中华绒螯蟹新型免疫调节剂及其酚氧化酶的纯化和性质研究

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英文题名：Studies on the Novel Immunomodulators and Purification and Characterization of Phenoloxidase of Chinese Mitten Crab Eriocheir Sinensis 作者：李义 论文级别：博士 学科专业名称：水产养殖 中文关键词：中华绒螯蟹 ; 免疫调节剂 ; 先天性免疫 ; 抗病力 ; 酚氧化酶原激活系统 ; 酚氧化酶 ; 信号转导 英文关键词：Eriocheir sinensis ; immunomodulator ; innate immune response ; resistance ; prophenoloxidase activating system ; phenoloxidase ; signal transduction 学位年度：2007 导师：夏德全 ; 吴婷婷 学科代码：090801 学位授予单位：南京农业大学 论文提交日期：2007-12-01

摘要

本文综述了甲壳动物免疫防御机制研究的现状及最新进展,首次比较系统地研究了左旋咪唑、米糠多糖、山药多糖及CpG寡脱氧核苷酸(ODN-2006)对中华绒螯蟹(Eriociteir sinensis)免疫功能和抗病力的影响;探讨了ODN-2006触发中华绒螯蟹血细胞酚氧化酶原激活系统的信号转导机制;从中华绒螯蟹血细胞破碎物上清液中分离纯化到了酚氧化酶并对其生物化学性质进行了研究。现将研究结果总结如下:
     (1)左旋咪唑对中华绒螯蟹免疫功能及抗病力的影响为研究饲料添加左旋咪唑(levamisole,LMS)对中华绒螯蟹的免疫调节作用,以0(对照)、100、200和300mg·kg~(-1)干饲料的剂量将LMS添加于基础饲料中,制成颗粒饲料投喂中华绒螯蟹7 d,然后投喂不含LMS的对照组饲料。在投喂含LMS的饲料之前(0周)和投喂后2、4、6、8周,采样测定中华绒螯蟹的血细胞总数(THC)、不同血细胞数量(DHC)、吞噬活性、呼吸爆发(超氧阴离子的释放)、酚氧化酶(PO)活性和溶菌酶(LSZ)活性;并在投喂含LMS的饲料后的第4周,按1.2×10~7 cfu·kg~(-1)体重的剂量,体腔注射接种中华绒螯蟹致病性嗜水气单胞菌(Aeromonas hydrophila)CL99920菌株,记录接种10d后中华绒螯蟹的累积死亡率。结果显示,各LMS处理组的THC、透明细胞(HC)数量、吞噬百分率(PP)、呼吸爆发、PO活性和LSZ活性均显著地高于对照组(P＜0.05);颗粒细胞(GC)数量、血细胞吞噬指数(PI)与对照组无显著差异(P＞0.05);LMS处理的中华绒螯蟹对嗜水气单胞菌的抵抗力明显增强。由此表明,饲喂适量的LMS可增强中华绒螯蟹的免疫力和抗病力;在试验条件下,200 mg·kg~(-1)干饲料的剂量为最适添加剂量;LMS可作为中华绒螯蟹的新型免疫调节剂在疾病预防中使用。
     (2)两种植物多糖对中华绒螯蟹免疫功能的调节作用为研究饲料添加米糠多糖(Rice bran polysaccharide,RBP)和山药多糖(Chinese yam polysaccharide,CYP)这两种高等植物多糖对中华绒螯蟹免疫功能的调节作用,将RBP、CYP按0(对照)、1.0、2.0和4.0 g·kg~(-1)干饲料的剂量添加于基础饲料中投喂中华绒螯蟹,采取间隔投喂的策略,即每投喂7天试验饲料后再投喂7天对照饲料,整个试验持续6周。在试验开始后1、2、4、6周采样,对中华绒螯蟹的一系列免疫学参数进行测定,并在试验开始后2周,按1.2×10~7 cfu·kg~(-1)体重的剂量,体腔注射接种中华绒螯蟹致病性嗜水气单胞菌CL99920菌株,记录接种7 d后中华绒螯蟹的累积死亡率。结果显示,饲料添加2.0～4.0 g·kg~(-1)的RBP、1.0～2.0 g·kg~(-1)的CYP能显著地提高中华绒螯蟹的THC和HC数量(P＜0.05),显著地增强血细胞的吞噬活性和HIS的PO活性(P＜0.05),显著地提高血清POD、SOD、ACP、ALP活性及抗菌活性(P＜0.05),并能明显地增强中华绒螯蟹对嗜水气单胞菌的抵抗力,但对血清LSZ活性的影响没有规律性。上述结果表明,饲喂2.0～4.0 g·kg~(-1)的RBP或1.0～2.0 g·kg~(-1)的CYP能显著地增强中华绒螯蟹的免疫功能和抗病力;两种植物多糖可作为中华绒螯蟹的新型免疫调节剂在养殖生产中试用。
