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为了解不同细菌刺激后仿刺参(Apostichopus japonicus)体腔液中免疫因子的应答变化,分别用灿烂弧菌(Vibrio splendidus)、哈维氏弧菌(V.harveyi)、假交替单胞菌(Pseudoalteromonas nigrifacien)、溶壁微球菌(Micrococcus lysodeikticus)和停乳链球菌(Streptococcus dysgadysgalactiae)注射刺激仿刺参,然后分别采用对硝基苯基磷酸酯(p NPP)底物法、氯化硝基四氮唑蓝(NBT)法、溶壁微球菌粉法和多巴络合物生成法对体腔液上清中的酸性磷酸酶(ACP)与碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、溶菌酶(LYZ)和酚氧化酶(PO)的活力进行了测定。结果显示,灿烂弧菌刺激后,酸性磷酸酶和碱性磷酸酶活力显著升高,而超氧化物歧化酶、溶菌酶和酚氧化酶活力显著降低;哈维氏弧菌刺激后,酸性磷酸酶、超氧化物歧化酶、溶菌酶和酚氧化酶活力显著升高,碱性磷酸酶活力变化不规律;假交替单胞菌刺激后,酸性磷酸酶、溶菌酶和酚氧化酶活力显著升高,超氧化物歧化酶活力先升高后降低,碱性磷酸酶活力变化不规律;溶壁微球菌刺激后,酸性磷酸酶和酚氧化酶活力显著升高,超氧化物歧化酶活力先升高后降低,溶菌酶活力先升高后降低,而后在72 h恢复至对照水平,碱性磷酸酶活力变化不规律;停乳链球菌刺激后,除碱性磷酸酶活力在4 h有所下降外,其余免疫相关酶活力均显著升高。研究结果表明,酚氧化酶是仿刺参非特异性免疫系统中最敏感、高效的免疫因子之一;革兰氏阳性细菌与革兰氏阴性细菌之间在诱导仿刺参免疫因子应答变化上无明显规律性差异;溶壁微球菌诱导溶菌酶的应答变化与灿烂弧菌、哈维氏弧菌、假交替单胞菌和停乳链球菌存在明显差异,溶菌酶可能是仿刺参清除入侵溶壁微球菌的主要免疫因子;灿烂弧菌诱导仿刺参免疫因子应答变化显著不同于其他4株细菌,显示出本研究选取的5个免疫指标在预警灿烂弧菌病害上具有潜在应用价值;停乳链球菌在仿刺参中具有作为免疫增强剂的潜在应用价值。
In order to understand the response changes of immune factors in the body cavity fluid of Apostichopus japonicus after different bacterial stimuli, we used Vibrio splendidus, V. harveyi, Pseudoalteromonas nigrifacien, Micrococcus lysodeikticus and Streptococcus dysgadysgalactiae were used to stimulate S. pseudoacacia, and then were treated with p-nitrophenylphosphate (p NPP) substrate method, nitrotetrazolium chloride The activities of acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) in the body fluid supernatant were determined by NBT method, Micrococcus solutes and dopa complex method. ), Lysozyme (LYZ) and phenoloxidase (PO) activity were determined. The results showed that the activities of acid phosphatase and alkaline phosphatase were significantly increased and the activities of superoxide dismutase, lysozyme and phenoloxidase were significantly decreased after Vibrio sinicus stimulated. After stimulation with Vibrio harveyi, the activity of acid phosphatase , The activities of superoxide dismutase, lysozyme and phenoloxidase were significantly increased, and the changes of alkaline phosphatase activity were irregular. The activities of acid phosphatase, lysozyme and phenoloxidase were significantly increased after pseudo- The activity of superoxide dismutase (SOD) increased first and then decreased, and the activity of alkaline phosphatase changed irregularly. The activity of acid phosphatase and phenoloxidase increased significantly after S. coimicus stimulation, and the activity of superoxide dismutase Lysozyme activity increased first and then decreased, and then returned to the control level at 72 h, alkaline phosphatase activity changes irregularly; Streptococcus dysforin stimulation, in addition to alkaline phosphatase activity decreased in 4 h, Other immune-related enzyme activity were significantly increased. The results showed that phenoloxidase was one of the most sensitive and efficient immune factors in the nonspecific immune system of A. japonicus. No difference was observed in the responses of gram-positive and gram-negative bacteria Significant differences were observed in the response of Lysozymemolyticus to lysozyme. There were significant differences in the response of lysozyme-induced Lysozyme from V. splendidum, V. harveyi, Pseudomonas aeruginosa and Streptococcus dysgalactiae. The main immune factors of Micrococcus paracasei; the changes of immune response to Agaricus viviparum were significantly different from those of the other 4 strains, indicating that the five immune indexes selected in this study had potential application value in early warning of Vibrio enhanus; Streptococcus agalactiae has the potential application value as immunostimulant in the imitation sea cucumber.