【作者】 张彦娇；

【导师】 麦康森；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2010， 博士

【摘要】 本论文主要包括二部分：一为盐度和温度对大菱鲆非特异性免疫力的影响；二为淡水螯虾相关免疫因子的研究。1.盐度对大菱鲆非特异性免疫力的影响。本实验主要研究不同盐度对感染鳗弧菌(Vibrio anguillarum)的大菱鲆(Scophthalmus maximus L.)非特异性免疫力的影响。大菱鲆(平均体重25±2 g)分别在不同盐度(8、20、32和40g/L)下饲养7d后注射感染鳗弧菌。在半致死浓度实验中,分别在每一个盐度处理组每一尾鱼的背鳍基部注射0.1 ml不同浓度的鳗弧菌液(3.75×107,3.75×108和3.75×109CFU/ml),统计4 d半致死浓度。在非特异性免疫实验中,在不同盐度处理组的每一尾鱼的背鳍基部注射1.1×108 CFU/ml鳗弧菌,然后分别在感染后24 h、48h和72 h取样,测定实验鱼血清中替代补体途径活力、溶菌酶活力及头肾吞噬活力。每一个处理组都有三个重复。实验结果表明,在养殖盐度为20g/L,时大菱鲆感染鳗弧菌4 d半致死浓度最高(8.88±0.17),显著高于40g/L处理组的半致死浓度(8.04±0.04)(P<0.05),而与其他两个处理组没有显著差异(P>0.05)。实验大菱鲆的替代补体途径活力、溶菌酶活力以及吞噬活力均在养殖盐度为20g/L处理组时具有最高值,而在40g/L具有最低值。除盐度40g/L处理组(溶菌酶活力从48 h到72 h降低)外,大菱鲆感染鳗弧菌后72 h内,随着感染时间的延长,各处理组的替代补体途径活力、溶菌酶活力以及吞噬活力均呈上升趋势。这些结果表明,生活在养殖盐度为20 g/L的大菱鲆具有较高的抗鳗弧菌能力。2.温度对大菱鲆非特异性免疫力的影响。本实验主要研究温度对感染鳗弧菌(Vibrio anguillarum)的大菱鲆(Scophthalmus maximus L.)非特异性免疫力的影响。大菱鲆幼鱼(平均体重25±2 g)分别在不同温度(10、15、20和25℃)下饲养7d后注射感染后。在半致死浓度实验中,分别在每一个温度处理组每一尾鱼的背鳍基部注射0.1 ml不同浓度的鳗弧菌液(3.75×107,3.75×108和3.75×109CFU/ml),统计4d半致死浓度。在非特异性免疫实验中,在不同温度处理组的每一尾鱼的背鳍基部注射5.0×107 CFU/ml鳗弧菌,然后分别在感染后24 h、48h和72 h取样,测定实验鱼血清中替代补体途径活力、溶菌酶活力及头肾吞噬活力。每一个处理组都有三个重复。研究结果表明,在养殖温度为15℃时大菱鲆感染鳗弧菌4d半致死浓度最高(8.94±0.12),并显著高于25℃处理组的半致死浓度(7.70±0.12)(P<0.05)。替代补体途径活力、溶菌酶活力以及吞噬活力均分别在养殖温度为15℃的处理组具有最高值,在25℃具有最低值。72 h之内,随着大菱鲆感染鳗弧菌时间的延长,不同温度处理的替代补体途径活力、溶菌酶活力以及吞噬活力均呈上升趋势。结果表明,养殖温度为15℃的大菱鲆具有较高的抗鳗弧菌能力。3.淡水螯虾(Pacifastacus leniusculus)发育中期的受精卵中相关免疫基因的表达。淡水鳌虾没有幼虫期,具有抱卵习性,通常产卵后在腹肢孵育直至小鳌虾破壳。本实验主要为了明确免疫相关基因是否在胚胎发育期间表达。所研究基因根据它们的功能和作用被分为以下三类：酚氧化酶原系统(proPO)、抗菌肽(AMPs)以及造血作用相关的因子。并进一步通过浸泡受精卵在高浓度的致病菌环境中检测胚胎的免疫反应。半定量RT-PCR分析表明：在此发育期的受精卵中,无法检测到proPO的mRNA,而其他所有proPO系统中相关的基因均可表达,其中血蓝蛋白的表达显著高于其他基因的表达。在抗菌肽基因的检测中,ALF和plcrustin-1的mRNA表达水平相近,但均高于plcrustin-2的mRNA水平,而在所有抗菌肽基因中plcrustin-3的表达最高。与造血组织细胞分化和增殖相关的Astakine 1、2和TGase的转录水平基本一致。因为proPO产生于血细胞中并且在甲壳类免疫反应中具有重要作用,这表明淡水螯虾的受精卵在此发育阶段没有完整有效的免疫系统,在保护胚胎不受细菌感染机制上,卵壳可能起到重要的屏障保护作用。4.Kazal型蛋白酶抑制剂转录本在淡水螯虾(Pacifastacus leniusculus)不同血细胞中的表达以及序列比较分析。甲壳类的血细胞可以产生大量的Kazal型蛋白酶抑制剂(KPI)转录本。在对淡水鳌虾(Pacifastacus leniusculus)和斑节对虾(Penaeus monodon)的研究中发现在它们的血细胞中分别至少存在26和20个不同的Kazal结构域。与其他种类的KPIs进行序列比较发现这一序列的高度变异性。尽管氨基酸P1(一个决定底物特异性的位置)会高度变化,但几个保守位置(例如6个不变的半胱氨酸)却存在于所有结构域。对单只淡水鳌虾的研究证明在个体水平上,血细胞产生的KPIs序列同样存在变异性。通过半定量RT-PCR分析,在淡水鳌虾造血组织细胞和不同类型血细胞中的4个KPI转录本表达分析表明部分KPI很可能参与造血或是成熟血细胞释放的过程,因为这些KPI只在特定血细胞或是成熟血细胞中产生。5.利用Bac-to-Bac杆状病毒表达系统表达淡水螯虾properoxinectin蛋白的研究。首先通过PCR方法扩增properoxinectin全长基因,N端融合6xHis序列,定向克隆入转移质粒pFastBacl中,经核苷酸序列测定正确后,转化感受态DH10Bac细胞,并在DH10Bac细胞内重组pFastBacl与杆粒发生转座筛选阳性克隆；提取重组杆粒,PCR方法鉴定后,转染sf9昆虫细胞株,连续感染sf9细胞,扩增病毒同时表达目的蛋白。此后,用Western Blot方法进行蛋白鉴定,成功获得His-properoxinectin重组蛋白,并通过Ni-NTA纯化目的蛋白。本研究利用Bac-to-Bac杆状病毒表迭系统成功表达和纯化properoxinectin,为进一步研究其生物学活性和功能以及蛋白结晶奠定了实验基础。更多还原