     (3) CpG寡脱氧核苷酸对中华绒螯蟹免疫功能的调节作用将ODN-2006(含3个CpG基序)、ODN-R(含1个反向CpG基序)按0(对照)、1、5和25μg·kg~(-1)体重的剂量体腔注射中华绒螯蟹(0.1 mL·只~(-1)),同时设注射等体积的0.85%生理盐水的对照组,研究了两种ODN对中华绒螯蟹免疫功能的调节作用。在注射ODNs或生理盐水之前(0 d)和注射后第1、3、5、8 d采样,对中华绒螯蟹的一系列免疫学参数进行测定,并在注射ODNs后第3 d,按1.2×10~7 cfu·kg~(-1)体重的剂量,体腔注射接种中华绒螯蟹致病性嗜水气单胞菌CL99920菌株,记录接种7 d后中华绒螯蟹的累积死亡率。结果显示,25μg·kg~(-1)ODN-2006能显著地提高中华绒螯蟹的THC、吞噬活性和呼吸爆发活性(P＜0.05),显著地增强血清PO、SOD、ACP、ALP、溶血素及抗菌活性(P＜0.05),但对中华绒螯蟹的DHC、血清POD及LSZ活性没有显著影响(P＞0.05)。此外,25μg·kg~(-1)ODN-2006能明显地增强中华绒螯蟹对致病性嗜水气单胞菌的抵抗力。与此相对,25μg·kg~(-1)ODN-R除对血清PO活性具有显著的增强作用外(P＜0.05),对其他免疫学参数没有明显的影响(P＞0.05)。上述结果表明,ODN-2006能显著地增强中华绒螯蟹的免疫功能和抵抗力,是中华绒螯蟹潜在的新型免疫调节剂,可进一步加以研发和利用。
     (4) CpG寡脱氧核苷酸触发中华绒螯蟹proPO激活系统的信号转导途径以0、5、10、15、20和25μg·mL~(-1)浓度的ODN-2006体外处理中华绒螯蟹血细胞30min,研究了ODN-2006对中华绒螯蟹PO活性的影响。结果显示,10μg·mL~(-1)以上浓度的ODN-2006能显著地增强血细胞胞内外受ODN刺激的PO(PO_S)活性及胞外总PO(PO_T)活性(P＜0.05),但胞内PO_T活性有所下降;ODN-2006诱导的胞内外PO活性的上升或下降均显示出剂量依赖性。由此表明,在试验条件下,ODN-2006能刺激中华绒螯蟹血细胞脱颗粒,但不能刺激血细胞合成新的proPO。进一步以10μg·mL~(-1)ODN-2006和一定浓度的特异性信号激活剂或抑制剂分别单独或联合体外处理中华绒螯蟹血细胞30 min,通过检测血细胞胞内外PO活性的增减情况,对ODN-2006触发proPO激活系统的信号转导途径进行了探讨。结果显示,用氟化钠(G蛋白激活剂)处理血细胞,胞内外PO_T活性均增加;用8-Br-cAMP(PKA激活剂)或咖啡因(磷酸二酯酶抑制剂)或白屈菜赤碱(PKC抑制剂)处理血细胞,胞内PO_T活性增加,胞外PO_T活性下降;用佛波酯(PMA,PKC激活剂)处理血细胞,胞外PO_T活性增加,胞内PO_T活性下降;用三羟基异黄酮(酪氨酸蛋白激酶抑制剂)处理血细胞,胞内外PO_S活性及胞外PO_T活性均上升,胞内PO_T活性下降。当用白屈菜赤碱和ODN-2006或PMA共同处理血细胞时,白屈菜赤碱对ODN或PMA诱导增强的胞内外PO_S活性及胞外PO_T活性具有较强的抑制作用。此外,试验发现氟化钠、PMA及三羟基异黄酮与ODN-2006对血细胞脱颗粒具有协同刺激作用,ODN-2006可部分地解除被8-Br-cAMP、咖啡因或白屈菜赤碱对血细胞脱颗粒的抑制作用。上述结果表明,ODN-2006可能经由G-蛋白介导的PKC途径活化中华绒螯蟹血细胞proPO系统,并通过酪氨酸蛋白激酶途径进行负调控。
     (5)中华绒螯蟹酚氧化酶的分离纯化及其生物化学性质以中华绒螯蟹的HLS为材料,利用凝胶过滤和离子交换层析等方法,对中华绒螯蟹的酚氧化酶(PO)进行了分离纯化和生物化学性质研究。试验发现,中华绒螯蟹酚氧化酶原(proPO)的相对分子质量约为76.4 kDa,在试验操作过程中极易发生降解或自身降解,变成有活性的大小约70.1 kDa的PO。以L-二羟苯丙氨酸(L-DOPA)为特异性底物对PO活性进行研究发现,中华绒螯蟹PO的最适温度为40℃,最适pH值为7.0,对L-DOPA和邻苯二酚的K_m值分别约为2.63 mmol·L~(-1)和6.25 mmol·L~(-1)。中华绒螯蟹PO对苯硫脲极为敏感,对硫脲、半胱氨酸、抗坏血酸及苯甲酸也很敏感。根据中华绒螯蟹PO对抑制剂的敏感性和对底物的亲和力,将其鉴定为邻二酚氧化酶(o-diphenoloxidase)。中华绒螯蟹PO对胰蛋白酶抑制剂SBTI较为敏感,但几乎不受另一种胰蛋白酶抑制剂TLCK的影响,推测该酶在蛋白酶性质上与胰蛋白酶存在着一定的相似性。此外,中华绒螯蟹PO的活性可被二乙基二硫代氨基甲酸钠(DETC)、乙二胺四乙酸(EDTA)、Cu~(2+)、Zn~(2+)及Ca~(2+)强烈抑制,Cu~(2+)能有效地恢复被DETC或EDTA抑制的PO活性,表明该酶很可能是一种Cu-金属酶(Cu-metalloenzyme)。
