中国对虾血细胞酶细胞化学的初步研究

运用电镜酶细胞化学技术 ,对中国对虾 (Penaeus chinensis)的血细胞和血清中的酚氧化酶、酸性磷酸酶、碱性磷酸酶和过氧化物酶的酶活性部位进行标记。实验结果显示 :酚氧化酶主要以酶原形式存在于大颗粒细胞的颗粒体中 ,酸性磷酸酶和碱性磷酸酶以酶原形式存在于小颗粒细胞的颗粒体中。健康虾和患病虾血细胞中酶活性的表现形式不尽相同 ,在病理情况下 ,颗粒细胞能以细胞解体的方式 ,排出细胞内的颗粒 ,转化成有活性的酶 ,以此提高血淋巴中这几种酶的活性。透明细胞可以吞噬排放颗粒后的颗粒细胞 ,透明细胞中由高尔基体合成的酸性磷酸酶等溶酶体酶主要用于透明细胞的细胞内消化作用。研究认为 :3种血细胞因含有不同的免疫酶以及酶的作用方式不同而具有不同的免疫功能     

菲律宾蛤仔(Ruditapes philippinarum)漆酶型酚氧化酶的纯化及特性分析

采用非变性电泳结合特异性底物发色方法从菲律宾蛤仔血细胞中分离出一种酚氧化酶,研究了该酶的酶促反应动力学参数,金属离子、抑制剂对酶活的影响。结果表明,分离获得的酚氧化酶分子量约为563kDa;对底物邻苯二酚、左旋多巴(L-DOPA)、多巴胺、对苯二酚的米氏常数Km分别为0.42、1.60、1.79和1.76mmol/L;酶的活性在Mg2+、Zn2+、Mn2+、Fe2+、Cu2+和Ca2+浓度为5、10、20、30mmol/L时受到抑制;酶的活性可被抑制剂抗坏血酸、二乙基二硫代氨基甲酸钠(DETC)、半胱氨酸、亚硫酸钠、柠檬酸、乙二胺四乙酸二钠(EDTA)、叠氮钠和硫代尿素等抑制。根据底物特异性、抑制剂等结果,所分离获得的酚氧化酶属于漆酶型的含铜酚氧化酶。     

四种免疫促进剂对日本蟳酚氧化酶和血细胞的影响

为查清免疫促进剂对日本蟳（Charybdis japonica）（俗称石蟹）自身免疫力的增强作用及机理，对脂多糖（LPS）、β葡聚糖（β—1，3-glucan）、灭活鳗弧菌和灭活哈维氏弧菌对日本蟳酚氧化酶（po）的产量与活性以及血细胞的数量与超微结构的影响进行了研究。结果表明，经4种免疫促进剂处理后，日本蟳PO产量和总酶活性都有显著增加，但PO的比活性与对照相比没有显著差异。日本蟳血细胞超微结构观察显示，多糖处理组小颗粒细胞和大颗粒细胞的糙面内质网和线粒体数量增加，颗粒数量减少、体积增大，而透明细胞的超微结构没有显著变化；灭活弧菌处理组小颗粒细胞中的颗粒数量明显减少，透明细胞中线粒体的数量显著增加。由此可见。多糖类和灭活弧菌类免疫促进剂对日本蟳非特异性免疫系统的影响不同，多糖类免疫促进剂主要提高酚氧化酶原激活组分PO的产量，进而提高PO总酶活性，而灭活弧菌类免疫促进剂主要提高透明细胞和小颗粒细胞的吞噬活性。     

注射病原菌对凡纳滨对虾血蓝蛋白合成、酚氧化酶活力的影响

研究注射病原菌对凡纳滨对虾血蓝蛋白合成、酚氧化酶活力的影响。结果表明:注射哈维氏弧菌对凡纳滨对虾血淋巴中血蓝蛋白含量、肝胰脏内血蓝蛋白mRNA和p75、p77亚基表达以及血淋巴、血细胞、血蓝蛋白酚氧化酶活力的影响显著(P<0.05),而对照组无显著变化。血蓝蛋白含量和p75、p77亚基表达在0～24 h内呈峰值变化,其中血蓝蛋白含量在6 h内逐渐下降、且6 h时达到最低值,而p75、p77亚基表达在3 h内无明显变化,然后均表现为逐渐上升趋势,各指标在12 h时达到最大值,而且亚基表达与注射哈维氏弧菌浓度呈正相关,24 h后各指标趋于稳定,与对照组无显著差异。在注射哈维氏弧菌短时间内血蓝蛋白mRNA表达呈迅速上升趋势,其中注射6×106cells/mL处理组mRNA表达在6h内呈峰值变化,3 h时达到最大值,6～48 h内保持稳定,但仍明显高于对照组水平(P<0.05),而注射6×105cells/mL处理组mRNA表达量虽然增加,但只在6 h达到最大值时与对照组差异显著(P<0.05),其余时间均与对照组差异不显著。血淋巴、血细胞和血蓝蛋白酚氧化酶活力分别在12 h,24 h内呈峰值变化,其中血淋巴、血蓝蛋...     

