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1.
WSSV具蛋白酶活性肽段的检测 总被引：2，自引：0，他引：2
在已有对虾白斑综合症病毒(WSSV)蛋白酶活性研究的基础上,从分子水平上确定WSSV呈蛋白酶活性的肽段。采用凝胶电泳技术的蛋白酶酶谱分析法确定WSSV蛋白酶活性,研究非变性条件下WSSV多肽裂解的条件,确定低温(20℃)裂解WSSV多肽可满足实验的要求。同时使用SDS-PAGE分离胶切割法结合蛋白洗脱分离出Vp19、Vp22、Vp24、Vp26和Vp28共计5个肽段,并对分离的5个组分进行蛋白酶活性的检测,初步结果表明,Vp19具有蛋白酶催化活性。 相似文献
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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 总被引：5，自引：0，他引：5
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] 相似文献
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通过差速离心法提取桡足类组织细胞膜，依照本实验室确立的细胞膜与WSSV特异性的结合关系，进行了桡足类细胞膜与白斑综合症病毒(WSSV)结合实验。结果表明，桡足类细胞膜与WSSV之间存在着特异性的结合，二者的结合说明了桡足类细胞膜上存在有WSSV的受体蛋白，其为确定桡足类属于WSSV感染宿主提供了重要证据。同时，比较了凡纳滨对虾(Litopenaeus vannamei)鳃细胞膜对桡足类细胞膜与WSSV结合的影响作用，证实了凡纳滨对虾鳃细胞膜对二者的结合具有明显的封闭作用，侧面说明了WSSV在二者细胞膜上存在有相同的受体蛋白或结合位点。桡足类细胞膜经表面活性剂Triton x-100处理后，与WSSV的结合活性没有受到影响，而经Tween-20处理能显著提高与WSSV的结合活性，提示受体蛋白在细胞膜上可能占有一段较小的跨膜区域。经SDS-PAGE分析确定了桡足类细胞膜由分子量为18～207kD范围的约33条蛋白条带组成。 相似文献
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利用非变性电泳与病毒铺膜印迹技术(VOPBA)分离了牙鲆(Paralichthys olivaceus)鳃细胞(FG)上淋巴囊肿病毒结合蛋白,结果显示在FG细胞膜上有分子量为135 kD的蛋白与淋巴囊肿病毒特异结合;对该蛋白切胶回收后进行SDS-PAGE与双向电泳,发现135 kD蛋白由3个蛋白组成,分子量分别为58.3 kD、44.6 kD及37.6 kD;135 kD蛋白SDS-PAGE的VOPBA显示,仅出现37.6 kD的蛋白带,而58.3 kD、44.6 kD蛋白皆不与淋巴囊肿病毒结合。结果表明牙鲆FG细胞上135 kD蛋白是淋巴囊肿病毒的结合蛋白,其37.6 kD蛋白具有病毒结合活性。 相似文献
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本研究采用RACE方法克隆了中国对虾Fenneropenaeus chinensis Rab 7基因(crab7)全长cDNA (GenBank∶JF742050).生物信息学分析显示,fcrab7的开放阅读框615bp,编码205个氨基酸,分子量23.2kD.FcRab7的氨基酸序列具有Rab蛋白家族的共同特征,与凡纳滨对虾和斑节对虾同源蛋白的同源性为100％.构建重组表达载体pBAD/ gⅢA-crab7转化到大肠杆菌E.coli进行诱导表达,诱导产物经SDS-PAGE检测和Western blot验证.本研究结果为进一步研究WSSV与FcRab7的相互作用提供了基础. 相似文献
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病鱼为威海水产养殖场感染淋巴囊肿病的牙鲆（Paralichthys olivaceus）,收集病鱼的囊肿组织,匀浆破碎,采用差速离心和蔗糖密度梯度离心方法,分离纯化淋巴囊肿病毒粒子.负染后,电镜观察证实获得的病毒纯度高,杂质极少,病毒粒子呈近似于圆形的多角形,结构完整.纯化的淋巴囊肿病毒粒子经SDS-PAGE,硝酸银染色后,电泳图谱清晰显示病毒结构蛋白带共有22条,且分子量主要集中在123～26 kD.应用Western blotting法分析病毒结构蛋白的抗原性,结果显示,分子量分别为123.55 kD、65.292 kD和54.438 kD的3条蛋白带发生了免疫反应,其中分子量为65.292 kD的蛋白带反应强度明显高于其他2条蛋白带.本研究旨为确定淋巴囊肿病毒主要衣壳蛋白提供基础依据.[中国水产科学,2006,13（3）：415-420] 相似文献
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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。 相似文献
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中国明对虾肌肉组织蛋白质双向电泳技术体系的建立 总被引：3，自引：2，他引：1
为了探索并建立中国明对虾的肌肉蛋白双向电泳体系,实验将中国明对虾的肌肉组织溶解处理后,通过固相pH梯度胶条等电聚焦、SDS-PAGE垂直电泳对蛋白质进行分离,对不同的裂解液配方、IPG胶条的pH范围及其长度、等电聚焦程序、上样量等进行了优化,并分别利用银染和考染方法进行染色,应用PDQuest软件对图谱进行了初步分析.结果显示,中国明对虾肌肉蛋白的等电点主要位于4～7之间,裂解液中添加硫脲、CHAPS、DTT等,可以增加对虾肌肉蛋白的提取率;采用17 cm,pH 4～7的中型IPG胶条,主动水化上样120μg,适当延长除盐时间和等电聚焦时间,搭建盐桥,采用浓度为12.5％的二向分离胶和硝酸银染色,能有效提高双向电泳(2-DE)图谱中蛋白点的分离度和分辨率;建立的中国明对虾肌肉蛋白双向电泳体系具有良好的重复性和稳定性. 相似文献
