论文标题:牙鲆免疫系统和抗病力的研究
Relationship between Endopeptidases and H_2O_2 during Wheat Leaves Aging and Characteristics of Endopeptidases Extracted from Chloroplast Stroma of Wheat Seedlings
论文作者 王宏田
论文导师 张培军,论文学位 博士,论文专业 海洋生物学
论文单位 中国科学院海洋研究所,点击次数 143,论文页数 87页File Size3693k
2000-06-03论文网 http://www.lw23.com/lunwen_99561472/ 牙鲆,免疫,抗病
Paralichthys olivaceus, Immunity, disease resistance
本文主要以一龄牙鲆为研究对象，研究了牙鲆免疫系统的组成，以初步探讨牙鲆的抗病机制，并为牙鲆生理状况的诊断建立快捷的方法；研究了牙鲆主要消化酶的活性，为牙鲆的营养强化进行基础性的研究；研究了含有大麻哈鱼生长激素基因的重组酵母菌对牙鲆的生长和非特异性免疫能力的影响；同时对牙鲆热激蛋白hsp70的cDNA进行了合成和扩增。 本论文共包括四个部分，由十个实验组成，每个实验的研究内容介绍如下： 实验1 利用姬姆萨染色技术观察了牙鲆血细胞的显微结构。结果表明，牙鲆的血细胞由红细胞和白细胞组成，两类细胞的显微结构具有明显的差异；红细胞有细胞核，在血细胞的组成中占较高比例；不同种类的白细胞其结构不同，实验中发现了血栓细胞、淋巴细胞、单核细胞；但是只凭借光学显微形态的差异难以对白细胞进行更确切的分类。 实验2 利用透射电镜技术对牙鲆外周血细胞的超显微结构进行了观察。牙鲆的血细胞可以分为红细胞和白细胞；白细胞包括淋巴细胞、血栓细胞、单核细胞、粒细胞；粒细胞包含两种类型；实验中还发现了类似巨噬细胞的一类细胞，但未发现浆细胞的存在。 实验3 利用区带毛细管电泳技术对牙鲆血清蛋白的组成进行了分析。实验条件下，牙鲆的血清蛋白主要包含三种类型，即P2.59，P1.60，P0.77。三种蛋白的分子量和稳定性各不相同，初步判断这三种蛋白主要为白蛋白、α球蛋白和β球蛋白。 实验4和实验5 对溶菌酶和抗蛋白酶物质在假雄牙鲆体内的分布以及不同在组织和器官中的比活性进行了研究。溶菌酶广泛分布于牙鲆的体表粘液、鳃、血清、消化组织和脾、肾等组织中；抗蛋白酶物质广泛分布于体表粘液、鳃、血清、肌肉中。在不同的组织和器官中，这两种酶的比活性不完全相同。体表粘液、血清、肝脏、前肠、肾、鳃中溶菌酶的比活性较高；胃、中肠、后肠、脾中溶菌酶的比活性较低；胆汁中溶菌酶的比活性最低。血清和体表粘液中抗蛋白酶物质的比活性较高，鳃和肌肉中抗蛋白酶物质的比活性较低。由实验结果可知，鳃和消化道在牙鲆抵抗病原微生物侵袭的过程中发挥着重要的作用。 实验6 对碱性磷酸酶在牙鲆体内不同消化器官中的分布进行了研究。碱性磷酸 牙稣免疫系统和抗病能力的研究 酶在牙蚜的胃、肝脏、肠中广泛分布。在前肠和中肠部位酶的比活性较其它部位的高。 胃中碱性磷酸酶的比活性随季节的不同没有显著变化，而肠和肝脏中酶的比活性随季 节的不同具有较显著的变化。 实验7 对牙鲜体内消化酶的比活性进行了研究。在牙鲫的胃、前肠、中肠、后 肠中可以检测出蛋白酶、脂肪酶、淀粉酶、纤维素酶的活性。胃中的蛋白酶主要为酸习 性蛋白酶，肠中的蛋白酶主要为碱性蛋白酶。肠中蛋白酶的比活性由前肠向后肠递减， 前肠与中肠酶的比活性没有显著差异：牙鲜肠中脂肪酶的比活性较胃中的高，在肠的 不同部位脂肪酶的比活性无显著差异；牙稣前肠中淀粉酶的比活性最高，胃中的次之， 中肠、后肠中淀粉酶的比活性较低；牙铃的胃及肠道不同部位处的纤维素酶的比活性 没有显著差异。随着牙鲜的生长，蛋白酶、脂肪酶、纤维素酶的比活性增强，淀粉酶 的比活性减弱；其中胃和中肠内纤维素酶的比活性增强得较显著。七 实验8和实验9 研究了重组酵母菌对牙鲜的生长、血清中某些激素的含量、－！血清蛋白的含量，以及血清中溶菌酶和抗蛋白酶物质的比活性等生理指标的影响。通 过投喂重组酵母菌，可以提高牙钎血清中生长激素的含量，影响牙解血清中甲状腺 激素和三碘甲状腺原氨酸的含量，促进牙蚜的生长，同时能够增加牙时血清蛋白的含 量，增强牙洱血清中溶菌酶和抗蛋白酶物质的比活性。牙醉体内生长激素含量的增加 以及酵母酉细胞壁中的多糖成分都能够增强牙醉的非特异性兔疫能力。 实验10 对牙钞肝脏中热激蛋白hsp70的CDNA序列进行了合成和PCR扩增。在 实验条件下能够合成750hP左右的核酸序列，为进一步利用热激蛋白hPP70作为生理 指标来诊断牙醉的生理状态，以及研究热激蛋白在牙稣抵抗外界胁迫中所发挥的作用 提供了基础。
In this thesis the immunity of one-year-old flounder, Paralichthys olivaceus, was studied in order to understand the mechanism of its resistance to diseases and to develop quick methods to measure the physical conditions of the fish. As the vegetative state is very important to the ability of the fish to resist diseases, the specific activities of certain digestive enzyme were studied too. The effects of recombinant yeast, in which salmon growth hormone gene is transferred, to the growth and non-specific immune parameters of Paralichthys olivuceus were studied In addition, the eDNA sequence of hepatic heat shock protein hsp7O was synthesized and amplified with PCR. This