中国对虾血清中酚氧化酶活力研究

１９９５年７－９月,以养殖中国对虾为材料,实验室用弧菌菌悬液注射感染对虾,血甭中测得一定量的酚氧化酶（ＰＯ）。正常状态下对虾血清中ＰＯ活力变化不大,而濒死对虾血清中ＰＯ活力变化较大。较低浓度的菌悬液能有效地提高ＰＯ活力,促使有机体对病原体的防御能力,这在实践中有重要意义。     

Effects of dietary vitamin C on the immune function of shrimps, Penaeus chinensis

Prepared in this experiment were six groups of diets, i.e. VC0, VC1, VC2, VC3, VC4 and VC5 with the contents of vitamin C (VCmg(100 g)⁻¹ diet) of 0, 100, 200, 400, 800, and 1200 respectively. It was found that vitamin C increased the concentrations of immunoglobulin-like (IgG-like, IgA-like and IgM-like) substances in the serum of Penaeus chinensis after a feeding period of 3 weeks. The differences among groups were significant (P<0.01), but there was no difference in the contents of complement3-like and complement4-like substances in the serum (P>0.05). Phenoloxidase (PO) activity in the serum of VC3 group shrimps was higher than that of VC0 and other groups, but no significant difference was observed between VC0 group and other groups. Furthermore, bactericidal activity of the serum to Vibrio parahaemolyticus in shrimps fed with the VC1 diet was higher than that in the other groups (P<0.01), while no difference was demonstrated among all groups for the bactericidal activity to Vibrio alginolyticus (P>0.05). It is, therefore, suggested that vitamin C (100–400 mg(100 g)⁻¹ diets) could be used as an immunostimulant of P. chinensis.     

日本蟳酚氧化酶的分离纯化及其部分生物化学性质研究

利用离子交换层析和凝胶过滤层析等方法,从日本(Charybdis japonica)血淋巴中分离纯化出了酚氧化酶,并以L-二羟苯丙氨酸(L-DOPA)作为特异性底物对其生化性质和酶性质进行了研究。结果表明,酚氧化酶和酚氧化酶原的分子质量分别为64.5 ku和69.5 ku。以L-DOPA为底物对酚氧化酶纯品进行研究发现,其最适pH值为6.0、最适温度为40℃。对底物L-DOPA和儿茶酚的米氏常数Km值分别为3.41和7.97 mmol/L。该酶对亚硫酸钠、苯硫脲极为敏感,对硫脲、苯甲酸非常敏感,表明该酶很可能是一种儿茶酚酶型的酶。此外,EDTA,DETC,Zn2 ,Mg2 和Cu2 均能显著抑制该酶活性,且10 mmol/L Cu2 能有效地回复该酶被DETC所抑制的酶活性,表明该酶确为一种Cu-金属酶。     

Effects of Several Immunostimulants on Phenoloxidase and Hemocytes of the Crab Charybdis japonica

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.     

Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species

Phenoloxidases(POs)are a group of copper proteins including tyrosinase,catecholase and laccase,which play crucial roles in the innate immune response of mollusks.In this research,POs were studied in cultured mollusk species,including scallop Chla-mys farreri,abalone Haliotis discus hannai and clam Scapharca subcrenata.The POs were isolated from hemocytes using linear-gradient native-PAGE combined with catechol staining.The PO activities and their characters were investigated.The molecular mass of PO in C.farreri was 576 kDa,and it was 228 kDa in H.discus hannai.In S.subcrenata,four POs were detected and their mole-cular masses were 391 kDa,206 kDa,174 kDa and<67 kDa,which were named as 391-PO,206-PO,174-PO and s-PO,respectively.Ki-netic analyses indicated that all of the POs,except for 391-PO had higher affinity to L-DOPA and catechol than to hydroquinone and dopamine.However,all of the POs failed to oxidize tyrosine.The effects of divalent metal ions on POs’activities were assayed,in-cluding Fe²⁺,Mg²⁺,Zn²⁺,Mn²⁺,Cu²⁺and Ca²⁺from FeCl₂,MgSO₄,ZnSO₄,MnCl₂,CuSO₄and CaCl₂.The POs were inhibited by Fe²⁺at all determined concentrations.Additionally,the inhibition assay showed that all of the POs were inhibited by cysteine,ascorbic acid,sodium sulfite,citric acid,ethylenediaminetetraacetic acid disodium(EDTA)and sodium diethyldithiocarbamate(DETC).The inhibition effects of critric acid and EDTA are dose-dependent.H.discus hannai PO and 391-PO were slightly inhibited by sodium azide,and H.discus hannai PO,391-PO and 174-PO were slightly inhibited by thiourea.In conclusion,the POs in the three cultured mollusks are copper-containing laccase-type phenoloxidases with similar biochemical characteristics even though their molecular masses are different.     

Effects of Dietary Vitamin C on the Immune Function of Shrimps, Penaeus chinensis

Prepared in this experiment were six groups of diets, i.e. VC0, VC1, VC2, VC3, VC4 and VC5 with the contents of vitamin C (VCmg(100g)-1 diet) of 0, 100, 200, 400, 800, and 1200 respectively. It was found that vitamin C increased the concentrations of immunoglobulin-like (IgG-like, IgA-like and IgM-like) substances in the serum of Penaeus chinensis after a feeding period of 3 weeks. The differences among groups were significant (P＜ 0.01), but there was no difference in the contents of complement3-1ike and complement4-1ike substances in the serum (P ＞0.05). Phenoloxidase (PO) activity in the serum of VC3 group shrimps was higher than that of VC0 and other groups, but no significant difference was observed between VC0 group and other groups. Furthermore, bactericidal activity of the serum to Vibrio parahaemolyticus in shrimps fed with the VC1 diet was higher than that in the other groups (P＜ 0.01), while no difference was demonstrated among all groups for the bactericidal activity to Vibrio alginolyticus (P ＞0.05). It is, therefore, suggested that vitamin C (100-400 mg(100 g) -1 diets) could be used as an immunostimulant of P. chinensis.