牙鲆皮肤黏液免疫球蛋白的纯化及部分特性分析

采用饱和硫酸铵分步盐析结合Sephacryl S-300凝胶过滤层析及DEAE Sepharose离子层析法,对牙鲆皮肤黏液中的免疫球蛋白(Ig)进行了分离纯化,并通过SDS-PAGE及Western-blotting技术对纯化蛋白的部分特性进行了分析。结果表明,30%、50%饱和硫酸铵分步盐析可以去除牙鲆黏液中除Ig外的很多杂蛋白,得到粗提黏液Ig;再经Sephacryl S-300纯化,Ig纯度较高,其中含有72和26 ku的条带;DEAE Sepharose层析法可进一步纯化Sephacryl S-300柱层析的产物,所提取黏液Ig经SDS-PAGE检测,只含有72和26 ku两个条带,初步认为是牙鲆黏液Ig的重链和轻链。Western-blotting结果显示,抗牙鲆血清Ig单克隆抗体可与黏液Ig重链72 ku条带发生发应。     

花鲈免疫球蛋白的分离纯化及部分特性分析

分离纯化了花鲈(Lateolabrax japonicus)血清免疫球蛋白(Ig),并对其特性进行初步研究与分析.采用山羊-IgG(Goat-IgG)免疫花鲈并制备血清,分别采用硫酸铵分级沉淀法、A蛋白亲和层析法及Goat-IgG偶联亲和层析法提取花鲈Ig,对提取组份进行了蛋白浓度测定、间接ELISA效价测定、SDS-PAGE和Western-blotting分析.结果表明,采用硫酸铵分级沉淀法提取的花鲈Ig主要集中在硫酸铵饱和度为30%～50%的区间,其中45%饱和度硫酸铵富集的的抗体比活最高,ELISA效价为2×104/mg蛋白,比血清ELISA效价提高2.2倍;A蛋白柱亲和层析可得单一蛋白峰,洗脱峰集中在洗脱体积0.8～1.6 mL,处,占洗脱蛋白总量的76.2%,峰值的ELISA效价为1.37×105/mg蛋白,抗体得率为2.57%;Goat-IgG偶联Sephrose 4B亲和柱层析也可得单一蛋白峰,洗脱峰集中在洗脱体积1.4～2.8 mL处,占洗脱蛋白总量的72.4%,峰值的ELISA效价为1.39×105/mg蛋白,抗体得率为2.63%.以上3种提取方法均可得到79 kD的重链和29 kD轻链,表明可得到纯化的免疫球蛋白;采用鼠抗花鲈Ig血清和单抗AA5经Western-blotting证实提取成份为花鲈Ig.本研究表明,A蛋白柱和Goat-IgG偶联柱亲和层析均可用于花鲈Ig的提取,其中A蛋白柱法获得的洗脱峰值更为集中,抗体效价高,且提取过程不需要Goat-IgG免疫鱼体,是一种更为有效的免疫球蛋白提取方法.     

欧洲鳗血清免疫球蛋白纯化及部分特性分析

采用饱和硫酸铵分步盐析法结合柱层析提取，纯化欧洲鳗血清免疫球蛋白（Ｉg) ,实验证实欧洲鳗Ｉg主要分布在硫酸铵饱和度３０％－５０％的区间内，Ｓephacry1-S200进一步提纯的Ｉg主要存在于第一个蛋白峰，而Ｓepharose-4B柱层析提纯的Ｉg则存在于第二个蛋白峰，ＤＥＡE-52脑子交换柱层析可进一步纯化Ｓepharose-4B柱层析的产物，ＥＬＩＳＡ和Western-blot分别证实上述提取物具有抗体的免疫学活性，ＳＤＳ－ＰＡＥＧ分析纯化的Ｉg，显示了洲鳗Ｉg重链约为６８kD，轻链约为２６kD.     

黄鳝血清、肠黏液免疫球蛋白的分离纯化及部分特性分析

采用硫酸铵盐析、SephadexG-200凝胶层析等方法从黄鳝血清和肠道黏液中分离纯化出黄鳝血清和黄鳝肠黏液免疫球蛋白。SDS—PAGE分析表明,二者的理化性质相同;经β-巯基乙醇处理后解离形成重链和轻链两个亚单位,分子量分别为74kD和29kD。试验结果表明,黄鳝血清和肠黏液中存在免疫球蛋白,二者具有相同的层析和SDS-PAGE电泳特性。     