thesis consists of four major parts which include ten experiments. In experiment 1, the fine structure of the peripheral blood cells of paralichthys olivaceus was studied by light microscopy. Examination of Giemsa抯 stained smears of the blood revealed the blood cells include erythrocyte. thrombocyte, lymphocyte and monocyte. Though the erythrocyte and leucocyte can be classified easily, it is difficult to classify the leucocyte further. In experiment 2, electron microscopy examination of the ultrastructure of the peripheral blood cells of Parallchthys olivaceus showed that the blood cells were classifiable as erythrocytes(red cells)and leucocytes(white blood cells). The leucocytes were classifiable as lymphocytes, monocytes, thrombocytes and two types of granulocytes. The phagocyte-like bllod cell was observed too. According to the structure, no plasma cell was found in the peripheral blood. In experiment 3, the serum protein components of Paralichthys olivaceus were analyzed by capillary zone electrophoresis (CZE). Three kinds of proteins can be analyzed with current experimental conditions. Based on the time that these proteins appeared, they were named as P 2.59, P 1.60 and P 0.77. After storage at room temperature (25C) for 24 hours, the protein P 2.59 remained stable , while the other two proteins were destroyed. Comparison of the results with those of the analysis of human being抯 serum , the protein P 3 L 2.59 is probably attributable to Albumin, the protein P 1.60 is probably attributable to Globulin- a , the protein P 0.77 is probably attributable to Globulin- P. In experiment 4, the specific activities of antiprotease in some tissues of pseudoniale Paralichthys olivaceus were studied in this experiment. The specific activities in gill, mucus, serum and muscle were 2.56?.79, 6.81?.95, 5.18?.35, 3.64?.07 (unit/mm mg protein). From the results it can be concluded that the antiprotease exists in these tissues, which is important for the fish to protect themselves from the infection of the bacteria in the environment. In experiment 5, the specific activities of lysozyme in different tissues of pseudomale Paralichthys olivaceus were studied. The specific activities of lysozyme in mucus, serum, kidney, spleen were 0.58?.13,6.54?.19, 5.87?.97, 4.54?1.75, 2.33 ?.98(x10~3 unit/mining protein) respectively. The specific activities of lysozyme in liver, stomach, foregut, midgut, hindgut~ bile were 6.49 ?.35, 1.57?.65, 4.32?1.46, 2.07? 1.13, 2.41 ?1.29, 0.58 ?0.l3(xl0~3 unit/mm mg protein). The specific activity of lysozyme in gills was 3.96 ?1 .47(x 1 o~ unit/mm mg protein). No lysozyme activity w
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