【作者】 张亚南；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2009， 博士

【摘要】 毛蚶(Scapharca kagoshimensis)是隶属于软体动物门瓣鳃纲的一种具有重要经济价值的海洋无脊椎动物,其缺乏真正意义上的抗体、没有免疫记忆能力,只能依靠非特异性免疫反应来抵御寄生虫和疾病,其中血细胞和免疫因子在其对多种病原微生物的免疫防御反应中起着决定性作用。血红蛋白(hemoglobin,Hb)是所有脊椎动物和部分无脊椎动物血液中所含有的一大类含铁的呼吸蛋白,除具有运输氧气的主要功能外,还具有稳定血压、抗菌、运输硫化物、调节酸碱平衡、氧化酶、过氧化酶活性等多种生物学功能,是最具有研究价值的蛋白质家族之一。在其发现无脊椎动物血蓝蛋白具有类酚氧化酶活性之后,本实验室首次发现毛蚶Hb也具有类酚氧化酶活性,从此揭开了毛蚶Hb非特异性免疫功能研究的序幕。本研究拟利用生物化学和分子生物学手段从其血淋巴中分离纯化出毛蚶Hb,鉴定其类酚氧化酶活性(PO-like activity)及其激活因子,确定其生物化学性质和酶性质,查清几种常用免疫促进剂刺激以及细菌感染对毛蚶Hb与血细胞的影响作用,克隆出毛蚶Hb的全长cDNA序列,并利用推测一级结构氨基酸序列进行初步的同源性分析,旨在证实毛蚶Hb的非特异性免疫功能,为揭示毛蚶血红蛋白在动物非特异性免疫中的作用及分子机理,鉴定其在呼吸蛋白家族中的分子进化地位等奠定基础。本文利用Sephacryl S-100凝胶过滤柱层析和Q sepharose Fast Flow离子交换柱层析技术成功从毛蚶血淋巴中分离纯化出了血红蛋白,并以L-二羟苯丙氨酸(L-DOPA)为特异性底物对其类酚氧化酶活性进行了鉴定,实验结果表明,所得毛蚶Hb纯化样品在SDS-PAGE电泳中为分子量约14.27 kDa的单一条带,胶内酶切肽指纹图谱分析结果证实所得纯化样品确为Hb;同时,所得毛蚶Hb纯化样品对L-DOPA具有很强的氧化活性,且Native-PAGE电泳条带的L-DOPA染色结果也证实其具有很强的氧化活性,表明毛蚶Hb确实具有类酚氧化酶活性。生化性质和酶性质研究结果表明,毛蚶血红蛋白纯化样品对L-DOPA发挥类酚氧化酶活性的最适pH值为7.0、最适反应温度为30℃,其Km值约为4.90 mmol/L,且其类酚氧化酶活性可为异丙醇所进一步激活;不同氧化酶抑制剂的研究结果发现,毛蚶Hb纯化样品的类酚氧化酶活性对苯甲酸非常敏感,对抗坏血酸、亚硫酸钠、柠檬酸和硫脲较为敏感,而对苯硫脲和半胱氨酸不敏感,表明毛蚶Hb发挥类酚氧化酶活性是具有酪氨酸酶类型的酚氧化酶性质;此外,金属离子螯合剂乙二胺四乙酸(EDTA),Zn2+和Cu2+均能强烈抑制毛蚶Hb纯化样品的类酚氧化酶活性,二乙基二硫氨基甲酸酯(DETC)、Ca2+和Mg2+的抑制作用较小,而Fe2+却具有激活作用,说明毛蚶Hb的类酚氧化酶活性还具有金属酶性质;毛蚶Hb纯化样品类酚氧化酶活性的回复实验结果显示,被EDTA抑制的类酚氧化酶活性可特异性地为Fe2+所回复,进一步证明其是一种含铁金属酶性质。综上所述,毛蚶Hb发挥类酚氧化酶活性具有酪氨酸酶类型的含铁酚氧化酶的性质。免疫促进剂处理毛蚶的实验结果显示,脂多糖、β-葡聚糖、灭活哈维氏弧菌和灭活鳗弧菌处理48 h后,与对照组相比,毛蚶Hb的产量分别增加了7.47%、6.17%、10.39%和27.92%,其类酚氧化酶总活性分别增加了20.72%、4.03%、11.40%和45.84%,而单位酶活性没有显著差异,表明上述四种免疫促进剂的刺激可不同程度地提高毛蚶Hb的合成量及其类酚氧化酶总活性,其中以灭活鳗弧菌的刺激效果最为显著;此外,脂多糖、β-葡聚糖、灭活哈维氏弧菌和灭活鳗弧菌处理48 h后,毛蚶的血细胞的总数分别增加了8.3%、6.3%、2.0%和20.8%,表明上述四种免疫促进剂的刺激可不同程度地增加毛蚶血细胞的数量,其中以灭活鳗弧菌的刺激效果最为显著。综上所述,免疫促进剂脂多糖、β-葡聚糖、灭活哈维氏弧菌和灭活鳗弧菌可不同程度地增加毛蚶血细胞的数量、提高毛蚶Hb的合成量及其类酚氧化酶总活性,暗示血细胞和血红蛋白的确参与了免疫促进剂刺激下毛蚶自身免疫力的提高。细菌感染毛蚶的实验结果显示,1×107个/mL鳗弧菌感染毛蚶48h后,与对照组相比,毛蚶Hb的产量增加了19.78%,类酚氧化酶总活性增加了5.29%,血细胞的总数却减少了37.71%;1×108个/mL哈维氏弧菌感染毛蚶48h后,与对照组相比,毛蚶Hb的产量增加了8.44%,类酚氧化酶总活性增加了6.36%,血细胞的总数却减少了1.75%;而浓度为1×105个/mL的哈维氏弧菌和鳗弧菌感染48h后毛蚶Hb的产量减少了1.99%和7.94%,类酚氧化酶总活性减少了1.15%和5.59%,血细胞数量减少了35.63%和16.67%;上述结果表明,细菌感染能不同程度地引起毛蚶血细胞数量的减少,且高浓度细菌感染可不同程度地引起毛蚶Hb产量和类酚氧化酶总活性的增加,但低浓度细菌感染反而会不同程度地引起毛蚶Hb产量和类酚氧化酶总活性的降低,暗示血细胞和Hb的确参与了毛蚶的非特异性免疫反应。从毛蚶血细胞中提取总RNA,利用反转录-聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术,分别克隆出了毛蚶血红蛋白HbI、HbII A和HbII B的全长cDNA序列,HbI的cDNA全长为756 bp,编码147个氨基酸残基,分子量为16.2 kDa,与不等壳毛蚶(Scapharca inaequivalvis)HbI的同源性为99.32%;HbII-A的cDNA全长为755 bp,编码150个氨基酸残基,分子量为16.5 kDa,与不等壳毛蚶(Scapharca inaequivalvis)HbII-A链同源性为98.67%;HbII-B的cDNA全长为745 bp,编码152个氨基酸残基,分子量为16.7 kDa,与不等壳毛蚶(Scapharca inaequivalvis)HbII-B链同源性为99.34%。研究毛蚶Hb的类酚氧化酶活性,对于揭示Hb在无脊椎动物非特异性免疫中的生物学功能具有重要的理论意义,在动物免疫和呼吸蛋白分子进化等方面也均具有重要的科学价值。更多还原

【Abstract】 Blood clam, Scapharca kagoshimensis, is one of a marine invertebrates which has an important economic value and belongs to Mollusca, Lamellibranchia. It lacks antibodies and immune memory and has to resist parasites and diseases with non-specific immune system. Haemocytes and humoral immune factors play an important roles in immune responses of S.kagoshimensis to many kinds of pathogenic microorganism. Hemoglobin (Hb), responsible for oxygen transport, are a kind of iron-containing respiratory proteins found in the hemolymph of both