Molecular cloning and expression analysis of interleukin - 1β from Japanese sea perch(Lateolabrax japonicus )

The technology of homology cloning and anchored PCR was used to clone the IL-1β gene from the Japanese sea perch （Lateolabrax iaponicus）. The full-length cDNA of sea perch IL-1β was 1 310 bp, including a 5＇ untranslated regiop （UTR） of 136 bp, a 3＇ UTR ot 430 bp, and an ORF of 774 bp encoding a polypeptide of 258 amino acids with an estimated molecular mass of 29.31 kDa. The searches for nucleotides and protein sequence similarities with the BLAST analysis indicated that the deduced amino acid sequence of sea perch IL-1β was homological to the IL-1β in other fish species and even the mammalian. Conserved signature sequences of the IL-1β gene family were found in the sea perch IL-1β deduced amino acid sequence. Temporal expressions of the IL-1β gene in LPS or iridovirus challenged group and in control group were measured by the semi-quantitative RT-PCR. The mRNA transcripts of IL-1β could be detected in head-kidney, spleen, liver, gill and heart of the healthy individuals, and the expression level of IL-1β in head-kidney, spleen and gill was higher than that in liver and heart, but it was hard to be detected in the brain. After being stimulated by the LPS or iridovirus, the IL-1β expression in most of examined tissues was up-regulated, and also could be detected in the brain. These results indicated that the expression of sea perch IL-1β was constitutive and could be up-regulated by immune effector stimulation. Therefore the sea perch IL-1β could play a critical role in the host-pathogen interaction.

Comparison for ecological economic performance of Chinese sea perch(Lateolabrax Maculatus)under different aquaculture systems

Chinese sea perch(Lateolabrax Maculatus)is one of the main marine fish culture species in China,its green development has received increasing attention and its ecological economic study is rare.Based on field survey,this paper takes water pollution tax of China as conversion indicator for aquaculture eutrophication to simulate aquaculture waste fee for further analyzing the ecological impact of Chinese sea perch’s aquaculture.For data analysis,data envelopment analysis model has been used.The comparison for Chinese sea perch’s economic performance and ecological economic performance under its main aquaculture systems has been done.The results show that:1.The simulated waste fee has no significant impact for the comparison.2.Ranking for ecological economic performance’s ecological economic,technical and scale efficiency is as follow:pond farming,offshore cage culture and cage culture.3.Offshore cage culture is the only system presenting positive increase for ecological economic performance,which makes its aquaculture activity more sustainable than other systems.4.The key point of green development is to maximize the ecological economic efficiency of main aquaculture systems.Thus,offshore cage culture is the most sustainable aquaculture system among China’s current marine fish culture systems,and other systems need innovation for achieving the green development of China’s aquaculture.

鲈鱼胚胎的玻璃化冷冻保存

本文对鲈鱼 (Lateolabraxjaponicus)胚胎进行了玻璃化冷冻保存研究 ,筛选出了浓度较低、玻璃化程度较稳定的 5种玻璃化液 ,冷冻时形成玻璃化的概率在 4 8 1%～ 10 0 % ,在 35～ 4 3℃的水浴中解冻时保持玻璃化的概率在 4 4 4 %～ 6 3 0 % ;玻璃化液VSD2在解冻时保持玻璃化的概率最高。对鲈鱼神经胚、 2 0对肌节胚、尾芽胚、心跳胚、出膜前胚在玻璃化液VSD2中的适应能力及适合于玻璃化冷冻的胚胎时期进行了比较 ,结果显示 :不同时期胚胎对玻璃化液的耐受能力不同 ,鲈鱼神经胚耐受能力最低 ,心跳胚耐受能力最强 ,出膜前期胚次之 ,心跳胚和出膜前胚适合于进行玻璃化冷冻。对 0 5mol/L蔗糖的洗脱时间进行了选择 ,结果显示 ,洗脱10～ 2 0min效果较好。利用玻璃化程度较好的VSD2对鲈鱼不同时期胚胎进行超低温 ( - 196℃ )冷冻 ,获得了2 1%～ 2 7 9%的透明胚。将鲈鱼心跳胚冷冻解冻后获 2粒复活胚 ,培养至出膜期 ,成活 4 2～ 5 0h ;出膜前期胚在冷冻解冻后有 1粒胚复活 。

