【作者】 严芳；

【导师】 李远友； 章跃陵；

【作者基本信息】 汕头大学 ， 海洋生物学， 2011， 博士

【摘要】 目前我国是世界上最大的甲壳动物(虾蟹)集约化养殖生产国。然而,与国际虾蟹养殖业相比,我国商业化的虾蟹养殖整体效益较低,可持续发展前景不容乐观。究其原因,主要与虾蟹病害和生物安全等有关。为此,学者们认为选育优质、抗逆优良虾蟹品种,建立安全、生态、环保的健康养殖模式,是解决目前虾蟹养殖疾病,提高虾蟹品质的有效途径。要成功解决这些问题,关键环节之一就是揭示虾蟹自身免疫防御机制,寻找虾蟹病害防治的有效方法。近年来研究表明,存在于对虾等无脊椎动物血淋巴中的血蓝蛋白是一种具有抗病毒和抗细菌等多种免疫活性的多功能蛋白。本研究以凡纳滨对虾、锯缘青蟹血蓝蛋白为研究对象,在国内外所获研究结果的基础之上,进一步对其免疫学活性及作用机理等进行了深入的研究,所获研究结果具体如下:1、发现凡纳滨对虾血蓝蛋白抑菌活性与其糖基化修饰有关,且OmpC为其抑菌作用靶标之一首先,在本课题组已分离得到的2种糖含量和抑菌活性存在显著差异的凡纳滨对虾血蓝蛋白(LHt和LH75)的基础之上,本研究采用凝集素印迹、Tricine-SDS-PAGE和HPLC等技术,进一步发现LH75、LHt糖基化修饰存在显著性差异,其中前者α-D-甘露糖、α-D-葡萄糖修饰程度比后者高;前者胰酶水解肽段分子量明显高于后者;前者胰酶酶解HPLC图谱表现为10个峰,而后者为24个峰。同时,2-DE显示,LHt主要蛋白点为7个,大小亚基分别为3和4个点,LH75主要蛋白点为6个,大小亚基分别为4和2个点。由此说明该2种血蓝蛋白不仅在蛋白质水平存在差异,其糖基化修饰也存在差异。继而,选用副溶血弧菌人工感染对虾,发现对虾刺激后虾血清中LHt、LH75表达均呈上升趋势,且LH75上升幅度明显高于LHt。结合前期研究发现LH75抑菌活性显著高于LHt抑菌活性的研究结果,提示LH75可能是血蓝蛋白发挥抑菌作用的重要组分,其抑菌活性应该与糖基化修饰有关。最后,利用亲和孵育获得3种与LH75相结合的副溶血弧菌外膜蛋白(Omp), p1、p2和p3,经质谱鉴定p2为OmpC,根据前期研究发现LH75对外膜蛋白敲除菌△OmpC的抑菌活性明显降低的结果,推测OmpC应为为LH75抑菌作用靶标之一,至于其是否还存在其他作用靶标还有待于进一步研究和证实。2、发现锯缘青蟹血蓝蛋白具有凝集活性,凝集作用靶标为OmpA和OmpX首先,采用亲和蛋白质组学策略,发现锯缘青蟹血蓝蛋白由分子量分别为70、72、75、76、80 kDa的5个亚基组成,其与岸蟹(Carcinus aestuarii)、蓝蟹(Callinectes sapidus)和黄道蟹(Cancer magister)血蓝蛋白亚基具有高度的同源性,且3个主要亚基(p75、p76和p80)与兔抗虾血蓝蛋白抗血清呈显著阳性,说明所获得的青蟹血蓝蛋白成分单一、可靠,适合于进一步研究。继而,采用凝集实验和凝集抑制实验,发现青蟹血蓝蛋白对副溶血弧菌、溶藻酸弧菌、河弧菌、哈维氏弧菌、嗜水气单胞菌、大肠杆菌K12和金黄色葡萄球菌等7种细菌具有凝集活性,且其凝集活性能被葡萄糖、半乳糖、木糖和N-乙酰神经氨酸部分或完全抑制。说明青蟹血蓝蛋白确实具有凝集活性。最后,运用SDS-PAGE、Western-blotting、凝集抑制实验、基因敲除凝集实验等发现青蟹血蓝蛋白凝集活性作用亚基为76 kDa亚基;大肠杆菌Omp能够抑制血蓝蛋白对7种细菌的凝集活性;与大肠杆菌K12野生菌株相比,血蓝蛋白对△OmpA和△OmpX凝集活性明显降低。由此推测,青蟹血蓝蛋白可能主要依赖其76 kDa亚基,通过与病原菌OmpA和OmpX相结合而发挥凝集活性。3、发现锯缘青蟹血蓝蛋白具有溶血活性,溶血作用机制为胶体渗漏机制采用溶血实验发现锯缘青蟹血蓝蛋白对鸡、鼠、兔、人等多种红细胞表现出依赖于钙离子的溶血活性。其溶血活性具有“剂量-效应”效应,同时对pH、温度敏感,在pH 5-8范围内,溶血活性随pH上升而降低;随着温度的升高,溶血活性逐渐增强, 37℃时溶血活性达到100%。在上述研究的基础之上,进一步采用SDS-PAGE、Western-blotting、渗透保护实验等,发现锯缘青蟹血蓝蛋白5个亚基均可与红细胞膜相结合,且青蟹溶血活性随着渗透保护剂分子的增大而降低。由此推测,青蟹血蓝蛋白可能依赖其全部亚基通过胶体渗漏机制而发挥溶血活性。综上所述,本课题研究主要发现:甲壳动物(虾、蟹)血蓝蛋白具有凝集活性、抑菌活性和溶血活性,其中凝集、抑菌活性作用靶标为细菌外膜蛋白,溶血作用机制为胶体渗漏机制。所获研究结果为揭示血蓝蛋白的免疫学分子作用机制,阐明血蓝蛋白与病原菌的识别模式奠定了良好的基础,同时为研究血蓝蛋白在虾蟹中的免疫抗病作用提供了较好的实验依据,对丰富和发展甲壳类动物免疫系统的基础研究及指导免疫学防治具有重要的意义。更多还原

【Abstract】 At present, crustaceans production of China is the largest in the world. However, the whole benefit of commercial crustaceans cultivation in our country is lower compared with the international shrimps and crabs farming, and the outlook of sustainable development is not optimistic. For the reasons related with disease and bio-security, most researchers consider that the effective strategies to prevent crustacean diseases and improve crustacean quality are breeding for stress tolerance species and establishing safe, ecotypic, protecting environmental and healthy culture model. It has been proved that one of the key to successfully resolve these problems is to reveal the self-defense mechanism of crustacean and search for the effective strategy to prevent crustacean diseases. Recently, hemocyanin of mollusks and arthropods was reported as an important non-specific immune protein present in the hemolymph with