【Abstract】 Two sections are included in the dissertation. One is effect of salinity and temperature on the non-specific immunity of turbot (Scophthalmus maximus L.); One is related to study on some immune related genes in Crayfish Pacifastacus leniusculus.1. Effect of salinity on the non-specific immunity of turbot. Studies were conducted to investigate the effects of salinity on the non-specific immune responses and disease-resistance of turbot Scophthalmus maximus L. challenged with Vibrio anguillarum. Four groups of turbot juveniles (mean weight 25±2 g) were cultivated at 8,20,32 and 40 g/L salinity, respectively, for 7 days to acclimate the experimental conditions before pathogen challenge. Three concentrations (3.75×107,3.75×108 and 3.75×109 CFU/ml) of V. anguillarum suspension were employed at each salinity to determine the 4-day LD50, in which each fish was injected with 0.1 ml of the pathogen suspension at the base of dorsal fin. For the observation of non-specific immune responses at the selected rearing salinities, each fish was injected with 0.1 ml of V. anguillarum suspension at 1.1×108 CFU/ml, then the activities of serum lysozyme, the alternative complement pathway (ACH50) and phagocytosis percentage of head kidney were recorded at 24 h,48 h and 72 h post-challenge. All treatments were conducted in triplicate. Fish reared at 20 g/L had lowest mortality, namely the highest 4-day LD50 value (8.88±0.16) that was significantly higher than that (8.04±0.04) of fish cultured at 40 g/L (P< 0.05), while no significant difference was observed compared with the other treatments (P>0.05). The activities of lysozyme, ACH50 and phagocytosis maintained the highest at the salinity of 20 g/L. In contrast, these activities at the salinity of 40 g/L were always the lowest at different sampling times (24 h,48 h and 72 h). Generally, the values of the non-specific immune parameters kept increasing within 72 h post-challenge, except that the lysozyme activity at 40 g/L significantly increased from 24 h to 48 h, and then decreased from 48 h to 72 h. The results indicate that besides growth improvement, rearing in intermediate salinity (20 g/L) is able to enhance the immunity and disease-resistance of turbot.2. Effect of temperature on the non-specific immunity of turbot. This experiment was conducted to investigate the effects of temperature on the non-specific immunity of turbot Scophthalmus maximus L. challenged with V. anguillarum. The juvenile healthy turbots (mean weight:25±2 g) were cultivated in the seawater with different temperature (10,15,20,25℃, respectively) for 7 d to acclimate the experimental conditions before pathogen challenge. The result of the 4-day LD50 showed that fish at the temperature of 15℃had lowest mortality, significantly lower than that of fish cultured at 25℃(P<0.05), while no significant difference was observed compared with the other treatments (P>0.05). The activities of lysozyme, complement of the alternative pathway (ACH50) and