海水网箱养殖鲈鱼皮肤溃疡病的防治药物

本文研究了海水养殖鲈鱼皮肤溃疡病病原菌—哈氏弧菌 (Vibrioharveyi)对 2 4种化学疗剂及 1 5种中草药的敏感性。结果表明 :环丙沙星、氯霉素、庆大霉素、头孢孟多、磺胺 +TMP、磺胺甲基异恶唑等 6种化学疗剂及地榆、石榴皮、五味子等 3种中草药抑菌作用较强 ,在此基础上进行了药物联用效果试验。

应用间接ELISA技术快速检测花鲈病原菌-鳗弧菌

以花鲈 (Lateolabraxjaponicus)弧菌病的病原菌———鳗弧菌 (Vibrioauguil larum)W 1为抗原 ,制备兔抗血清 ;利用辣根过氧化酶标记的羊抗兔血清 (HRP IgG)为酶标二抗 ,建立了检测鳗弧菌的间接ELISA快速检测法。结果表明 ,应用间接ELISA技术检测鳗弧菌有较高的灵敏度 ,最低的检测量为 10 5个 /ml,即 10 4 个 /孔。同时交叉反应表明其具有较高的特异性。对 4 6份人工感染后的花鲈组织样品 ,包括肌肉、鳃、肠、肝、肾等组织的检测表明 ,阳性检测率为 76 .1%。

鲈鱼鳗弧菌病疫苗的制备及免疫防治效果

以从患出血、烂尾病的鲈鱼 ( L ateolabrax japonicus)中分离的 1株致病性鳗弧菌 W- 1( Vibrio anguillarum)为材料 ,进行了鳗弧菌全细胞灭活疫苗研制 ,比较了福尔马林、苯酚、氯仿等不同灭活剂在不同温度下的灭活效果。最终确定以 0 .5 % ( v/ v)的福尔马林溶液在 2 8℃下处理 4 8h为最佳灭活条件 ,所用的菌悬液浓度为 2 .5× 10 9cfu/ m L。用福尔马林灭活的 W- 1( Vibrio anguil-larum)疫苗 ,分别浸泡免疫鲈鱼苗和注射成鱼 ,并检测了疫苗对鱼体的免疫保护力。结果表明 ,经疫苗浸泡免疫 4周后的鲈鱼苗免疫保护力达 64.7% ,90 d后同批次的鲈鱼再注射免疫 ,其免疫保护力为 78.9% ,血清中抗体凝集效价为 1∶ 192。仅进行注射免疫组 ,其免疫保护力为 5 7.9% ,血清中抗体凝集效价为 1∶ 2 2 4。

低盐度可诱导鲈鱼胞浆型PEPCK基因表达

磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达.

鲈鱼口服生物胶囊疫苗的研究

以暴发弧菌病的鲈鱼 (Lateolabraxjaponicus)鱼苗体内分离的 1株病原菌W 1(Vibrioanguillarum)为材料 ,分别以直接浸浴法、创伤后浸浴法和肌肉注射法人工感染鲈鱼鱼苗。结果表明 ,这三种感染方式均能使鲈鱼发病 ,其半致死浓度 (LD50 )分别为2 75× 10 7cfu/ml ,4 .68× 10 6cfu/ml和 3.72× 10 5cfu/尾。在此基础上 ,分别以福尔马林灭活法和喷雾干燥法制备了全细胞疫苗和微胶囊疫苗。将全细胞疫苗及微胶囊疫苗以直接拌入饵料法和卤虫携带法等两种方法口服免疫接种鲈鱼鱼苗 ,一周后以W 1活菌攻毒( 2 50× 10 6cfu/尾 )。结果表明 ,各免疫组一周的累积死亡率远低于两组对照组 ( 95% )。其中 ,以卤虫携带的生物微胶囊疫苗组的一周累积死亡率最低 ,为 2 5.0 % ;以卤虫携带的生物全细胞疫苗组的一周累积死亡率较高 ,为 60 .0 %。因而 ,以卤虫携带的生物胶囊疫苗组的免疫保护力最高 ,为 73.7% ,其次为微胶囊疫苗直接投喂组 ,保护力为 56.8%。直接投喂和卤虫携带法的全细胞疫苗也显示了一定的免疫效果 ,其免疫保护力分别为 52 .6%和 36.8%。