指状拟舟虫诱导牙鲆抗血清免疫球蛋白分析

对引起牙鲆体表溃烂的原纤毛虫－指状舟虫诱导牙鲆免疫反应产生的免疫球蛋白ＩｇＭ进行分析,结果表明,病原纤毛虫免疫注射牙鲆 ,可诱导牙鲆发生特异性免疫反应,产生抗体型上鲆抗指状拟舟虫血清的凝集试验,发现纤毛虫停止游动并发虫体聚集;抗血清经与标准分子量和鼠ＩｇＭ单克隆抗体以及对照血清的ＳＤＳ－ＰＡＧＥ电泳比较分析证明,牙鲆抗指状拟舟虫血清免疫球蛋白ＩｇＭ重链分子量为７１０００,轻链分子量为２３０００;Ｉ     

欧洲鳗鲡血清免疫球蛋白纯化及其结构分析

应用亲和层析技术提纯欧洲鳗鲡免疫球蛋白（Ig）,并对欧鳗Ig的结构和抗原性进行分析。层析结果表明：Ig蛋白呈现一个锐形曲线,蛋白峰与抗原活性峰高度重叠。SDSPAGE分析表明：欧鳗Ig重链分子量为68 kD,轻链有3条,分子量分别为21 kD、23 kD和26 kD。凝胶电泳结果显示：在非变性非还原条件下,欧鳗Ig有790 kD和350 kD 2条蛋白带,在变性非还原条件下有790 kD、593 kD和350 kD 3条蛋白带。Western-blotting试验证实：兔抗欧鳗Ig能识别欧鳗Ig的多种不同聚合体和Ig的重链,但不能识别Ig的轻链。结论：欧鳗Ig在自然条件下可能以四聚体和二聚体的形式存在,这与其它硬骨鱼的Ig形式有差异。欧鳗Ig链间二硫键不健全,在SDS作用下可解聚产生多种不同分子量的聚合体,首次揭示欧鳗Ig的轻链有3种异型。     

中华鲟、史氏鲟及达氏鳇血清免疫球蛋白的纯化及部分特性分析

采用饱和硫酸氨分步沉淀和Sephadex G200凝胶层析的方法,首次分别纯化制备了健康非免疫状态下中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrenckii）和达氏鳇（Huso dauricus）的血清免疫球蛋白（Ig） ,并采用聚丙烯酰胺凝胶电泳（PAGE）、SDS-聚丙烯酰胺凝胶电泳（SDSPAGE）、蛋白免 疫印迹（Western blotting）及免疫琼扩实验等方法对其Ig及Ig亚单位的分子量和部分特性进行了分析。PAGE及SDSPAGE的结果显示：史氏鲟,中华鲟和达氏鳇IgM的相对分子量分别为867 kD, 896 kD和924 kD;3种鲟鱼Ig的重链分子量均为88 kD,都具有29 kD的轻链,其中达氏鳇还另有一分子量约为26 kD的轻链蛋白。分子量的测定及计算结果显示鲟鱼的Ig为四聚体。Western-blotting的检测结果表明,3种鲟鱼Ig的重链与其Ig具有同样的抗原性,在硝酸纤维素膜上可被兔抗鲟Ig多克隆抗体所识别,而轻链的Western blotting检测结果则呈阴性。免疫沉淀反应的结果显示,3种鲟鱼的血清及其Ig与相互之间的兔抗Ig血清有免疫沉淀反应,但与兔抗鲤Ig血清无免疫沉淀反应,这表明3种鲟科鱼类的Ig在结构和序列上是较为相似的,而与鲤鱼等高等硬骨鱼类的Ig存在较大的差别。     

人工感染淋巴囊肿病毒牙鲆的免疫球蛋白水平变化

用患淋巴囊肿病牙鲆体表肿瘤纯化的淋巴囊肿病毒对健康牙鲆进行人工感染,应用抗牙鲆免疫球蛋白单克隆抗体通过间接酶联免疫法（ELISA）检测感染之后的牙鲆血清总抗体水平和抗LCDV特异性抗体水平的变化规律。结果表明,感染7d后,牙鲆血清中特异性抗体和总抗体水平都显著升高。抗LCDV特异性抗体水平在405nm处的OD值从对照的0.129上升至0.238;随着感染时间延长抗体水平持续升高（第14天0.247,第21天0.410）,在第28天达到最高值0.436,然后开始缓慢降低（第35天0.385,第98天0.357）;总抗体水平在405nm处的OD值从对照的0.135上升至0.250;随着感染时间延长抗体水平持续升高（第14天0.266,第21天0.561）,在第28天达到最高值0.613,然后开始缓慢降低（第35天0.480,第98天0.475）。通过间接免疫荧光技术（IFAT）在感染第14天以后的牙鲆的肠、胃中检测到病毒。     