The current research advancement on immune defense mechanism of crustacean was reviewed based on the data that have been issued in domestic and overseas journals.Effects of levamisole(LMS),rice bran polysaccharide(RBP),Chinese yam polysaccharide(CYP) and CpG oligodeoxynucleotide(ODN) on immune response and disease resistance of Chinese mitten crab Eriocheir sinensis,signal transduction of the prophenoloxidase activating system of haemocytes of E.Sinensis triggered by CpG oligodeoxynucleotides, and purification and biochemical characterization of phenoloxidase from E.sinensis were studied for the first time in this paper.The results are summarized as follows:
     (1) Effects of levamisole on immune response and disease resistance of E.sinensis
     In order to determine the immunomodulatory effect of the dietary intake of levamisole on the E.sinensis,the crabs were fed diets containing 0(control),100,200 and 300 mg levamisole kg~(-1) dry diet for 7 days.The total haemocyte count(THC),differential haemocyte count(DHC),phagocytic activities,respiratory burst(release of superoxide anion),phenoloxidase(PO) and lysozyme(LSZ) activities were examined at 0,2,4,6,and 8 weeks after administration of levamisole.Crabs were challenged at 1.2×10~7 colony forming units(cfu) kg~(-1) crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E.sinensis at 4 weeks after administration of levamisole,and mortalities were recorded over a 10-day period.The results demonstrate that crab treated with levamisole showed significantly higher THC,the amount of hyaline cells(HC),phagocytic percentage(PP),respiratory burst,PO and LSZ activities than those of the control group (P＜0.05).No significant differences were observed in the amount of granular cells(GC) and phagocytic index(PI) of haemocyte among the crabs fed with diets containing 0 (control) and those fed with diets at 100,200 or 300 mg levamisole kg~(-1) dry die(P＞0.05). Furthermore,the levamisole treated E.sinensis were the more resistant.It was concluded that E.sinensis were fed with diets at 100,200 or 300 mg levamisole kg~(-1) dry die showed increased immune ability as well as resistance to A.hydrophila infection.Under the experimental condition,the optimum dose of dietary intake of levamisole should be 200 mg levamisole kg~(-1) dry diet.