应用间接荧光抗体技术快速检测花鲈病原菌——鳗弧菌

以花鲈 (Lateolabrax japonicus)弧菌病的病原菌——鳗弧菌 (Vibrio anguillarum) W-1为抗原 ,制备兔抗血清 ;利用异硫氰酸荧光素标记的羊抗兔免疫球蛋白 (FITC-Ig G)为荧光标记二抗 ,并以罗丹明标记的牛血清白蛋白为背景染色 ,建立检测鳗弧菌的间接荧光抗体快速检测技术。应用该技术对人工感染后的花鲈组织 (肌肉、鳃、肠、肾 )样品和养殖水体样品进行了鳗弧菌检测 ,结果显示间接荧光抗体技术不仅可以用于诊断发病的感染花鲈 ,也可用于检测带菌状态或未发病的感染花鲈。

控制海水网箱养殖鲈鱼皮肤溃疡病的药物研究

研究了海水养殖幼鱼皮肤溃疡病病原菌──哈氏弧菌 Ｖ ｈａｒｖｅｙｉ对２４种化学疗剂及１５种中草药的敏感性，结果表明：环丙沙星、氯霉素、庆大霉素、头孢孟多、磺胺＋ ＴＭＰ、磺胺甲基异恶唑等６种化学疗剂及地榆、石榴皮、五味子等３种中草药抑菌作用较强，在此基础上进行了药物联用效果试验。

海水网箱养殖鲈鱼的主要疾病与防治

通过对浙江省海水网箱养殖鲈鱼Lateilabrajaponicees疾病的调查，分别介绍了13种疾病的病原，疾病特征及防治措施。

鲈鱼胚胎程序化冷冻保存的研究

对鲈鱼(Lateolabraxjaponicus)胚胎的程序降温冷冻保存进行了研究。通过稀释液的筛选实验 ,选择了一种培养效果好的稀释液DS1液。研究了不同发育阶段胚胎对抗冻液的毒性耐受力 ,表明肌肉效应期胚胎的耐受力最强。测定了心跳期胚胎在抗冻液A1 、A2 、B1 、B2 中的平衡处理时间 ,为进行鲈鱼胚胎冻前平衡提供了参考时间。比较了植冰和不植冰对鲈鱼胚胎冷冻保存结果的影响及不同洗脱方法洗脱胚胎的效果。结果表明 ,在冷冻过程中诱导植冰的胚胎成活率高于未植冰组 ,两步法洗脱效果优于一步法和三步法。用不同降温速率进行了鲈鱼胚胎的超低温冷冻保存实验 ,结果表明 ,采用 1 .5℃ /min的降温速率降温 ,在液氮中冷冻保存 30min的 72粒心跳期胚胎中有 3粒成活 ,成活率为 4.2 %。

花鲈胚胎干细胞移植及嵌合体的构建

旨在通过花鲈胚胎干细胞（LJESl）移植构建嵌合体，证实LJES1细胞的体内分化能力和发育全能性。采用脂质体介导法将线性化pCMV-EGFP质粒导入到长期培养的LJES1细胞内，细胞绿色荧光蛋白（GFP）的表达率为5％～10％，以此为供体，通过显微注射方法把20—50个LJES1细胞移植到花鲈囊胚中，共移植囊胚478枚，获得了20枚表达GFP的嵌合体胚胎，其中的15枚胚胎发育成鱼苗。荧光显微观察和PCR检测显示了LJES1细胞可在宿主胚胎内片状嵌合和单细胞分散嵌合，嵌合部位可分布于胚体的三个胚层；同时也证明GFP是一种优良的遗传标记。本实验结果为证实LJES1细胞的发育全能性提供了有力证据。