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为分离日本鳗鲡脾脏中的抗菌肽,实验采用10 ku和3 ku切向流中空纤维柱对脾脏醋酸粗提物进行分级分离,并结合阳离子交换层析和反相高效液相层析技术对小于10 ku和小于3 ku 2部分样品分别进行分离纯化。结果显示,2种分子量截留纤维柱超滤获得了大于10 ku、小于10 ku和小于3 ku 3部分样品。小于10 ku和小于3 ku蛋白样品抗菌活性较大于10 ku样品强。对小于10 ku样品经阳离子交换层析以及采用pH 9缓冲液进行反相高效液相层析分离纯化,获得一个抗菌肽,质谱分析其分子量为1 391.82 u;对小于3 ku样品经阳离子交换层析并采用pH 9和pH 2两种缓冲液进行反相高效液相层析分离纯化,获得一个抗菌肽,质谱分析其分子量为839.19 u。研究表明,日本鳗鲡脾脏中起抗菌活性的主要成分是一些小分子蛋白。 相似文献
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虾类是海洋生态系统功能群的重要组成部分,其生物量变化受到多重因素的影响。本研究在开展黄海和东海北部水域虾类白斑综合征病毒(white spot syndrome virus, WSSV)流行病学调查的基础上,利用梯度随机森林模型(gradient random forest model, GFM)和广义加性混合模型(generalized additive mixed models, GAMM),分析了2016—2018年间黄海和东海北部水域WSSV流行对虾类生物量的影响。分子检测结果显示,调查所获取的26种虾类中,11种被检测为WSSV阳性;2016、2017和2018年WSSV阳性采样站点的比率分别为48.40%、38.75%和21.74%,虾类样品中WSSV阳性检出比率分别为16.86%、9.60%和4.80%。GFM模型分析显示,解释变量“阳性样品数的对数(ln_posi)”对响应变量“虾类生物量的对数(ln_Abu)”的重要性最高。GAMM分析中,根据赤池信息准则(Akaike information criterion, AIC)最小原则筛选出的最优模型为：ln_Abu~WSSV阳性率(P_rate)+ln_posi+经度(Long),该模型中ln_posi和P_rate是影响虾类生物量的极显著相关因子,ln_Abu随着P_rate的升高而降低。研究表明,WSSV在黄海和东海北部水域虾类中流行,推测对该海域的虾类生物量存在影响。 相似文献
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Victor Stalinraj Koyadan Kizhakedath Vijayan Mohandas Sanjuktha Changaramkumarth Paran Balasubramanian Shankar Vinayakarao Alavandi Thairiyam Chinnappan Santiago 《Aquaculture Research》2009,40(2):129-138
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. 相似文献
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Protection of crayfish, Cambarus clarkii, from white spot syndrome virus by polyclonal antibodies against a viral envelope fusion protein 总被引：8，自引：0，他引：8
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. 相似文献
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Molina-Garza ZJ Galaviz-Silva L Rosales-Encinas JL Alcocer-González JM 《Journal of fish diseases》2008,31(3):197-203
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. 相似文献
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中国对虾几个产卵场群体携带白斑综合征病毒状况调查 总被引：5，自引：1，他引：4
白斑综合征可以导致养殖对虾短时间内大面积死亡,是迄今为止对虾养殖业面临的最大挑战。本试验采用巢式PCR法对2001年采自黄渤海的中国对虾几个产卵场群体进行白斑综合征病毒检测,旨在较全面地了解黄渤海野生中国对虾携带病毒状况。各群体的阳性检出率分别为：朝鲜半岛南海岸群体55％;渤海湾群体35％;辽东湾群体94．7％;海州湾群体47．4％。结果显示,中国对虾几个产卵场群体均不同程度地携带白斑综合征病毒。辽东湾产卵场群体阳性检出率明显高于其他群体,推测人工孵化苗种放流、海湾的地理和水质条件与中国对虾的WSSV感染率相关。而中国对虾野生群体携带病毒对于对虾养殖业的影响是不容忽视的,笔者认为,只有从无特异病原(SPF)及抗特异病原(SPR)对虾养殖群体的建立着手才能从根本上避免由于对虾携带病毒而可能导致的病毒性疾病的暴发。同时,应该重视海区污染的治理,减少病毒病暴发的诱因。本试验建立了快速检测WSSV的PCR方法,1pg病毒核酸仍可检测到,为白斑综合征病毒病的防治及早期诊断提供了有效的手段。 相似文献