     (3) Regulation of two plant polysaccharides on the immune response of E.sinensis
     In order to determine the immunomodulatory effect of the dietary intake of RBP or CYP in plant polysaccharides on the E.sinensis,the crabs were fed diets containing 0(control), 1.0,2.0 and 4.0 g polysaccharides kg~(-1) dry diet for 7 consecutive days,alternated with 7 days without polysaccharides throughout a 6-weeks test period.A series of immune parameters of E.sinensis were examined at 1,2,4 and 6 weeks after the experiment started. Crabs were challenged at 1.2×10~7 cfu kg~(-1) crab weight with a virulent strain of A. hydrophila(CL99920) of E.sinensis at 2 weeks after the experiment started,and mortalities were recorded over a 7-day period.The results showed that the THC and the amount of HC; the phagocytic activities of hacmocytcs;the polysaccharidcs-stimulated PO(POs) activities, total PO(PO_T) activities in haemocyte lysate supematant(HLS);the SOD,ACP,ALP and antibacterial activities in serum of E.sinensis fed with diets containing 2.0,4.0 g RBP kg~(-1) or 1.0,2.0 g CYP kg~(-1) were higher than those of the control group(P＜0.05).The effect of RBP or CYP on LSZ activity in serum of E.sinensis was irregular.Furthermore,the two plant polysaccharides treated E.sinensis were the more resistant.It was concluded that E. sinensis that were fed with diets at 2.0,4.0 g RBP kg~(-1) or 1.0,2.0 CYP kg~(-1) dry die showed increased immune ability as well as resistance to A.hydrophila infection.
     (3) Regulation of CpG oHgodeoxynucleotides on the immune response of E.sinensis
     The immunomodulatory effects of two ODNs on the E.sinensis were studied when the crabs were coelom injected individually with ODN-2006(containing 3 CpG motifs) or ODN-R(containing 1 inverted CpG motif) at 0(control),1,5 and 25μg kg~(-1) weight(0.1 mL crab~(-1)),and the crabs in another group were injected individually with saline of an equal volume was also set as a control.A series of immune parameters of E.sinensis were examined at 0,1,3,5,and 8 days after administration of the ODNs or saline.Crabs were challenged at 1.2×10~7 cfu kg~(-1) crab weight with a virulent strain of A.hydrophila (CL99920) of E.sinensis at 3 days after administration of the ODNs or saline,and mortalities were recorded over a 7-day period.The results showed that the THC,the phagocytic activities and respiratory burst of haemocytes,the PO,SOD,ACP,ALP, homolysin and antibacterial activities in serum of E.sinensis injected with ODN-2006 at 25μg kg~(-1) weight were higher than those of the control group(P＜0.05).However,No significant differences were observed in the DHC,POD and LSZ activities in serum of E. sinensis among the crabs injected with ODN-2006 at 0(control),1,5,25μg kg~(-1) weight and injected with saline.Furthermore,the crabs injected with ODN-2006 at 25μg kg~(-1) weight were the more resistant.Surprisingly,the PO activities in serum of E.sinensis that injected with ODN-R at 25μg kg~(-1) weight were higher than the control group(P＜0.05).It was concluded that ODN-2006 could enhance the non-specific immune response and resistance to potential infection of E.sinensis.