欧洲鳗皮肤黏膜免疫球蛋白纯化及其结构组成分析

应用亲和层析技术提纯欧洲鳗(Anguilla anguilla)黏膜免疫球蛋白(Ig),并在结构和抗原性上进行分析.柱层析结果表明,欧洲鳗黏膜Ig经亲和层析分离后出现1个锐形的蛋白峰,蛋白主要分布于收集物的第2-6管,蛋白峰和抗原活性峰重叠.凝胶电泳结果显示,在变性还原条件下,欧洲鳗黏膜Ig重、轻链分子量分别为68 kD和26 kD,还有1条分子量约为18 kD的蛋白带;在非变性非还原条件下,黏膜Ig只有1条蛋白带,分子量约为350 kD,略旱扩散拖带现象,这一结果提示自然条件下欧洲鳗黏膜Ig可能以二聚体形式存在;在变性非还原条件下,黏膜Ig有3条蛋白带,分子量约为350 kD、138 kD和135 kD,表明欧洲鳗黏膜Ig在SDS作用下可发生不同程度的解聚.Western-blotting试验证实,兔抗血清能识别欧洲鳗黏膜Ig的二聚体、解聚体、重链和81 kD、84 kD处的蛋白带,显示出黏膜Ig的抗原特异性.欧洲鳗黏膜Ig与血清Ig在结构和抗原性上的差异.为进一步探讨欧洲鳗是否存在相对独立的黏膜免疫系统奠定了基础.     

奥尼罗非鱼血清免疫球蛋白的纯化及分子量的初步分析

通过饱和硫酸铵盐析结合 Sephadex G-200柱层析的方法,纯化制备了健康非免疫状态下奥尼罗非鱼的免疫球蛋白,进行变性还原条件下的聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹试验对血清的免疫球蛋白进行初步分析,发现SDS-PAGE电泳条件下血清重链分子量为85 kD,轻链分子量为30 kD.如果罗非鱼血清免疫球蛋白在自然状态与其他硬骨鱼类一样也为四聚体,那么其总分子量的理论值应为920 kD.     

牙鲆头肾巨噬细胞的分离培养与鉴定

本研究使用密度梯度离心法从牙鲆(Paralichthys olivaceus)头肾中分离得到了巨噬细胞,通过差速贴壁法对获得的细胞进行纯化,后续经过优化培养条件和培养过程,采用L-15培养基(Gibco)、5％胎牛血清、1％青–链霉素、1％非必需氨基酸、30％L929细胞培养基在24℃、无CO2的条件下进行培养.显微镜...     

牙鲆体质量与形态性状的异速生长分析

为分析牙鲆（Paralichthys olivaceus）体质量与形态性状之间异速生长的遗传规律,通过人工受精的方法建立牙鲆19个全同胞家系,测量了牙鲆不同日龄的体质量和形态性状。采用逐步回归方法建立最优联合异速生长模型,将此模型镶嵌到体质量动物模型的每个遗传和环境效应中,进一步分析对多个形态性状异速生长遗传规律。静态异速生长分析表明：体质量与全长之间存在最大异速生长指数（1.415 5）,表现为正异速生长,剩余形态性状与体质量之间的异速生长指数为0.061 5~0.718 0,皆表现为负异速生长。全长与尾柄高之间异速生长指数的正遗传相关最大（0.907 8）;全长与尾柄长之间的负遗传相关最大（0.946 8）。不同模型通过统计标准比较,确定模型Ⅰ为进行牙鲆动态异速生长分析的最优随机回归模型。     

Development of microparticle diets for Japanese flounder Paralichthys olivaceus larvae

ABSTRACT:   Two newly designed microparticle diets (MD), with two kinds of peptide (C700 and C800) as a protein source were developed. Microparticle diet Q (MD-Q) contained C700 (molecular weight ∼30 000 Da) and C800 (molecular weight 1000–2000 Da); in contrast, microparticle diet T (MD-T) contained C700 only. Two separate trials, representing larvae from different hatches, were conducted. Japanese flounder Paralichthys olivaceus larvae were fed newly designed MD or a combination of them with live food (LF) between 3 and 10 days after hatching (d.a.h.) in experiment I, or between 11 and 20 d.a.h. in experiment II, and compared them to LF and a commercial diet (CD) feeding groups. The growth and survival rates in both experiments were the highest in the LF treatment. But in the microparticle treatment larvae fed MD-Q had a higher survival rate and better growth than those fed MD-T, either alone or in combination with LF. First-feeding larvae fed on MD-Q had a 20.5% survival rate and 1.12 mm gain by 10 d.a.h. Unfed larvae died within 6 d.a.h. These results indicate that using a mixture of different molecular weight peptides is good protein sources and also this type of microparticle diet can be applied to flounder from larval to juvenile stages.     

