赤点石斑鱼神经坏死病毒MCP基因原核表达条件优化(英文)

[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.     

不同饵料对点带石斑鱼幼鱼生长、营养成分及组织消化酶活性的影响

将点带石斑鱼幼鱼(15±0.5) g,随机分为3组,每组投喂不同饵料,分别为鱼肉组、配合饲料组和混合饵料组(小杂鱼和配合饲料交替投喂),每组3个重复,养殖6周后测定各组幼鱼生长指标、全鱼营养成分、肠胃消化酶活性及肝脏磷酸酶活性以研究这3种饵料对点带石斑鱼幼鱼生长、消化功能及全鱼营养成分的影响。结果表明:鱼肉组和混合饵料组的相对增重率和特定生长率均显著高于配合饲料组(P<0.05)。肠、胃蛋白酶活性呈现鱼肉组>混合饵料组>配合饲料组的趋势且差异显著(P<0.05),肠淀粉酶活性趋势相反且差异显著(P<0.05);各组肠、胃脂肪酶活性之间没有显著差异(P>0.05);鱼肉组肝脏碱性磷酸酶活性显著高于配合饲料组(P<0.05),鱼肉肝脏组酸性磷酸酶活性显著高于混合饵料组和配合饲料组(P<0.05)。各组全鱼水分和灰分含量没有显著差异(P>0.05),粗蛋白含量为鱼肉组>混合饵料组>配合饲料组且差异显著(P<0.05);粗脂肪含量趋势相反,为鱼肉组<混合饵料组<配合饲料组且差异显著(P<0.05)。因此,小杂鱼在各方面都要优于配合饲料,交替投喂小杂鱼和配合饲料是一种较好的投喂方式。     

Immunization of grouper, Epinephelus coioides, confers protection against a protozoan parasite, Cryptocaryon irritans

The present study aimed to determine whether protection is conferred by immunization of grouper, Epinephelus coioides, against a protozoan parasite, Cryptocaryon irritans. The immunization of E. coioides was carried out by a low level exposure of fish to live C. irritans theronts from predetermined number of tomonts and by an intraperitoneal injection of a vaccine consisting of formalin-killed C. irritans theronts.

Mucus titers detected by ELISA were significantly higher in fingerling and adult grouper subjected to the low level of exposure to C. irritans theronts at 3-week post-exposure compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced from adult grouper after three successive exposures than the tomonts produced after a single exposure to the parasite.

In the vaccine-immunization experiment, no mortality was monitored in fish that received high dose vaccine (100 μg/fish), while 40% cumulative mortality and 100% cumulative mortality were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively. In the succeeding replicate, the vaccine-immunized group (high dose) had 37.5% cumulative mortality and 100% cumulative mortality for the control. In addition, a total of 1830 tomonts were collected at 5-day post-challenge from the control group while none from the vaccine-immunized group. Significantly fewer trophonts and tomonts were enumerated at 5-day and 7-day post-challenge, respectively, in the vaccine-immunized group than the control.

Results suggest that a protective immunity has been conferred on the immunized grouper as indicated by high antibody titers in the mucus of C. irritans-exposed fish and higher survival and fewer parasites in vaccine-immunized fish than the control groups. The conferred immunity played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts on the skin of the immunized grouper.     

循环水养殖密度和氨氮对斜带石斑鱼生长和免疫力的影响

选取平均体重为146.3 g/尾的斜带石斑鱼450尾分成3个密度组进行饲养,分别为低密度组D1(100尾/m³,14.6 kg/m³)、中密度组D2(150尾/m³,21.7 kg/m³)和高密度组D3(200 尾/m³,29.5 kg/m³),比较不同养殖密度引起的水质变化对斜带石斑鱼的生长性能、摄食率和免疫力的影响。结果表明：水体中氨氮含量随斜带石斑鱼养殖密度的增加而增加(P<0.05);斜带石斑鱼的摄食率随水体中氨氮浓度的增加而显著下降(P<0.05)。随着养殖密度的增加,斜带石斑鱼的生长速度呈现负相关性,各试验组的特定生长率分别为1.07、0.66、0.47;各实验组斜带石斑鱼体重也出现显著性差异(P<0.05),各实验组的尾均增重量分别为118.5 g、63.3 g和44.1 g;养殖密度对斜带石斑鱼的饵料系数和成活率无显著影响(P>0.05);斜带石斑鱼的免疫指标血清溶菌酶(LZM)、碱性磷酸酶(AKP)和超氧化物歧化酶(SOD)随养殖密度的增加而下降,随养殖时间的延长呈现出先上升后下降的趋势。阐明了养殖密度引起养殖水质状况的改变,继而影响斜带石斑鱼摄食率的变化,从而造成石斑鱼的生长性能出现显著性差异,可能是养殖密度对斜带石斑鱼生长性能影响的机制之一。     

Maximum prey size estimation of longtooth grouper,Epinephelus bruneus,using morphological features,and predation experiments on juvenile cannibalism

In fish culture, cannibalism, which induces critical damage, is separated into two types: type I, which occurs in early larval stages when size heterogeneity is low; and type II, which starts in later larval and/or early juvenile stages following type I when size heterogeneity is high. In type II, the cannibal's maximum prey size is limited by its gape size relative to the prey fish body size. Ratios of prey to cannibal body size changes are associated with allometric factors and feeding behaviour with growth. In a previous study on grouper, the maximal prey size was estimated morphologically (i.e., the relationship between gape size and body height). This method is very simple and useful; however, it does not consider allometric factors and feeding behaviour changes; therefore, it probably causes a large estimation error. To overcome this, we present a new method for estimating maximal prey size to determine the size ranges for size grading of longtooth grouper, Epinephelus bruneus, juveniles using predation experiments. It is shown that grouper type II cannibalism is affected by two factors; in early juvenile stages by morphology and in later (TL over 50 mm) by prey size selectivity.     