vertebrates and some invertebrates. This study indicate that hemoglobin not only have oxygen-bearing function, but also have functions as follows: antimicrobial activity , adjusting blood pressure, transporting sulfide, adjusting acid-base balance, oxidase, peroxidase activity and so on. It is one of the most valuable respiratory proteins to worth researching. Many studies have indicated the hemocyanins can be functionally converted into a kind of phenoloxidase-like enzyme and display PO activities under certain conditions. In this study, phenoloxidase-like activities of the hemoglobin from blood clam S.kagoshimensis was found and it lay solid foundation in non-specific immune of invertebrates. In order to characterize the properties of PO-like activities of the hemoglobin from blood clam S.kagoshimensis, Hb from S.kagoshimensis was purified by biochemical and molecular biology methods, and characterized its biochemistry and enzymatic properties. To study the stimulating mechanisms of immunostimulates, the immunostimulating effects ofβ-glucan, lipopolysaccharide (LPS), inactivate Vibrio harveyi and Vibrio anguillarum on the PO-like activities of Hb from S. kagoshimensis were investigated and full-length cDNA sequence has been obtained. This study has not only great significance in understanding its roles in invertebrate non-specific immune system, but also helps us to fully cognize the multiple biological functions of Hb.In this study, Hb from hemolymph of S. kagoshimensis was purified by sephacryl S-100 gel-filtration and Q sepharose Fast Flow ion-exchange chromatography, and characterized its biochemistry and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate to study its PO-like activity. The result indicate that the purified Hb had a molecule weight of only 14.27kDa in sodium dodecyl sulfate polyacrylemide gel electrophoresis(SDS-PAGE), and it is testified by peptide mass fingerprint(PMF). The purified Hb can specific oxidate L-DOPA and dyed by L-DOPA after Native-PAGE .The result indicate that the Hb from S. kagoshimensis have the PO-like activities.The biochemical and enzymatic properties of Hb from S. kagoshimensis using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate was studied and the result indicated that the optimum pH and temperature was 7.0 or so and 30℃and using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate to study its PO-like activity. The Km values was 4.90 mmol/L against L-DOPA. The PO-like activities of Hb from S. kagoshimensis can be activated by isopropanol, and was extremely sensitive to some effective inhibitors of benzoic acid; sensitive to ascorbic acid,urea and sodium sulfite,citric acid; but not sensitive to cysteine and1-phenyl-2-thiourea. All these indicate that the PO-like activities of Hb from S. kagoshimensis in this study is a kind of tyrosinase. The PO-like activity of Hb from S. kagoshimensis was very sensitive to EDTA, Cu2+ and Zn2+, but not sensitive to DETC ,Ca2+ and Mg2+, and Fe2+ activate the PO activity effectively. Then it can be concluded that the Hb of S. kagoshimensis was a type of iron-containing protein.To study the stimulating mechanisms of immunostimulates, the immunostimulating effects