南美白对虾类Ig的定性、功能和免疫分子进化的研究

【作者】 章跃陵；

【导师】 彭宣宪；

【作者基本信息】 厦门大学 ， 动物学， 2003， 博士

【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(IgM、IgG、IgA)在单向扩散板和硝酸纤维膜上可分别形成明显的沉淀环和沉淀斑。对虾血清的Western-blotting实验结果显示，与羊抗人Ig发生特异性结合的对虾类Ig的表观分子量为70 kDa(Wp70)，其结合反应可以被特异性阻断和中和。这些与羊抗人IgM、IgG、IgA结合的蛋白质依次称为对虾血清类IgM、IgG和IgA，其在虾血清中的含量依次为0.1578±0.0427、1.2255±0.6844和0.3178±0.1243mg／mL。免疫组化研究证明，对虾组织和血细胞中同样存在与羊抗人Ig发生特异性结合的类Ig物质。对虾类Ig的定性分析，为类Ig的进一步研究奠定了扎实基础。 将免疫亲和层析和蛋白质组学技术相结合建立了亲和蛋白质组学技术。采用该技术对对虾血清中的类Ig进行分离、纯化和鉴定。结果显示，采用羊抗人IgM、IgG、IgA层析得到的对虾血清类Ig在1-D和2-D电泳上均表现为表观分子量分别为75kDa、73kDa、62kDa的三种蛋白(Ap75、Ap73、Ap62)，其中Ap75蛋白与羊抗人IgG反应呈阳性，而Ap73蛋白与羊抗人IgM和IgA反应呈阳性。经MALDI-TOF／MS分析，Ap75、Ap73和Ap62均与南美白对虾血清中的血蓝蛋白具有同源性，其匹配率分别为22％、26％和24％。运用ESI-MS／MS对其进行进一步鉴定，结果发现Ap75和Ap73分别与南美白对虾血蓝蛋白Hcl单体(gi|7414468)和血蓝蛋白前体(gi|1085839)匹配。由此认为，对虾血清中与羊抗人Ig发生特异性结合的蛋白为血蓝蛋白及前体。 选择不同物种范围，对血蓝蛋白及前体PMF进行Mascot检索。结果表明，血蓝蛋白及前体与高等生物的免疫分子和低等生物的识别分子具有同源性。进一厦门大学博士学位论文步利用生物信息学方法对血蓝蛋白及前体进行保守区的搜索、序列比对、三维结构同源建模及进化树的构建，发现血蓝蛋白及前体中含有一个长252个残基，位于其c端的19一l议e区。在血蓝蛋白及前体氨基酸序列中存在多个与人19高度同源的模序，其中血蓝蛋白前体和Hol单体19一like区的motifoll-625(HPCR611一动和motifelg-633在三维结构中的位置与人Ig重链相对应moti玩.55的位置相似。由于两者在此模序中的氨基酸序列仅有1个氨基酸不同，故仅选取血蓝蛋白前体HPcR6，:一625模序进行序列比对分析，结果发现该模序序列EYNI开GSHGvYPDKR、与不同生物的免疫分子(如人Ig、TCR、MHq和识别分子(如细菌粘附分子)具有较高的同源性，是一个新发现的在免疫分子进化过程中的保守模序。尤其值得重视的是，在血蓝蛋白前体与脊椎动物IgSF的分子进化树中，血蓝蛋白前体与兔IgM和人Ig重链等IgSF分子关系非常密切。这些结果提示，血蓝蛋白及前体是一类与人Ig密切相关的新的免疫球蛋白超家族(195玛分子。 利用基因工程技术，对血蓝蛋白基因(Hc基因)及其19一like区(Hc*I基因)、19一like+CuB区(He一IB基因)和。+euA区(Hc一NI基因)进行克隆和原核表达。取各重组蛋白进行以羊抗人Ig为一抗的immunoblot分析。结果表明，Hc，Hc一I和Hc一IB的重组蛋白能与羊抗人Ig反应，而Hc一NI的重组蛋白不能与羊抗人Ig反应。说明血蓝蛋白与羊抗人Ig的结合区域为其C端的Ig一like保守区。进一步研究显示，其两者之间的特异性结合是血蓝蛋白与人Ig的抗原决定簇相似所致。 在对对虾血清类Ig进行了分析和鉴定的基础上，进一步探讨了血蓝蛋白及前体的免疫学功能。结果发现，血蓝蛋白能直接与细菌相互作用，其结合位置在血蓝蛋白为19一like保守区，在细菌为膜蛋白ompx和ompc。血蓝蛋白的lg一like区重组表达蛋白与细菌外膜蛋白的结合可直接导致血清中酚氧化酶活性的变化，其作用机制可能是一种免疫分子与病原微生物之间的分子互作。实验还发现，血蓝蛋白前体对鸡红细胞和10种细菌表现出明显的凝集活性，其凝集活性仅被N一乙酞神经氨酸强烈抑制。研究结果说明，血蓝蛋白是一种重要的抗感染分子。 为进一步阐明血蓝蛋白的免疫防御功能，采用副溶血弧菌人工感染南美白对虾。结果发现，大部分受感染的对虾血清中条带明显增加，其中分子量为28.5kD a的血蓝蛋白裂解片段帅28．5）在12k前后死亡对虾中出现的比例分别为25％和56．5％。经卡方检验，两者之间具有极显著性差异O叙对1卜这些结果提示，该蛋白可能与对虾抗感染免疫的能力有关。 综上所述，本文发现对虾类Ig为血蓝蛋白或其前体，具有免疫防御功能，可能是Ig更多还原

