Advances, problems and prospect of inland shrimp farming
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摘要:
文章综述了目前对虾内陆养殖的主要品种、养殖方式和发展现状，并对其优点和存在的问题进行了分析。内陆地下咸水的离子组成比例失调是目前对虾内陆养殖遇到的最大的障碍，文章详述了地下咸水的成因和分布，介绍了有关水体离子浓度和比值对对虾生理生态学影响的研究现状。最后讨论了克服内陆对虾养殖存在的问题并使之成为可持续发展的产业的途径。
Abstract:Main cultured species, culture methods and developing status of inland shrimp farming in the world were reviewed, and the advantages and problems of this type of aquaculture were analyzed. The imbalance of ionic composition of the inland saline groundwater was the main obstacle for the development of inland shrimp farming. The formation and distribution of inland saline groundwater were explained, and the study advances on the effects of ionic concentrations and ratios on the ecophysiology of shrimps were concluded. At the end, a discussion on the sustainable development of inland shrimp farming was made.
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Keywords:
- marine shrimp /
- inland aquaculture /
- ionic composition /
- salinization
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鱼类诺达病毒也称神经坏死病毒(nervous necrosis virus，NNV)[1]，鱼类脑炎病毒(fish encephalitis virus, FEV) [2], 分类上属于诺达病毒科(Nodaviridae)，Beta诺达病毒属(Betanodavirus)，病毒粒子无外膜，只有一种衣壳蛋白[1]。由鱼类诺达病毒引起的病毒性神经坏死症(viral nervous necrosis, VNN)[3]，又称空泡性脑视网膜炎(vacuolating encephalopathy and retinopathy, VER)[4]，是一种世界范围内流行的海水鱼类病毒病，主要发生在海水鱼类种苗生产阶段，鱼苗死亡率高达90%以上，给海水鱼类养殖业的发展带来严重的危害[5]，目前已有8目18科30多种经济鱼类的人工繁殖鱼苗和部分成鱼受到感染而致病, 而且有向淡水鱼类蔓延的趋势[6-7]。
2001年，我们在我国大陆首次报道了石斑鱼神经坏死病毒[8]，并克隆了该病毒的衣壳蛋白基因[9]，研究了该病毒株的基因型[10]。为在细胞株上分离该病毒，以进一步研究该病毒基因的体外表达和调控机制、病毒的感染和致病机理等，需得到高度纯化的病毒。本文报告了病毒的纯化方法和对病毒衣壳蛋白的Western-blot分析，为后续的研究奠定基础。
1. 材料和方法
1.1 材料
2000年广东省大亚湾水产试验中心生产的赤点石斑鱼(Epinephelus akaara)鱼苗。采集具有病毒性神经坏死症症状的垂死的鱼苗，全长1.5~2.0 cm，日龄20~30 d，保存在-85℃的超低温冰箱中。
Western-blot所用试剂：
4×Laemmli buffer：0.125 mol·L-1 Tris-Cl，pH 6.8；40 g·L-1 SDS；100 g·L-1β-巯基乙醇；0.2 g·L-1溴酚蓝。