ofβ-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on the PO-like activities of Hb from S. kagoshimensis were investigated in this study. Under the stimulation ofβ-glucan, LPS, inactivated Vibrio Harvey and inactivated Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis increased about 7.47%、6.17%、10.39% and 27.92%, respectively and PO-like activities increased about20.72%、4.03%、11.40% and 45.84%, but the unit activity of PO-like remains almost unchanged. The immunostimulation of Vibrio anguillaru is the most efficient on the amount of Hb synthesized and PO-like activity. Further more, the number of all hemocytes increased about 8.3%、6.3%、2.0% and 20.8%. It is concluded that under the stimulation ofβ-glucan, LPS, inactivated Vibrio Harvey and inactivated Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis, PO-like activities and the number of all hemocytes are all increased, The result indicated that the hemocytes and Hb play an important roles in Scapharca kagoshimensis’s immune responses.Under the infection of 1×107ind/mL Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis increased about 19.78%, respectively and PO-like activities increased about5.29%, the number of all hemocytes reduced 37.71%. Under the infection of 1×108ind/mL Vibrio Harvey, the amount of Hb synthesized from S.kagoshimensis increased about 8.44%, respectively and PO-like activities increased about6.36%, the number of all hemocytes reduced 1.75%. Under the infection of 1×105ind/mL of Vibrio Harvey and Vibrio anguillaru, the amount of Hb synthesized from S.kagoshimensis reduced about 1.99% and 7.94%, respectively and PO-like activities reduced about1.15% and 5.59%, the number of all hemocytes reduced 35.63% and 16.67%. The result indicated that the infection results in reduction of the number of all hemocytes. Infection with high concentration pathogeny results in increase of the amount of Hb synthesized, PO-like activities and the number of all hemocytes. Otherwise, infection with low concentration pathogeny results in reduction of the amount of Hb synthesized, PO-like activities and the number of all hemocytes.Total RNA is isolated from the hemocytes of S.kagoshimensis and full-length cDNA sequence of HbI ,HbII-A and HbII-B has been obtained using reverse transcriptase(RT)-PCR and rapid amplification of cDNA end(RACE).The full-length cDNA of HbI from Scapharca kagoshimensis is 756 bp, encoding a 147-amino acid protein, the molecular weight is about 16.2 kDa, the identity with HbI from Scapharca inaequivalvis is 99.32%; HbII-A from Scapharca kagoshimensis is 755 bp, encoding a 150-amino acid protein, the molecular weight is about 16.5kDa, the identity with HbII-A from Scapharca inaequivalvis is 98.67%; HbII-B from Scapharca kagoshimensis is 745 bp, encoding a 152-amino acid protein, the molecular weight is about 16.7kDa, the identity with HbII-B from Scapharca inaequivalvis is 99.34%.In conclusion, the study of the PO-like activities of Hb from S. kagoshimensis has great significance in not only understanding the roles and mechanisms of Hb in invertebrate non-specific immune system, but also identification the molecular evolution status of Hb in the family of respiratory protein.更多还原