大黄鱼肝脏免疫保护蛋白的鉴定

本研究以大黄鱼(Larimichthys crocea)肝脏为研究对象,采用比较蛋白质组学技术分析大黄鱼免疫保护后蛋白表达水平的变化。结果发现,对照组和免疫保护组中共有20个表达水平差异显著的蛋白点,经MALDI-TOF-MS分析,获得18个差异蛋白点肽质量指纹图谱(peptide mass fingerprint,PMF),通过数据库查询,与免疫型受体、新型抗原受体等蛋白具有高度同源性。在此基础之上,进一步选取4个蛋白差异点(1、10、14和18号)进行Westen blot验证。结果表明,该4个蛋白差异点可以与所制备的抗体发生特异性的结合,且变化趋势与蛋白质组学分析结果一致。由此推测,免疫型受体等18个蛋白应该与大黄鱼免疫保护密切相关,所获研究结果为揭示大黄鱼免疫保护机理奠定了良好的基础。教育部新世纪优秀人才支持计划项目(NCET-11-0922)~

Advancement on the function of hemocyanin

广东省自然科学基金(博士启动基金)资助项目(04300750);; 厦门市科技发展资助项

虾类免疫学的基础和应用研究

国家海洋局第三海洋研究所海洋生物工程重点实验室开放课题基金资助项

Study on Ig-like protein of identification, function and evolution of immune molecule in Penaeus vannamei

摘要 随着淡海水养殖业的迅速兴起，对虾等养殖动物的病害问题显得日益突出。人们尝试了多种途径来防治虾病，但收效甚微。由此认为虾类免疫学研究是最终战胜虾类病害的重要基础。一般认为，无脊椎动物体液中不具有免疫球蛋白。但近年来，许多学者在无脊椎动物中证实了免疫球蛋白超家族分子，甚至初级适应性免疫的存在。关于虾内是否存在类Ig物质，尚存在争论。本文运用免疫学，蛋白质组学，基因克隆和生物信息学等现代生物学技术，以我国南方主要养殖虾种——南美白对虾为研究对象，对对虾血清中的类Ig物质进行分离、纯化、鉴定和功能分析，为丰富虾类免疫系统研究，探索对虾疾病免疫学防治奠定基础。 结果表明，对虾血清与羊抗人Ig(...Abstract Shrimp aquaculture has been developed rapidly since it serves as an industrial activity. However, shrimp farms have suffered from dramatic decreases in production due to infectious diseases within the past decade. Interest has been focused on immune molecules and defense mechanisms for the establishment of strategies for disease control. Invertebrate animals lack immunoglobulin, but ...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：B20002600

Approach on Adaptive Immune in Invertebrate

汕头大学博士启动基金资助项目(130-934088)

STUDY ON THREE CLASSES OF IMMUNOGLOBULIN-LIKE COMPONENTS IN Penaeus japonicus

采用单向免疫扩散法 (SRID)和免疫酶斑点法 (Dot ELISA)对日本对虾 (Penaeusjaponicus)血清中类IgM ,IgG ,IgA样物质进行了较系统的研究分析。结果发现 ,虾血清中存在能与羊抗人IgM ,IgG ,IgA发生结合且能被特异性阻断试验确证的物质。文中还对12例对虾血清类IgM ,IgG ,IgA样物质的含量进行了检测。结果提示 ,虾血清中确实存在与人免疫球蛋白 (Ig)特异性抗原决定簇极为相似的物质 ,它的存在对研究虾的抗病机制具有重要意义。Both methods of single radial immunodiffusion(SRID) and dot ElISA were used to characterize three kinds of immunoglobulin like proteins (IgM, IgG and IgA) in serum of Penaeus japonicus. The results showed that there were components reacted with anti human μ,γ or αchain in the serum, which could be blocked by specific tests. Furthermore, the IgM like, IgG like and IgA like components were measured in 12 cases of Penaeus japonicus. These findings suggest that there were materials with similar isotopes in serum from both the shrimp and human. And Ig like proteins in the shrimps were important to understand their disease free mechanism.国家海洋局第三海洋研究所海洋生物工程重点实验室 开放课题基金!资助项

Circulating immune complexes of some fresh water fishes

采用SDS -PAGE技术和免疫酶斑点法 (Dot -ELISA) ,对罗非鱼、草鱼、鲫、鳙和鲤 5种淡水鱼的血清免疫复合物 (IC)进行了研究。结果表明 ,鲤科的 4种鱼之间的电泳条带差异较小 ,丽鱼科的罗非鱼与鲤科的 4种鱼之间的电泳条带有差异但较小。 2 %PEG能较好地沉淀 5种鱼血清IC ,且不同鱼血清IC含量有一定的差异。Circulating immune complexes of Tilapia, Ctenopharyngodon idellus, Carassius auratus, Aristichthys nobilis and Cyprinus carpio were characterized by SDS-PAGE and Dot-ELISA. The results show that the circulating immune complexes of the 5 fishes can be isolated by 4% PEG, and the differences between the 4 fishes of Cyprinidae are not very obvious in the bands of electropherogram, and neither does the difference between Tilapia and the 4 Cyprinidae fishes, but there are some obvious difference between these immune complexes, which may be related to the molecular weight of their antibodies.国家自然科学基金资助项目 ( 39770 5 85 ) ;; IFS基金资助项目 (A/2 338-2

Quick identfication for pathogenic vibrio of aquacultivated creatures by UPPCR-SSCP