multifunction such as antiviral and antibacterial activities. In this paper, an attempt was made to investigation the immunological functions and their mechanisms of hemocyanins from Litopenaeus vannamei and Scylla serrata. The main findings are as follows:1. Hemocyanin from L. vannamei showing antibacterial activity was related to its glycosylation and OmpC was appeared to be one of the antibacterial targetFirstly, on the basis of our previous findings that two L. vannamei hemocyanins (named LHt and LH75) presenting different carbohydrate content and antibacterial activity were purified, in the current study, the methods of lectin-blotting, Tricine-SDS-PAGE and HPLC were used to analyse the variation of the two hemocyanins. The results indicated that the glycosylation level ofα-D-mannose /α-D-glucose content of LH75 was higher than that of LHt. And molecular weights of main peptides digested by trypsin,of LH75 were also higher than those of LHt. In additional, 10 and 24 peaks of digested LH75 and LHt were observed in HPLC map, respectively. Furthermore, 7 and 6 protein spots were appeared in the 2-DE map of LHt and LH75, respectively, in which, the former contained 3 big subunit spots and 4 small subunit spots, while the later contained 4 big subunit spots and 2 small subunit spots. Thus, these results suggested two hemocyanins were probably discrepant with glycosylation.Secondly, more LH75 increased than that of LHt in serum after injecting Vibro parahaemolyticus. Combined to the previous results that antibacterial activity of LH75 was obvious higher than that of LHt, these results suggested that LH75 was a important component of hemocyanin with antibacterial activity, which was related to glycosylation.Finally, three V. parahaemolyticus outer membrane proteins (Omps) binding to LH75 was separated by affinity incubation, which named p1, p2, p3 respectively. The p2 was identified as OmpC by MALDI-TOF-TOF MS. Our previous research showed that antibacterial activity of LH75 with E. coli Omps gene-deleted mutants,△OmpC, decreased obviously in comparison with that of wild type. Thus, the results manifested that OmpC appeared to be one of the antibacterial target of LH75 and its other antibacterial target need further examination.2. S. serrata hemocyanin mediates agglutination by recognizing OmpA and OmpX of bacteria.First of all, we identified the S. serrata hemocyanin using affinity proteomics and the results showed that S. serrata hemocyanin consisted of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. All five subunits showed high homology with hemocyanin subunits from other crab species including Carcinus aestuarii, Callinectes sapidus and Cancer magister, and three mainly subunit p75、p76 and p80 reacted with the rabbit anti-hemocyanin IgG. Together, these results suggested that S. serrata hemocyanin was purified to homogeneity and suitable for further investigation.Furthermore, seven bacterial species including