12.5%浓缩胶：1.565 mL 40% Acrylamide，2.135 mL dH2O，1.25 mL 1.5 mol·L-1 Tris pH 8.8，0.05 mL 10%SDS pH 7.2，7 μL TEMED，80 μL 10%APS。
5%分离胶：0.125 mL 40% Acrylamide，0.74 mL dH2O，0.125 mL 1.0 mol·L-1 Tris pH 6.8，0.01 mL 10%SDS pH 7.2，6 μL TEMED，6 μL 10%APS。
1×Transfer：25 mmol·L-1Tris-Cl，pH 8.1~8.5；192 mmol·L-1 glycine；20%甲醇(V/V)。
1×TBS：Tris-buffered saline，50 mmol·L-1 Tris-Cl，pH 7.5；150 mmol·L-1NaCl。
1×TBST：50 mmol·L-1 Tris-Cl，pH 7.5；150 mmol·L-1 NaCl；1 mL·L-1 Tween 20。
封闭液：含有30 g·L-1脱脂奶粉(BIO-RAD)的1×TBST。
1.2 方法
1.2.1 病毒的纯化
取保存的样品120 g，剪碎后于液氮中研磨成粉，加入10倍体积1×PBS (pH 7.4)，于玻璃匀浆器冰浴匀浆后，4℃，10 000×g离心10 min，上清用Beckman SW28转子于4℃，150 000×g离心45 min，沉淀用1×PBS重悬。将病毒重悬液铺于20%，35%，50% (W/V，sucrose：1×PBS) 不连续蔗糖梯度上，用Beckman SW41转子4℃，100 000×g离心1 h。用Beckman回收器在35%~50%蔗糖梯度中间收集病毒带(收集到样品1~13号)，加适量1×PBS重悬后, 各回收产物用Beckman SW50转子于4℃，150 000×g离心45 min，沉淀用氯化铯(ρ﹦1.32，溶于1×PBS)重悬，用Beckman SW41转子于4℃，100 000×g离心12 h，各沉淀颗粒保存于-85℃的超低温冰箱中。用紫外测定仪, 在波长254 nm处测定各纯化产物的光吸收值(表 1，图 1)。用折射仪测定各个样品的折射指数值η，然后用公式ρ=10.2402η-12.6483计算氯化铯的密度值(表 2)。
表 1 纯化产物在254 nm紫外线波长的吸收峰Table 1. Ultraviolet absorbance of purified products at 254 nm wave length样品
sample吸收值
absorbance样品
sample吸收值
absorbance1 733.0 8 520.0 2 1039.5 9 770.0 3 783.0 10 766.5 4 666.0 11 597.0 5 633.0 12 598.0 6 615.0 13 995.5 7 590.0 ![]()
图 1 纯化产物在254 nm紫外线波长的吸收峰Figure 1. Ultraviolet absorbance of purified products at 254 nm wave length表 2 纯化产物的折射指数和密度值Table 2. Refractive index and density data of purified products样品
sample折射指数η
refractive index密度ρ
density1 1.3700 1.3809 2 1.3695 1.3755 3 1.3689 1.369 4 1.3675 1.3538 5 1.3665 1.3428 6 1.3661 1.3385 7 1.3658 1.3352 8 1.3648 1.3243 9 1.3645 1.3211 10 1.3640 1.3158 11 1.3635 1.3102 12 1.3630 1.3049 13 1.3610 1.2723 1.2.2 病毒衣壳蛋白的Western-blot分析