phagocytosis maintained the highest at the salinity of 15℃, however, they were always the lowest at different sampling time (24 h,48 h and 72 h) at the salinity of 25℃. All results indicated that turbot cultured at temperature of 15℃had relatively higher anti-infection of V. anguillarum and higher anti-disease compared with higher or lower temperature.3. RT-PCR was used to analyse the expression of a number of immune-related genes in the middle phase of crayfish Pacifastacus leniusculus embryo. Crayfish do not have larval stage as other crustacean such as penaeid shrimp, they brood their eggs until hatching and what hatches out from the eggs are miniature crayfish known as juveniles. In order to address the question whether immune genes are initially expressed during the embryo development in the egg stage, the expression of some immune-related genes:prophenoloxidase (proPO), peroxinectin, hemocyanin, anti-lipopolysaccharide factor (ALF), plcrustin, astakine 1,2 and transglutaminase (TGase) were determined in the middle phase of crayfish embryo development. Furthermore, immune challenge was used to determine the immune response of eggs by immersing them in a solution of the highly pathogenic bacterium Aeromonas hydrophila. Semi-quantitative RT-PCR analysis showed that all tested genes are present except proPO in this phase of crayfish embryo development and none of the genes tested changed their expression following immersion in A. hydrophila. The proPO transcript has been reported from hemocytes in crustaceans and it plays crucial roles in crustacean immune response. This may indicate that the development of immune-competent hemocytes in this stage of crayfish embryo is not completed and the egg shell as such plays an important role as a shield in protecting the embryo from bacteria and maybe also other pathogens.4. Expression of Kazal-type proteinase inhibitors in different types of hemocytes in ccrayfish and sequence alignment. Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of PI amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.5. Expression of recombiant properoxinectin from crayfish Pacifastacus leniusculus in Bac-to-Bac Baculovirus Expression System. The full-length cDNA of preoperoxinectin was amplified by PCR with 6×His Tag on 5’, cloned the gene into pFastBacTM1, sequencing. Transform purified plasmid DNA into DH10BacTM E. coli for transposition into the bacmid. Use blue/white selection to identify colonies containing the recombinant bacmid, analyzing recombinant bacmid DNA by PCR, transfecting insect cells to produce recombinant baculovirus, harvest the virus from the cell culture medium to amplify and express the protein, the recombinant properoxinectin expressed in the insect cell. It’s analysed with SDS-PAGE and identified by WesternBlot, and purified by Ni-NTA.更多还原