V. parahaemolyticus, V. alginolyticus, V. harveyi,V. fluvialis, Aeromonas hydrophila, Staphylococcus aureus and Escherichia coli K12 were selected for agglutination and agglutination inhibition analysis. S. serrata hemocyanin presented agglutinative activities against the seven pathogenic bacteria ranging from 7.5-30 g/mL, which was inhibited by N-acetylneuraminic acid,α-D-glucose, D-galactose and D-xylose, respectively. These results suggested that S. serrata hemocyanin possessed agglutination properties.Additionaly, SDS-PAGE, Western-blotting, agglutination inhibition and gene knock-down analysis were performed. The results showed that the 76 kDa subunit of S. serrata hemocyanin was responsible for its agglutinative activities. Moreover, Omps of bacteria could completely stop the agglutination. The agglutinative activities of hemocyanin with△OmpA and△OmpX were obviously decreased. Together, the data suggest that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination by recognizing OmpA and OmpX of bacteria.3. S. serrata hemocyanin mediated hemolysis by binding tightly to erythrocyte membranes and following a colloid-osmotic mechanism.Hemolysis assays was performed to investigate the hemolytic activity of S. serrata hemocyanin. The results showed that S. serrata hemocyanin exhibited Ca2+-dependent hemolytic activities against vertebrate erythrocytes including chicken, mouse, rabbit and human erythrocytes. The hemolysis assay of hemocyanin showed the“dose-response”positive relativity, and the hemolysis was sensitive to pH and temperature. The hemolytic activity of hemocyanin decreased with increasing pH from 5 to 8, and increased with the increasing temperature, the 100% hemolysis was occured at 37 oC. Further evidence revealed that the five subunits of hemocyanin were presented in the solubilized incubation products of erythrocytes with hemocyanin, and the hemolysis could be inhibited to different degrees by osmoprotectants with various molecular masses, indicating that S. serrata hemocyanin mediated hemolysis by binding tightly to erythrocyte membranes and following a colloid-osmotic mechanism. Thus, these finding suggested that S. serrata hemocyanin also can act as a hemolysin.In summary, our results indicated that hemocyain of crustacean (shrimps and crabs) showed agglutinative activity, antibacterial activity and hemolytic activity. And bacterial Omps were acted as the agglutinative and antibacterial targets of pathogen recognized by hemocyanin. Importantly, the mechanism of hemocyanin-induced hemilysis was colloid-osmotic. These findings lay the foundation for revealing the mechanism of immunological functions of hemocyanin and elucidating the pattern of recognizing pathogenic bacteria by hemocyanin. Also, they can provide evidence on the anti-diseases activitives of hemocyanin in crustacean, and will help us understand the immune status of hemocyanin in shellfish such as shrimps and crabs and develop the knowledge of shellfish’s immune system and immune prevention.更多还原