A comprehensive study on the viral proteins leading to the development of safe and potent antiviral therapeutic is of adverse need. The novel synthesized compound 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 is proved to have potent antiviral activity against WSSV. The compound antiviral activity is validated in freshwater crabs (Paratelphusa hydrodomous). An in silico molecular docking and simulation analysis of the envelope proteins VP26 and VP28 with the ligand 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 are carried out. The docking analysis reveals that the polar amino acids in the pore region of the envelope proteins were involved in the ligand binding. The influence of the ligand binding on the proteins is validated by the molecular dynamics and simulation study. These in silico approaches together demonstrate the ligand's efficiency in preventing the trimers from exhibiting their physiological function.     

Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb

White spot disease (WSD) is an important viral disease of penaeid shrimp caused by white spot syndrome virus (WSSV). WSSV isolated from WSD outbreaks in commercial shrimp (Penaeus monodon) farms in India were propagated in the laboratory in healthy shrimp. The virus was purified from the infected tissues by sucrose gradient centrifugation. The VP28 was electroeluted from SDS-PAGE gels and was used to immunize Balb/c mice to produce hybridomas secreting monoclonal antibodies (MAb) against WSSV. A total of five hybridoma clones secreting MAbs to VP28 were produced. The MAbs were of the isotypes IgG1, IgG2b and IgM. The MAbs reacted with VP28 of WSSV and not with any other viral or shrimp protein in western blot. The MAbs were used to develop dot immunoblot assay using an immunocomb to detect WSSV from field samples. The test developed had an analytical sensitivity of 625 pg and a diagnostic sensitivity of 100% compared to single step polymerase chain reaction (PCR). The test can be used as an alternate for first step PCR to detect WSSV from field samples.     

Virulence status,viral accommodation and structural protein profiles of white spot syndrome virus isolates in farmed Penaeus monodon from the southeast coast of India

The objective of this study was to investigate the reason for variation in the virulence of white spot syndrome virus (WSSV) from different shrimp farms in the Southeast coast of India. Six isolates of WSSV from farms experiencing outbreaks (virulent WSSV; vWSSV) and three isolates of WSSV from farms that had infected shrimps but no outbreaks (non‐virulent WSSV; nvWSSV) were collected from different farms in the Southeast coast of India. The sampled animals were all positive for WSSV by first‐step PCR. The viral isolates were compared using histopathology, electron microscopy, SDS‐PAGE analysis of viral structural proteins, an in vivo infectivity experiment and sequence comparison of major structural protein VP28; there were no differences between isolates in these analyses. A significant observation was that the haemolymph protein profile of nvWSSV‐infected shrimps showed three extra polypeptide bands at 41, 33 and 24 kDa that were not found in the haemolymph protein profile of vWSSV‐infected shrimps. The data obtained in this study suggest that the observed difference in the virulence of WSSV may not be due to any change in the virus, rather it could be due to the shrimp defence system producing certain factors that help it to accommodate the virus without causing any mortality.     

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。

     

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。     

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.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV‐positive farms: post‐larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7–100% nucleotide sequence identity with four strains in the GenBank database – China ( AF332093 ), Taiwan ( AF440570 and U50923 ) and Thailand ( AF369029 ). This is the first broad study of WSSV infection in L. vannamei in Taiwan.     

Protection of Fenneropenaeus chinensis (Osbeck, 1765) against the white spot syndrome virus using specific chicken egg yolk immunoglobulins by oral delivery

Two kinds of specific chicken egg yolk immunoglobulins (IgYs), IgY‐WSSV and IgY‐VP28, were, respectively, raised against the 2 mM binary ethylenimine (BEI)‐inactivated white spot syndrome virus (WSSV) and a principal envelope protein VP28. The activity of purified specific IgYs was stable under the conditions of 20–70 °C, pH 3.0–10.0 and 0–700 g L^?1 sucrose solution. In the neutralization assay, these high‐affinity IgY antibodies can specifically bind with the virus particles to protect shrimp (Fenneropenaeus chinensis) against WSSV infection. After oral delivery for 20 days, the IgY‐WSSV exerted a higher protection effect (RPS: 71.5%) than IgY‐VP28 (RPS: 63.7%). Moreover, an increase in RPS (79.2%) was found on addition of IgY‐WSSV:VP28 (0.1% IgY‐VP28 plus 0.2% IgY‐WSSV). This may indicate that neutralization of WSSV refers to the multiple‐hit model. By time‐course study of the levels of the specific IgYs in vivo, the data showed that the titre was enhanced to a relatively high level (P/N=8.35±0.45) at 3 days post administration, declined slightly (P/N=7.13±1.01) at 7 days post administration and then remained stable for further investigation. The stable antibody level potentially contributes towards blocking a large number of WSSV particles from entering and infecting on the major tissues at the early and late stages after challenge in shrimp.     

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.