【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 many molecules of the immunoglobulin super family (IgSF) and a peculiar form of adaptive immune response have been documented to be present in an invertebrate. In shrimp, one of invertebrates, there are no direct evidences of an Ig-like molecule. An attempt was made to identify the protein bound to anti-human Ig (Ig-like protein) in shrimp Penaeus vannamei using immunological, proteomic, genetic and bioinformation technologies in the current study.Using immunological methods we found that there were proteins which reacted specifically with goat anti-human Ig (IgM, IgG and IgA), namely Ig-like (IgM-like, IgG-like and IgA-like) proteins, in shrimp serum, haemocyte and tissues. The reactivity could be strongly blocked and neutralized by anti-human Ig and human Ig, respectively. Based on Western blotting analysis we estimated that there was an Ig-like protein about 70 kDa (Wp70) in shrimp serum. And the average concentrations of IgM-like, IgG-like and IgA-like protein were determined to be 0.1578 ?0.0427,1.2255 ?0.6844 and 0.3178 ?0.1243 mg/ml by direct Dot-ELISA, respectively. These findings suggest that there are Ig-like proteins with similar epitopes to human Ig, which was important to purify and identity the Ig-like proteins from shrimp.Therefore, we applied affinity chromatography to isolate IgM-like, IgG-like and IgA-like proteins from shrimp serum with the purified antibody of goat anti-human IgM, IgG and IgA respectively. Three kinds of purified Ig-like proteins were subsequently analysed using 1-DE, 2-DE and their immunoblottings. The results showed that three proteins with 75 kDa (Ap75), 73 kDa (Ap73) and 62 kDa (Ap62) were isolated, of which Ap75 was documented to be strongly reacted with goatanti-human IgQ while Ap73 was bound to both of goat anti-human IgM and IgA. The three proteins were cut out from 2-D gel and subjected to MALDI-TOF mass analysis. The database search results indicated that the Ap75, Ap73, Ap62 proteins showed homology with Litoenaeus vannimei hemocyanin of 22%, 26% and 24%, respectively. To increase our confidence peptides from the digested proteins were also analysed by tandem mass spectrometry. When the ESI tandem MS data were subjected to the database search, Ap75 and Ap73 were identified to be Penaeus vannimei hemocyanin Hcl monomer (gi|7414468) and hemocyanin precursor (gi| 1085839), respectively. Thus, the shrimp Ig-like proteins were determined to be hemocyanin and its precursor.Peptide mass fringerprints (PMF) of hemocyanin and its precursor were subjected to Mascot search, the results indicated that the two proteins shared homology with many of immune molecules of high levels of organisms or recognition molecules of low levels of organisms. Then bioinformatics technologies were used to analyse the structure and evolution of hemocyanin and its precursor. Results searched of conserved domain showed that there was an Ig-like conserved domain of 252 amino acid residues in the C terminus of hemocyanin and its precursor. Amino acid sequence alignment indicated that hemocyanin and its precursor shared seven conserved motifs with human Ig, in which both of motif 611-625 of hemocyanin precursor (HpCR 511-625 motif) and motif ei9-633of hemocyanin Hcl, one amino acid difference between them, were very similar to motif 41.55 of human Ig in their 3D structures respectively. Then we chose the HpCRei 1-625 motif to analyse its evolution status. The results showed that the HpCR6n-625 motif shared high homology with many immune molecules (eg.human Ig, TCR and MHC) or recognition molecules (eg. bacterial adhesion protein) of organisms. It lead to be indicated that HpCR6ii-625 motif is a novel conserved更多还原