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In the present study, we used TaqMan real‐time polymerase chain reaction to quantify and compare infection of white spot syndrome virus (WSSV) with shrimp production of Fenneropenaeus chinensis cultured in outdoor ponds along the west coast of the South Korea. In 2007, a total of 60 specimens in summer and 116 specimens in autumn were collected from 12 growing‐out ponds and 12 harvest ponds respectively. Pond harvest data were obtained from farmers. Of the summer samples, all specimens were WSSV positive, with a wide range of 12.4–7.0 × 107 (mean 7.5 × 106) copies ng?1 DNA; shrimp production was 1.7 metric tonnes per hectare (mt ha?1). Of the 116 autumn‐sample specimens, 81 (69.8%) were WSSV positive; WSSV infection had been decreased dramatically, to 0–7.2 (mean 3.5) copies ng?1 DNA. Shrimp production of autumn ponds was 2.1 mt ha?1. Statistical analysis indicated that the difference in WSSV infections detected in summer and autumn was highly significant (P<0.01). In summer, seven ponds (58.3%) with low‐WSSV infection loads (0–1000 WSSV copies ng?1 DNA) had shrimp production of 2.7 mt ha?1; the others had shrimp production of only 0.2 mt ha?1. The mean shrimp production between the two infection levels showed a highly statistically significant difference (P<0.01). 相似文献
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Carlos R. Pantoja Xiaoling Song Lee Xia Hui Gong Josh Wilkenfeld Brenda Noble Donald V. Lightner 《Aquaculture (Amsterdam, Netherlands)》2005,250(3-4):573-578
A total of 36 gravid female shrimp Fenneropenaeus chinensis, captured off the coast of Qingdao, Peoples Republic of China, from April 20 through April 24, 2001, were examined by PCR for the presence of WSSV and HPV, the two most prevalent viral diseases affecting this species of shrimp in this region of China. Eight spawns, derived from 8 different females, were selected from shrimp where no WSSV or HPV had been detected. Nauplii obtained from 7 individual spawns and unhatched eggs from one other shrimp were transported to the University of Arizona, in Tucson, Arizona, USA, for larval rearing and production of postlarvae under quarantine protocols. The resultant postlarvae were kept in Primary Quarantine for approximately 2 months, while additional tests were performed to determine their specific pathogen-free (SPF) status according to criteria set by the United States Marine Shrimp Farming Program (USMSFP). By the time the shrimp had reached an average weight of approximately 0.4 g, no USMSFP specific listed pathogens had been detected in any of the 7 hatches and the shrimp were released into Secondary Quarantine at the Oceanic Institute in Kailua-Kona, Hawaii for production of a F1 generation. This F1 generation may be used as a SPF founder population for propagation and selection programs. 相似文献