取纯化样品1~13号各75 μL，加入25 μL 4×Laemmli buffer，充分混合，在99℃中热变性5 min，然后取20 μL进行电泳。先在电压80 V下电泳，待样品跑到同一直线时，改用120 V电泳，直到样品条带刚刚跑出分离胶为止。标准蛋白分子量采用BenchMark (Invitrogen)。电泳完毕后，用转膜仪(BIO-RAD)以半干法将蛋白质转移到硝化纤维素膜PROTRAN (Schleicher & Schuell, Germany)上，12V转移1 h。膜用封闭液封闭30 min，然后用兔抗巨石斑神经坏死病毒衣壳蛋白血清(第一抗体)反应1 h，用1×TBST溶液洗去非特异性结合的第一抗体，共3次，每次5 min。再用山羊抗兔辣根过氧化酶藕联抗体(Dianova，Germany)反应30 min，用1×TBST洗去非特异性结合的第二抗体，共3次，每次5 min。以上反应在室温中进行。用显色剂SuperSignal(Perbio Science, Germany)进行显影、定影，拍照。
2. 结果
2.1 纯化产物的紫外吸收
纯化产物在254 nm紫外线波长处有吸收峰。
2.2 纯化产物的密度
各纯化产物的折射指数和密度值见表 2。
2.3 病毒衣壳蛋白的Western-blot分析
在8、9、10、11四个纯化样品中检测到病毒的衣壳蛋白，与紫外吸收光谱的结果基本一致。根据氯化铯密度值，病毒粒子(包含空病毒粒子)的密度范围为：1.3102~1.3243 g·cm-3。病毒的衣壳蛋白含有2条带，大小分别为37和31 kDa(图 2)。通过成像扫描系统分析，小带的含量约为大带的10倍。
3. 讨论
为了研究病毒的本质并对病毒性疾病进行诊断，必须对病毒颗粒的物理化学性质和病毒遗传物质的结构进行研究，需要高度纯净的病毒制品。利用超速离心法提纯各种大小的颗粒和病毒是一种有效的手段，主要有差速离心法、密度梯度离心法、等密度梯度离心法等[11]。在鱼类诺达病毒的研究中，通常将上述3种超速离心法结合起来，对病毒进行纯化。Breuil等[12]采用差速离心和氯化铯(25%~38%，W/W)密度梯度离心，纯化了欧洲鲈(Dicentrarchus labrax) 脑炎病毒。经氯化铯梯度离心可见2条病毒带，浮力密度为1.30~1.34 g ·cm-3。Comps等[2]同样采用差速离心和氯化铯(25%~38%，W/W)密度梯度离心对感染尖吻鲈(Lates calcarifer)和欧洲鲈的2种鱼类脑炎病毒进行了纯化分析。完整病毒粒子在氯化铯中的浮密度为1.35~1.36 g·mL-1。Mori等[1]采用差速离心、蔗糖(10%~40%，W/V)密度梯度离心和氯化铯(30%~40%，W/W)等密度梯度离心纯化了3 mm拟鲹仔鱼的神经坏死病毒。Chi等[13]用差速离心和氯化铯(10%~40%，W/W)密度梯度离心对经GF-1(来源于斜带石斑鱼鳍细胞)细胞培养的石斑鱼神经坏死病毒(GNNV)进行纯化。可见，不同种类的宿主、同一宿主不同生长阶段的个体所感染的鱼类诺达病毒和不同方法制备的鱼类诺达病毒需用不同的方法和条件进行纯化。本文借鉴了不同的纯化方法，经过反复摸索和改进，纯化了赤点石斑鱼诺达病毒大亚湾株(RG-CN)，测定其浮密度为1.3102~1.3243 g·cm-3，与上述研究结果相一致。
在病毒结构蛋白的分析中经常采用SDS-PAGE和Western-blot法。而后者因用抗原抗体反应检出蛋白质，因此灵敏度、特异性都很高，是特定蛋白质分子高灵敏检出的方便而有效方法。Mori等[1]用SDS-PAGE的考马斯亮蓝染色法对纯化的拟鲹神经坏死病毒结构蛋白进行分析，得到2条蛋白带，分别为42和40 kDa。Comps等[2]对感染尖吻鲈和欧洲鲈的纯化脑炎病毒也采用SDS-PAGE的考马斯亮蓝染色法进行分析。结果表明这2种病毒粒子的结构多肽各有2种，尖吻鲈脑炎病毒的结构多肽分子量为42、36 kDa，欧洲鲈脑炎病毒的为42、40 kDa。Chi等[13]也用同样的方法分析了纯化的石斑鱼神经坏死病毒，得到分子量为43和41 kDa的结构蛋白带。Hegde等[14]用Western-blot法对感染SB(sea bass)细胞的纯化巨石斑神经坏死病毒的结构蛋白的分析表明，只有1条约42 kDa的主带。由此可见，不同来源的鱼类诺达病毒的结构蛋白大小不尽相同。本文采用Western-blot法对纯化的赤点石斑鱼诺达病毒的结构蛋白进行分析，所用的一抗为兔抗巨石斑神经坏死病毒衣壳蛋白血清，这2种病毒的衣壳蛋白(结构蛋白)均由338个氨基酸组成，其氨基酸同源率达99%[9]，因此本文所用的一抗能很好地识别所检测的结构蛋白。结果赤点石斑鱼诺达病毒的衣壳蛋白含有2条带，大小分别为37和31 kDa，小带的含量较大带多，说明衣壳蛋白以31 kDa的组分为主，与上述几种鱼类诺达病毒的结构蛋白大小不同，这和纯化病毒的浮密度差异相一致。
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