卵形鲳鲹 JAK3 蛋白的原核表达与纯化

【目的】纯化获取卵形鲳鲹(Trachinotus ovatus)JAK3(TroJAK3)重组蛋白,为了解TroJAK3蛋白功能、相互作用及抗体制备奠定基础。【方法】应用分子生物学技术构建重组质粒pET-32a-TroJAK3,转化大肠杆菌BL21感受态细胞,经IPTG诱导表达,SDS-PAGE和Western blot检测TroJAK3重组蛋白表达情况。【结果】PCR扩增片段长度为3 333 bp,经双酶切鉴定、测序确认序列和开放阅读框正确,结果显示成功构建重组质粒pET-32a-TroJAK3。经终浓度为1 mmol/L IPTG 37℃诱导4 h后进行SDS-PAGE检测,结果表明TroJAK3重组蛋白以包涵体形式大量表达,分子量约为140 ku。经Ni-IDA树脂柱纯化,获得纯化的重组蛋白。Western blot检测结果显示有140 ku的条带,表明TroJAK3重组蛋白能被抗His抗体识别。【结论】成功构建了重组质粒pET-32a-TroJAK3,纯化得到高纯度的TroJAK3融合蛋白。     

羽叶决明代木栽培鸡腿菇的物质转化

研究羽叶决明(Chamaecrasta nictitans)代木栽培鸡腿菇(Coprinus ovatus)对其绝对生物学效率、基物失重、呼吸消耗及木质纤维素转化的影响.结果表明,羽叶决明牧草替代60%木屑(A60)栽培鸡腿菇的绝对生物学效率最高,均为10.77%.覆土前和采收后的基物失重和呼吸消耗都以A0(CK)为最高,分别为11.47%、11.47%和31.75%、25.55%,其木质素转化率也最高,为79.21%;纤维素转化率以A100(羽叶决明牧草替代100%木屑)为最高,达50.93%;半纤维素转化率以A40(羽叶决明牧草替代40%木屑)为最高,为49.74%.回归分析结果表明,绝对生物学效率和半纤维素转化率与替代比例呈抛物线相关,而基物失重、呼吸消耗及木质素、纤维素转化率与替代比例呈线性相关.     

Nutrients removed by Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in integrated cultivation with fishes in re-circulating water

The potential of the red alga Kappaphycus alvarezii to remove nutrients was tested to treat effluents of Trachinotus carolinus fish cultivation, and the production of carrageenan in this condition was analyzed. Experiments were conducted in four tanks of 8000 L with approximately 1200 fishes of 30 g each integrated with three tanks of 100 L with 700 g of K. alvarezii, as initial biomass per tank. Seawater was re-circulated between tanks with seaweed and with fish. As a control, three tanks with seawater circulating in an open system were utilized. Seawater samples were collected daily for 10 days and concentrations of nitrate, nitrite, ammonium and phosphate were determined in the inflow and outflow water of the tanks. Significant differences between both collecting points were considered as nutrient removal by the seaweed. Growth rates and carrageenan yields were also analyzed in seaweed cultivated in seawater and in effluents. Growth rates of seaweed cultivated in tanks were lower than those obtained in open sea and in laboratory cultivation. Effluents had concentrations of nitrate and nitrite ca. 100 times higher than in the control. Maximum values of nutrient removal on effluents were: nitrate = 18.2%; nitrite = 50.8%; ammonium = 70.5% and phosphate = 26.8%. All plants survived throughout the experimental period, but some developed “ice–ice”, a disease associated with physiological stress. After the experimental period, some plants selected and cultivated in open sea presented higher growth rates in 40 days, indicating nutrient storage. No significant differences between carrageenan yields of K. alvarezii cultivated in seawater and in the effluents were observed. Our results show that K. alvarezii can be utilized as a biofilter for fish cultivation effluents, reducing the eutrophication process and can also be processed for carrageenan production, which provides an additional benefit to the fisheries.     