采用通用引物PCR配合SSCP分析即UPPCR SSCP技术 ,对弧菌科弧菌属中的副溶血弧菌、溶藻酸弧菌、费尼斯弧菌、鳗弧菌、哈维氏弧菌的 1 6S RNA基因进行检测。结果发现 ,UPPCR SSCP技术能较好地鉴别细菌的不同种 ,但对同种不同株细菌的区分较困难。认为PCR SSCP是将病原菌判定至种的有力工具。此研究对水产养殖病原菌的快速诊断与及时防治有重要意义。By universal primer PCR (UPPCR) amplification and SSCP, 16S DNA gene fragments of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio furnissii, Vibrio anguillarum, Vibrio harveyi had been detected in this paper. It was found that different species of bacteria could been easily identified by this technique, but the same species could not. The findings suggest that UPPCR SSCP may be a potential tool for identifying the species of pathogenic bacteria. Therefore, this study will be of benefit in quick diagnosis and precaution in aquacultivated creatures.国家自然科学基金!(39770 5 85号 );; 国家海洋局第三海洋研究所海洋生物工程重点实验室开放课题;; IFS基金资助项 目!(A/ 2 338 2号

On immunolocalization of Ig-like proteins in shrimp Litopenaeus vannamei

以凡纳滨对虾(Litopenaeus vannamei)为研究对象,运用Western-blotting和免疫组织化学等现代免疫学方法,对对虾中的类Ig(immunoglobulin,免疫球蛋白)蛋白进行定性和定位研究。结果发现,虾血清中有两种分子质量约为70 ku和62 ku的蛋白可与抗人Ig发生特异性的结合。同时,虾血细胞——无颗粒细胞的外膜及虾类淋巴器、肝胰脏、心脏、胃和肠等5种组织器官的石蜡切片与抗人Ig反应呈不同程度的阳性,其中类淋巴器和肝胰脏显色较深。提示,虾体内确实存在能与抗人Ig发生特异性结合的类Ig蛋白,其不仅存在于虾血清中,还存在于无颗粒细胞、类淋巴器和肝胰脏等组织中,为对虾的免疫系统的研究和疾病的防治奠定基础。In the paper, an attempt was made to analysis the immunolocalization of Ig-like proteins that bound to anti-human Ig (immunoglobulin, Ig) in shrimp L. vannamei using modern methods of Western-blotting and immunohistochemistry. The results showed two types of protein which reacted strongly with goat an-ti-humanIg in the shrimp hemolymph, whose molecular weights were about 70 ku and 62 ku respectively. And the outer membrane of the hyalocyte and five tissues including lymphatic-like organ, hepatopancreas, heart, intestine and stomach were also bound with anti-human Ig specially, of which, the lymphatic-like organ and hepatopancreas were stained stronger than other samples. These results indicated that there do exist Ig-like proteins in the shrimp, which localized not only in hemolymph but also in haemocytes, lymphatic-like organ and hepatopancreas.国家“863”计划项且基金(2002AA629050);; 广东省自然基金——博士启动基金(130—122187

Variation of phenoloxidase activity affected by hemocyanin in shrimp Penaeus vannamei

以采自厦门的南美白对虾(Penaeusvannamei)为研究对象,运用病原菌人工感染、血蓝蛋白浓度和酚氧化酶(phenoloxidase,PO)活性测定等方法探索南美白对虾血蓝蛋白在体内、外对其血清酚氧化酶活性的影响。结果表明,南美白对虾人工感染副溶血弧菌后,其血淋巴中血蓝蛋白浓度和PO活性的变化趋势基本一致。在体外实验中将一定量的血蓝蛋白重组蛋白添加至血清中,其PO活性可明显升高;但同时添加血蓝蛋白重组蛋白和E.coliK12超声破碎液后,其PO活力反而降低。研究提示,血蓝蛋白在一定范围内确实可以正反馈调节PO活性,但在细菌等干扰之下,其又可表现出一定的负反馈抑制活性。It has been confirmed that hemocyanin is a kind of important multifunctional protein found in the hemolymph of both arthropod and mollusk. And phenoloxidase is a type of vital immune protein in invertebrate. Recently,interest has been focused on comparative research between hemocyanin and phenoloxidase. In the paper,a preliminary study was made to characterize the variation of phenoloxidase activity affected by in vivo hemocyanin and in vitro hemocyanin recombinant protein in shrimp Penaeus vannamei obtained from Xiamen. The methods of artificial infection by V. parahaemolyticus and measurement of both concentration of hemocyanin and phenoloxidase activity were used. The results showed that the variational trend of hemocyanin's concentration was similar to that of phenoloxidase activity after the shrimp was infected by V. parahaemolyticus,both of which increased to their maximum in 24?h,then descended slowly,finally came back to normal level in 36?h. In comparison with control group in vitro, the shrimp serum's phenoloxidase activity increased obviously after hemocyanin recombinant protein was added in at the concentration of 200?μg/mL or ultrasonic solution of E. coli K12 at the concentration of 35?mg/mL. However,when both hemocyanin recombinant protein and ultrasonic solution of E. coli K12 were added into the shrimp serum, its phenoloxidase activity descended immediately. It suggests that hemocyanin can adjust the phenoloxidase activity by means of positive feedback,but it also can adjust the phenoloxidase activity by means of negative feedback when its reactive solution is involved in some bacteria. In summary,these results are the latest discovery in the field of the immune function of hemocyanin and phenoloxidase,which will be helpful for elucidating the immune mechanism of hemocyanin and interrelation between hemocyanin and phenoloxidase.国家“863”计划项目基金(2002AA629050);; 广东省自然科学基金(博士启动基金)资助项目(130-122187).~