荧光定量PCR法检测点带石斑鱼神经坏死病毒研究

根据己公布的神经坏死病毒全基因序列,应用引物设计软件依据Gene Bank所收录的各种石斑鱼神经坏死病毒RNA2基因组全序列选择保守区域,根据Taqman探针引物设计原则,设计Taq Man探针及其引物。采用Taq Man探针技术,并且将假病毒技术构建的含有神经坏死病毒RNA2基因组的MS2噬菌体假病毒作为阳性质控品,建立Taq Man探针检测点带石斑鱼神经坏死病毒的荧光定量PCR方法,拟合标准曲线,能够在病毒检测中进行绝对定量,能检测到10个病毒拷贝模板数,在临床检测中得到很好运用。本研究建立实时荧光定量RT-PCR,有助于日常检验工作,为鱼类神经坏死病毒的筛选和监测提供基础,有助于控制神经坏死病的疫情传播。     

急性低盐胁迫对斜带石斑鱼幼鱼存活、血清离子浓度和内分泌激素水平的影响

为探究急性低盐胁迫对斜带石斑鱼(Epinephelus coioides)幼鱼存活、血清离子浓度和激素水平的影响,将斜带石斑鱼幼鱼从盐度30(对照组)的水体直接转移至盐度0、5、10、20的水体中,于2 h、6 h、12 h、48 h和72 h检测幼鱼血清中的钠离子(Na~+)、钾离子(K~+)、氯离子(Cl~-)浓度和生长激素(GH)、催乳素(PRL)、皮质醇(COR)水平的变化,并记录幼鱼存活情况。结果显示,血清Na~+和Cl~-浓度随盐度增加显著上升(P0.05)。盐度0组和对照组血清Na~+浓度随实验时间变化不大,较稳定(P0.05);而盐度5和盐度10组血清Na~+浓度在6 h降低,随后升高并保持稳定(P0.05);盐度20组血清Na~+浓度随时间的延长出现显著波动(P0.05)。随时间延长Cl~-浓度在盐度0组呈显著下降趋势(P0.05),在盐度5、10和20组和对照组变化不明显(P0.05)。K~+浓度在盐度0组显著高于其它组(P0.05)。随时间延长K~+浓度在盐度0组先升后降(P0.05),而在盐度5、10和20组以及对照组则先降后升(P0.05)。GH水平在盐度20和对照组显著高于其它3组(P0.05)。随时间延长GH水平在盐度0、5和10组呈先下降至6 h处达到最低点,而后上升的趋势(P0.05),而在盐度20和对照组无显著变化(P0.05)。PRL在各试验组显著高于对照组(P0.05)。随时间延长各试验组血清PRL水平先下降后上升至12 h达到最大值后又下降(P0.05),最后趋于稳定。COR水平在盐度0、5和10组显著高于盐度20和对照组(P0.05)。随时间延长在盐度0、5和10组的变化规律与PRL水平类似,而在盐度20和对照组无显著变化(P0.05)。随时间延长在盐度0组幼鱼死亡率逐渐升高,72 h内全部死亡;盐度5组幼鱼在实验期间仅有极少数死亡,而其它组幼鱼无死亡情况,这表明斜带石斑鱼幼鱼能够适应在盐度低至5的急性胁迫下存活,但在淡水急性胁迫条件下不能长时间存活。     

盐度与pH对云纹石斑鱼胚胎发育和仔鱼活力的影响

为探讨盐度、pH对云纹石斑鱼Epinephelus moara胚胎发育和仔鱼活力的影响,采用单因子实验的方法,在水温22±0．2℃条件下,将受精卵在不同盐度（5、10、15、20、25、30、35、40和45）、不同pH（5．5、6．0、6．5、7．0、7．5、8．0、8．5、9．0和9．5）下孵化,观察比较其孵化率HR、畸形率DR以及对初孵仔鱼活力和仔鱼培育周期的影响。并在上述设定条件下,进行了初孵仔鱼耐受试验,测定其存活率SR及生存活力指数（SAI）。结果表明,云纹石斑鱼胚胎发育的最低和最高临界盐度分别为10和45,最低和最高临界PH分别为6．O和9．5;盐度、PH的适宜范围分别是20～40、6．5～9．0,最适范围分别是30～35、7．5～8．0。仔鱼生存盐度、pH的适宜范围分别是20～35、6．0～9．0,最适范围分别是25～30、7．5～8．0。     

Effects of dietary live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.