二种佐剂对哈氏弧菌OMPC-溶藻弧菌LPS偶联疫苗的增效作用

制备哈氏弧菌SpGY020601外膜蛋白复合物(OMPC)和溶藻弧菌EpGS021001脂多糖(LPS)偶联疫苗,设置偶联疫苗组、偶联疫苗 ISA763佐剂组、偶联疫苗 β-葡聚糖佐剂(DEAE)组以及生理盐水对照组,并以相同程序免疫卵型鲳鲹。溶菌酶活性检测、微量凝集反应试验结果显示:各疫苗组血清中的溶菌酶含量无显著差异(P>0.05),但均远高于生理盐水对照组(P<0.05);ISA763佐剂组抗哈氏弧菌SpGY020601抗体滴度较其他组出现的早,抗溶藻弧菌EpGS021001抗体滴度较其他组持续时间长;β-葡聚糖佐剂组次之,其次是无佐剂组,对照组始终未检测到抗体。各组分为腹腔注射攻毒与浸泡攻毒,对EpGS021001的攻击,ISA763佐剂组相对保护率分别为90%和70%,高于β-葡聚糖佐剂组的85%和55%,以及无佐剂组的80%和55%;对SpGY020601的攻击,ISA763佐剂组相对保护率分别为95%和80%,高于或相当于β-葡聚糖佐剂组的95%和75%,以及无佐剂组的90%和75%。     

卵形鲳鲹消化道的形态学、组织学和组织化学

采用形态解剖、组织学和组织化学方法,研究了卵形鲳鲹Trachinotus ovatus消化道结构的特点。结果表明：卵形鲳鲹口咽腔较小,上下唇有许多绒毛状突起;上下颌无齿,鳃耙内侧具有绒毛状细齿,口咽腔具有多种类型齿;食道粗短,内表面分布有许多较为宽大的黏膜褶;胃呈＂U＂形,盲囊部明显,贲门部和盲囊部具有丰富的胃小凹和胃腺;前、中、后肠黏膜褶丰富,后肠黏液细胞密度最大,肠长比为0.61;幽门盲囊细长,有25～35条。消化腺为肝胰脏。食道以Ⅰ型黏液细胞为主,贲门部以Ⅰ型和Ⅲ型黏液细胞为主,盲囊部、幽门部、前肠、中肠和幽门盲囊以Ⅲ型黏液细胞为主,后肠以Ⅱ型黏液细胞为主。     

卵形鲳鲶消化酶活性的研究I．成鱼和幼鱼消化酶活性在不同消化器官中的分布及其比较

比较研究了卯形鲳够（Trachinotus ovatus）成鱼和幼鱼阶段消化酶（蛋白酶、淀粉酶、脂肪酶）在不同消化器官中的活性。结果表明：1）成鱼蛋白酶在不同消化器官中的活性大小依次为胃〉前肠〉中肠〉幽门盲囊〉后肠〉肝;淀粉酶活性为前肠〉后肠〉幽门盲囊〉中肠〉肝〉胃;脂肪酶活性为前肠〉中肠〉后肠〉幽门盲囊〉肝〉胃。2）幼鱼蛋白酶在不同消化器官中的活性大小依次为胃〉肠〉幽门盲囊〉肝;淀粉酶活性为肠〉幽门盲囊〉肝〉胃;脂肪酶活性为肠〉幽门盲囊〉肝〉胃。3）成鱼不同消化器官中蛋白酶和淀粉酶的活性均小于幼鱼,成鱼胃和幽门盲囊的淀粉酶活性与幼鱼的差异显著;幼鱼胃脂肪酶活性大于成鱼,但其他器官的活性均小于成鱼。卵形鲳鳕幼鱼不同消化器官中的3种消化酶活性大小顺序与成鱼基本相似。     

Development of plant‐based diets and the evaluation of dietary attractants for juvenile Florida pompano,Trachinotus carolinus L.

Sustainable and profitable commercial aquaculture production of marine fish species is dependent on the development of sustainable protein sources as substitutes for expensive animal meals such as fishmeal (FM). Previous Florida pompano Trachinotus carolinus studies have indicated that poultry by‐product meal (PBM) and meat and bone meal can be used to produce a FM‐free diet if suitable levels of nutrients (such as taurine) are included in the diets. In this study, we attempted to develop an all‐plant protein diet by removing the animal proteins in practical diets for pompano by substituting back select ingredients. A series of eight FM‐free diets were formulated, four systematically replaced soybean meal (SBM) with soy protein concentrate (SPC) and four replaced PBM with SPC. Based on the results, there is no clear disadvantage to the use of SPC as a replacement for SBM. However, the complete removal of PBM resulted in reduced performance. Two additional growth trials were conducted to supplement additional amino acids including glycine, valine and histidine, a proprietary chemical attractant mix, fish protein concentrate and squid hydrolysate to improve the growth of pompano when fed all‐plant protein diets. The only improvement in performance occurred with the squid hydrolysate. These results demonstrate that using soybean meal, soy protein concentrate and corn gluten meal as the primary protein sources, a plant‐based feed formulation can be developed, but the removal of all animal proteins is not yet feasible.     