重组鲈鱼生长激素的分离纯化及抗体制备

用IPTG诱导鲈鱼生长激素基因工程菌E coli RV30 8,使重组鲈鱼生长激素得以表达并分泌到周质空间。利用渗透休克法从菌液中提取间质蛋白 ,经DEAE SepharoseCL 6B、聚丙烯酰胺凝胶电泳及SephadexG 75纯化后得到单一电泳纯的重组鲈鱼生长激素 ,并将此激素作为抗原制备抗体 ,可应用于鱼类血液中生长激素含量的测定。

CHARACTERIZATION OF LACTATE DEHYDROGENASE ISOZYME PATTERN AND MORPHOLOGY OF THREE MARINE FISH CELL LINES

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder ( Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch ( Lateolabrax japonicus ) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream ( Pagrosomus major ) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns was related to morphological changes of fish cells in vitro .

野生鲈鱼亲鱼的驯养

The study of acclimation and breeder nursing of wild parent Japanese sea perch (Lateolabrax japonicus) has been done in 1994 and 1997. The result is that after 25 to 30 days of acclimation, the wild perch can be fed on trash fish for about 35 days feeding normally. The daily feeding rate is about 7.2% to 10%. The methods of induction of the wild perch feeding and how to choose the parent perch were also related in this paper.

花鲈Syntaxin 4基因及其在免疫应答反应中的表达特征

采用RT-PCR技术,从花鲈(Lateolabrax japonicus)中克隆得到膜蛋白Syntaxin 4基因近85%的CDS序列。同源性分析表明,该序列与斑马鱼以及哺乳动物的Syntaxin 4基因的有较高相似性。利用实时定量PCR技术分析Syntaxin 4基因在mRNA水平上的相对表达量。结果表明,该基因在脑组织和免疫相关组织中都有表达,以脑组织的表达量最多,在免疫相关组织中的表达量由多到少依次是脾脏、头肾和血液。在LPS刺激24 h后,脾脏和头肾中的表达量分别是对照组的7.83倍和10.41倍,表明免疫组织中Syntaxin 4基因的相对表达量随诱导增强,提示该基因在花鲈免疫应答反应中可能起重要作用。

重组鲈鱼生长激素的酶联免疫测定方法

建立了重组鲈鱼生长激素 (jspGH)的间接ELISA ,以 jspGH的小鼠多克隆抗体作为一抗 ,HRP羊抗鼠IgG作为二抗 ,在抗体及酶标二抗的稀释液中加入 4 %PEG ,从而提高检测灵敏度至 4 0ng/ml,抗原浓度在 4 0～ 10 0 0ng/ml范围内与OD4 50nm 值成线性关系。此法较简便、灵敏 ,可用于检测基因工程菌发酵液中的jspGH含量。

壳聚糖-咖啡酸衍生物涂膜对鲈鱼的保鲜效果研究

以鲈鱼为试材,用0.20%咖啡酸、2%壳聚糖、0.20%咖啡酸+1.8%壳聚糖、2%壳聚糖-咖啡酸衍生物、无菌水5个不同溶液浸泡处理,通过测定4℃冷藏4 d内挥发性盐基氮、菌落总数、pH、持水力以及感官评价等指标的变化,探讨壳聚糖-咖啡酸衍生物复合涂膜对鲈鱼的保鲜效果。结果表明,4℃下贮藏4 d后,相较于以无菌水处理的样品,4种不同处理均能够在一定程度上延缓鲈鱼的挥发性盐基氮含量、菌落总数、pH的升高,降低样品持水力的下降。在5种处理中,2%壳聚糖-咖啡酸衍生物涂膜对鲈鱼的保鲜效果最好。