     (4) Signal transduction of the prophenoloxidase activating system of haemocytes of E. sinensis triggered by CpG oligodeoxynucleotides
     Effects of ODN-2006 on the PO activities of haemocytes of E.sinensis were studied when haemocytes were separately treated in vitro with ODN-2006 at 0(control),5,10,15, 20 and 25μg mL~(-1) for 30 min.The results showed that the intracellular and extracellular stimulated PO(PO_s) activities and extracellular total PO(PO_T) activities of haemocytes treated with ODN-2006 at 10μg mL~(-1) or more were higher than control group(P＜0.05), but the intracellular PO_T activities were decreased,and that the increase or decrease of the intra- and extracellular PO activities of haemocytes was a dose-dependent following the addition of the ODN-2006.These results suggested that the degranulation of haemocytes of E.sinensis could be stimulated by ODN-2006,but new proPO were not synthesized under the test conditions.In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system,haemocytes were treated in vitro with 10μg mL~(-1) of ODN-2006 and a suitable concentration activators or inhibitors of specific signalling components by alone or together for 30 min.And then,the intra- and extracellular PO activities of these haemocytes were measured.The results showed that there was an increase in both intra- and extracellular PO_T activities of haemocytes treated with sodium fluoride(a G-protein activator);the intracellular PO_T activities of haemocytes were increased and the extracellular PO activities of haemocytes were decreased when 8-bromo-cAMP(a phosphokinase A(PKA) activator) or caffeine(a phosphodiesterase inhibitor) or chelerythrine(a PKC inhibitor) was added to haemocytes,in contrast,the intracellular PO_T activities of haemocytes were decreased and the extracellular PO activities of haemocytes were increased when haemocytes were treated with phorbol-12-myristate-13-acetate (PMA;a phosphokinase C(PKC) activator).On the other hand,the intra- and extracellular PO_s activities and extracellular PO_T activities of haemocytes were increased and the intracellular PO_T activities of haemocytes were increased when haemocytes were treated with genistein(an inhibitor of protein tyrosine kinase).When haemocytes were treated with chelerythrine and ODN-2006 or PMA together,the increase of the ODN-2006 or PMA-induced intra- and extracellular PO_s activities and extracellular PO_T activities of haemocytes were greatly decreased.In addition,sodium fluoride or PMA or genistein with ODN-2006 on the degranulation of haemocytes of E.sinensis have a synergistic action.Furthermore,the 8-bromo-cAMP or caffeine or chelerythrine induced inhibition on the degranulation of haemocytes could be partially relieved by ODN-2006. These results suggest that the activation of the proPO system of haemocytes of E.sinensis, including degranulation and PO activities,are induced by ODN-2006 via a G-protein mediated PKC-activating signaling pathway,but negatively regulated via the tyrosine kinase pathway.
     (5) Purification and biochemical characterization of phenoloxidase from E.sinensis
     PO from HLS of E.sinensis was purified by gel-filtration and ion-exchange chromatography,and characterized in terms of its molecular mass and biochemical and enzymatic properties by using L-dihydroxyphenylalanine(L-DOPA) as the specific substrate.It was found that proPO,isolated as a monomeric protein,had a molecular mass of 76.4 kDa,and a 70.1 kDa PO molecule was often contained in preparations.The PO activities showed optimal temperature of 40℃,optimal pH of 7.0,and an apparent K_m value of 2.63 mmol L~(-1) on L-DOPA,and 6.25 mmol L~(-1) on catechol.The PO was extremely sensitive to 1-phenyl-2-thiourea,very sensitive to thio urea,cysteine,ascorbic acid and benzoic acid.Based on its inhibition characteristics and the substrate affinity,the PO was classified as a kind of o-diphenoloxidase.The PO was very sensitive to trypsin-specific inhibitors SBTI,but almost not affected by trypsin-specific inhibitor TLCK at all,indicates that it is like a tyrosine-type protease.Besides,the PO was strongly inhibited by diethyldithiocarbamate(DETC),ethylenediaminetetraacetic acid(EDTA),Cu~(2+),Zn~(2+) and Ca~(2+).The results on DETC,EDTA,and some metal ions,combined with the perfect recovery effect of Cu~(2+) on DETC,EDTA-inhibited PO activities,indicate that Eriociteir PO is most probably a copper-containing metalloenzyme.

引文

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