Cytological studies on induced meiogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major . After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.     

双列杂交分析比较两个养殖群体杂交牙鲆抗病力

采用双列杂交设计对牙鲆Paralichthys olivaceus两个养殖群体：日本群体（J）和中国抗病选育群体（C）进行群体间杂交和群体内自繁,获得C（♀）×C（♂）、J（♀）×J（♂）、J（♀）×C（♂）和C（♀）×J（♂）4个交配组合的子一代。针对受精率、孵化率、白化率和存活率4个性状进行了组合间的比较,计算了各项性状的杂种优势率,对组间各性状进行了方差分析,并进行了相关分析。结果表明,杂交组在受精率、孵化率及存活率上存在着不同程度的杂种优势,其中C（♀）×J（♂）后代在孵化率和存活率两个性状上的杂种优势率分别为33.99%和31.37%。对4个性状的表型相关分析表明,受精率和孵化率的相关系数为0.657,受精率和存活率相关系数为0.432,分别达到了极显著水平（P〈0.01）。分析结果认为,两个不同群体间的种内杂交是牙鲆优良品种培育的有效途径。     

牙鲆养殖群体遗传变异的微卫星标记研究

应用10对微卫星引物对中国牙鲆一个养殖群体的30个个体进行了群体遗传结构分析。结果显示，微卫星标记比其他标记具有更高的多态性，10个微卫星座位的等位基因数在4～10之间，有效等位基因数在2．23～5．82之间，平均等位基因数为7．6，群体平均杂合度为0．6960，Hardy-weinberg遗传偏离指数的平均值为0．1774。     

Dietary vitamin A requirements of juvenile Japanese flounder Paralichthys olivaceus

A 70‐day feeding experiment was conducted to assess the dietary vitamin A (VA) requirements of juvenile Japanese flounder (Paralichthys olivaceus). Six semi‐purified diets with VA supplementations of 0, 5000, 10 000, 15 000, 20 000 and 25 000 IU kg^?1 were fed twice a day to triplicate groups of 20 juveniles per tank with an initial weight of 1.59 ± 0.01 g (mean ± SE). Weight gain (WG) and specific growth rate (SGR) increased as dietary VA increased up to 10 000 IU kg^?1. Significantly lower WG and SGR were observed for the 0 IU kg^?1 treatment than for treatments of 5000, 10 000 and 15 000 IU kg^?1. Highest WG and SGR were observed in fish fed 10 000 IU kg^?1; slightly lower values were recorded in fish fed 15 000, 20 000 or 25 000 IU kg¹. No significant difference was observed in survival rate among treatments. Whole body total lipid was significantly higher in fish fed 0 and 5000 IU kg^?1 than for other levels. Reduced growth and small livers were observed as signs of VA deficiency in fish fed 0 IU kg^?1. Slightly reduced growth and pale fragile livers were observed as effects of VA excess in fish fed 25 000 IU kg^?1. Total retinol contents in liver and eyes increased with increasing levels of dietary VA. No retinol was detected in livers, and significantly lower total retinol content was observed in eyes, of fish fed 0 IU kg^?1. WG analysed by the broken line method indicated that an optimum dietary VA requirement of 9000 IU kg^?1.     

Fast development of genetically uniform strains by gynogenesis in Japanese flounder (Paralichthys olivaceus)

The Japanese flounder is a major species in both aquaculture and research. Inbred strains of Japanese flounder were developed efficiently in our laboratory by meiotic and mitotic gynogenetic reproduction techniques. To determine the induction efficiency of gynogenesis and the rate at which full homozygosity is produced, six meiotic gynogenetic females (G1–G6) that had experienced meiotic gynogenesis once and three common females (C1–C3) were selected for production of mitotic gynogenesis in our experiments. Of the nine adult females, all six gynogenetic fish successfully produced viable offspring. However, only one of the three common fish did. Using microsatellite markers, we estimated the homozygosity of gynogenetic fish induced by mitotic gynogenesis. We found that the homozygosity of seven lines (C1, G1–G6) increased quickly, individually from 43%, 72%, 69%, 71%, 78%, 69% and 58% to 100%, 100%, 100%, 90%, 100%, 100% and 90% offspring complete homozygosity. Under mitotic gynogenesis, individuals with higher homozygosity had a higher induction rate. The G4 line showed the highest induction rate, achieving 44.59% convert hatching rate and 29.28% convert normality rate. We conclude that meiotic gynogenesis may be a feasible method to produce DH genetic material in Japanese flounder.