【作者】 彭博；

【导师】 邵健忠； 项黎新；

【作者基本信息】 浙江大学 ， 细胞生物学， 2008， 博士

【摘要】 本研究在鲫鱼中分离鉴定了5类免疫球蛋白基因,包括IgM基因、IgZ基因和嵌合型的IgMZ1、IgMZ2及IgZ2基因。除了IgZ2基因只分离出分泌型外,其他基因都具有分泌型和膜型两种存在形式。对这些基因序列和分子结构进行系统分析后发现鲫鱼IgZ基因也具有与斑马鱼IgZ类似的结构特点,不同的是,鲫鱼IgZ还能以“分子间”嵌合(IgMZ1和IgMZ2)和“分子内”嵌合(IgZ2)的形式存在;同时在IgM分子中还可能存在类铰链区和补体位点。进一步对嵌合基因形成机制进行研究,发现IgZ类嵌合基因的形成可能发生在DNA水平。IgZ作为一类在硬骨鱼中新发现的抗体类型,其应答方式还未有深入研究。因此,本研究选取典型的胸腺依赖性抗原BSA和胸腺非依赖型抗原LPS以及致病菌嗜水性气单胞菌(Aeromonas hydrophila)免疫鲫鱼,分别研究了IgM、IgZ和IgMZ1在不同刺激条件下脾脏、头肾和外周血中的基因表达变化及在血清、体表和肠道的应答规律。结果显示IgMZ和IgZ主要在体表和肠道等黏膜系统发挥免疫作用,而IgM则在不同器官和组织中都广泛存在,尤其在血清中是主要的抗体类型。本研究还首次在鲫鱼外周血中鉴定出IgM~+、IgZ~+和IgM~+/IgZ~+等不同标记的B淋巴细胞。进一步的研究发现淋巴细胞表面的IgM可能与淋巴细胞的吞噬作用有关,而IgZ则不然,表明不同标记的淋巴细胞可能具有不同的功能。结合以上的研究结果,本研究还对鲫鱼等硬骨鱼类及其他物种中的免疫球蛋白基因进行了进化分析,发现硬骨鱼IgZ类基因可能与哺乳动物IgA有一定的进化关系,并提出了免疫球蛋白基因起源与进化的假说。更多还原

【Abstract】 Recently,a unique immunoglobulin isotype had been identified in teleost namedζ(in zebrafish)orτ(in rainbow trout)besides two universal immunoglobulin heavy chain isotypesμandδ.However,in crucian carp(Carassius auratus),the situation was remain mysterious.In present study,five isotypes were identified and characterized in Carassius auratus in total,including two chimeric isotypes(μ-ζ-1 andμ-ζ-2),twoζisotypes(ζ-1 andζ-2)andμ.Among these isotypes,ζ-2 represent an "intra-chimeric" pattern as Cζ3-Cζ4-Cζ3-Cζ4 structure whileμ-ζ-1 andμ-ζ-2 were showed different "inter-chimeric" splicing patterns as Cμ1-Cμ2-Cζ3-Cζ4(μ-ζ-1)and Cμ1-Cζ2-Cζ3-Cζ4(μ-ζ-2).This diversification was first mentioned in early vertebrate evolution.In addition,a unique hinge-like region was noticed between Cμ1 and Cμ2 inμgene as well as a possible Clq binding site located in Cμ3 domain.To comprehend different Igs’ positions and responsibilities in immune response, crucian carps were immunized with LPS(a typical TI antigen),BSA(a typical TD antigen)and Aeromonas hydrophila(a pathogenic bacteria for crucian carp), respectively.Skin mucus and serum were collected from body surface and caudal vein weekly while intestine protein was obtained at intervals of two weeks during the following 16 weeks.During these phase,a secondary stimulation was performed after 49 days since the first immunization.Sensitive monitoring of the antibody responses was achieved by ELISA and Real-time PCR methods.The results suggested that most secretary IgZ and IgMZ were detected in mucosa-associated immune system while IgM was expressed in all tissues.This expression pattern was much more like IgX in Xenopus and IgA in mammalians.The location of IgM and IgZ in B lymphocyte surface in crucian carps were also been investigated using polyclonal antibodies after peripheral blood lymphocytes isolated from caudal vein.Three markings of lymphocytes were detected in PBL, including IgM+,IgZ+ and IgM+/IgZ+ lymphocytes.In addion,IgM was demonstrated to be involved in lymphocytes phagocytosis of crucian carps by antibody blocking tests.According to these results,the molecular origin and evolution analysis of immunoglobulin heavy chain constant region gene in vertebrates were performed and a hypothetical evolutionary relationship was constructed.更多还原