卵形鲳鲹幼鱼饲料中适宜蛋白能量比的研究

以鱼粉、豆粕为蛋白源,豆油、鱼油为脂肪源,配制9种不同蛋白能量水平的饲料,蛋白质3个水平(38%、43%、49%),每个蛋白水平设3个脂肪梯度(6%、10%、14%),其蛋白能量比为19.3～27.4mg/kJ。卵形鲳鲹初始质量为(25.02±0.16)g,每个处理设3个重复,每个网箱(1.5 m×1.0 m×2.0m)放养卵形鲳鲹苗20尾,养殖试验持续8周。结果显示,卵形鲳鲹特定生长率随饲料蛋白水平的升高而显著升高(P<0.05)。投喂含43%或49%蛋白质、6%脂肪饲料的卵形鲳鲹表现出较好的特定生长率。饲料中不同的脂肪水平未表现出蛋白质节约效应,较高的脂肪水平反而抑制了卵形鲳鲹的特定生长率、饲料效率和蛋白质效率。卵形鲳鲹鱼体蛋白和脂肪含量分别随饲料蛋白质和脂肪水平的上升而显著升高(P<0.05)。在该试验条件下,卵形鲳鲹幼鱼饲料中最适蛋白质、脂肪水平和蛋白能量比分别为43%、6%和24.4 mg/kJ。     

饲料盐藻水平对卵形鲳鲹体色和生长的影响

以初始体质量为(13.88±0.28)g的卵形鲳鲹为试验对象,研究不同盐藻添加量(0.0%、0.1%、0.2%、0.4%、0.8%、1.6%)饲料对其体色和生长的影响。每组设3个平行,每个平行饲养20尾鱼,每日饱食投喂2次(8:00;17:00),试验周期为56d。结果显示,盐藻添加量为0.1%~1.6%时,随着饲料中盐藻水平的提高,卵形鲳鲹背部和腹部黄色值(b*)呈显著上升趋势(P0.05),饲料中添加盐藻可明显改善卵形鲳鲹体色;与此同时,卵形鲳鲹质量增加率和特定生长率随饲料盐藻水平的上升呈下降趋势(P0.05),未添加盐藻的对照组具有最高蛋白质效率和最低饲料系数,且与盐藻添加量为0.4%的试验组差异显著(P0.05),饲料中添加盐藻对卵形鲳鲹生长性能和饲料利用没有明显的促进作用;此外饲料盐藻水平对卵形鲳鲹体成分也没有显著影响(P0.05)。     

卵形鲳鲹TLR13基因的克隆、表达分析和亚细胞定位研究

Toll样受体(Toll-like receptor, TLR)是一种古老的先天性免疫受体,参与病原体相关分子模式识别,对维持免疫稳态和预防感染至关重要。本研究克隆和鉴定了卵形鲳鲹(Trachinotus ovatus) TLR13基因(命名为ToTLR13),其开放阅读框(ORF)为1 269 bp,编码422个氨基酸,等电点为8.13。保守结构域分析显示,ToTLR13含有跨膜结构域(TM)、LRR结构域和TIR结构域,符合TLR家族的典型特征。通过建立TLR13保守域三级结构发现,ToTLR13与小鼠(Mus musculus)和大黄鱼(Larimichthys crocea) TLR13功能结构域的蛋白三级结构具有较高重叠性。多序列比对显示,ToTLR13与其他硬骨鱼TLR13具有较高的相似性,与其他纲物种的序列相似性较低。系统进化树结果显示,ToTLR13与硬骨鱼TLR13聚在一起,其中与鞍带石斑鱼(Epinephelus lanceolatus)最为接近,与哺乳动物、两栖类和贝类相分离。实时荧光定量PCR (Real-time fluorescence quantitative PCR, RT-qPCR)分析显示,ToTLR13在健康卵形鲳鲹的心、鳃、肾、头肾、肝、脾、脑和肌肉中普遍表达,其中鳃的表达量最高,其次是脾。ToTLR13在其鳃、脾、肝和肾免疫相关组织中的表达情况呈现出不同程度的上调,提示其经无乳链球菌(Streptococcus agalactiae)和溶藻弧菌(Vibrio alginolyticus)免疫刺激后可能激活了炎症反应,启动了先天性免疫反应。亚细胞定位显示,ToTLR13定位于A549细胞质。本研究表明,ToTLR13在抵御病原菌免疫应答过程中可能发挥重要的作用,研究结果可为阐明脊椎动物TLRs的功能进化史提供基础资料。