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题名: 欧鳗免疫球蛋白基因克隆与表达的研究
作者: 冯建军
答辩日期: 2008-06-14
导师: 关瑞章
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 欧鳗 ; 免疫球蛋白 ; 免疫球蛋白阳性细胞 ; 重链 ; 轻链 ; 基因克隆 ; 基因表达 ; Real-time PCR
其他题名: Cloning and Expression of the Genes Encoding the Immunoglobulin of European Eel, Anguilla.anguilla
摘要: 欧洲鳗鲡(Anguilla anguilla Linnaeus 1758)简称欧鳗,是我国重要的水产养殖鱼类,对其免疫系统的研究在理论和应用方面都具有重要意义。本文研究了欧鳗血清免疫球蛋白(Immunoglobulin, Ig)的分离纯化、 Ig 阳性细胞的组织定位、Ig 重链(Immunoglobulin heavy chain, IgH)、Ig 轻链(Immunoglobulin light chain, IgL) cDNA 的克隆与分析、原核表达以及时空表达等。研究结果为进一步阐明欧鳗免疫应答机理、制备基因工程抗体以及抗体基因转移育种等提供重要的参考依据。主要研究内容及结果如下: 1. 采用蛋白 A 亲和层析和山羊 IgG-琼脂糖免疫亲和层析两种方法对欧鳗血清 Ig 进行分离纯化,同时制备了相应的兔抗欧鳗 Ig 抗血清。SDS-PAGE凝胶电泳显示:经蛋白 A 亲和层析纯化后的 Ig 有两条分别为分子量约 68 ku 的重链和 26 ku 的轻链清晰蛋白条带,经山羊 IgG-琼脂糖免疫亲和层析纯化后的 Ig 在 SDS-PAGE 凝胶有三条清晰的蛋白条带,分别为分子量约68 ku的重链、分子量为26 ku和28 ku的两条轻链;兔抗欧鳗 Ig 血清经酶联免疫吸附法测定的效价均达到1:51200。Western-blotting结果显示兔抗欧鳗血清能清晰识别欧鳗血清的68 ku的重链,约在56 ku也出现一条信号较强的蛋白带,但轻链与兔抗鳗鲡 Ig 抗血清反应较弱。 2. 运用免疫组织化学技术研究了嗜水气单胞菌(Aeromonas hydrophila) 感染的实验组欧鳗和未经感染的对照组欧鳗脾脏、肾脏和肝脏 Ig 阳性(Ig+) 细胞定位与分布特点。结果表明:对照组欧鳗脾脏 Ig+ 细胞数较少,肾脏相对较多,肝脏未发现。实验组欧鳗脾脏 Ig+ 细胞数量明显比对照组增多,肾脏有所增多但不明显,肝脏未发现。在实验组和对照组中,黑色素巨噬细胞在脾脏和肾脏中均见明显聚集并与淋巴细胞形成黑色素巨噬细胞中心(Melano-macrophage centres, MMCs),其中脾脏 MMCs 多数位于血管附近,脾脏 Ig+ 细胞均主要分布在 MMCs 及其周边,且在实验组中数量多而密集,而肾脏 Ig+ 细胞在对照组中明显分布于 MMCs 及其周边,但在实验组不明显。与对照组相比,实验组中脾脏和肾脏黑色素巨噬细胞数量和体积均有明显增多。本试验结果说明欧鳗肾脏和脾脏是具有 Ig+ 细胞的重要免疫器官,其中黑色素巨噬细胞数量和体积变化很可能与免疫应答过程密切相关。 3. 采用RT-PCR和RACE方法克隆到的欧鳗 IgH 全长cDNA共2,089 bp,其中所编码的 IgH 蛋白分子含有 581 个氨基酸残基,由前导肽、可变区、恒定区1、恒定区2、铰链区、恒定区3、恒定区4以及 C 末端序列构成。经氨基酸序列分析表明恒定区 3 区存在其它鱼类未见报道的两个连续糖基化位点,该位点与形成链内二硫键半胱氨酸邻近,且在半胱氨酸下游 3 bp 处发现结合补体 C1q 的氨基酸基序‘His-X-Asp-X-X-X-Pro’。IgH恒定区的氨基酸序列与海鲢(Elops saurus)、大西洋鲑 (Salmo salar)、虹鳟 (Oncorhynchus mykiss)、 草鱼 (Ctenopharingodon idella)、 鲤鱼 (Cyprinus carpio)、斑点叉尾鮰 (Ictalurus punctatus)和斜带石斑鱼(Epinephelus coioides) 的同源性分别为 46.1%、 39.7%、 38.9%、 32.4%、 32.3%、 31.7% 和 30.7%。 4. 根据已经克隆的欧鳗 IgH 全长cDNA序列构建了欧鳗IgH链重组表达质粒,并在大肠杆菌中实现了高效表达。经Western blotting分析,目的基因表达产物都能被兔抗欧鳗 Ig 的抗血清特异性识别,表明原核表达的欧鳗 IgH 融合蛋白具有天然 Ig 的免疫原性。 5. 采用RT-PCR和RACE方法克隆到的欧鳗 IgL全长cDNA共1016 bp,其中所编码的 IgL 蛋白分子含有238 个氨基酸残基,由前导肽、可变区和恒定区组成。经氨基酸序列分析后发现两个对 IgH 和 IgL 结构域的聚合作用必须的两个基序,分别是位于可变区 FR2的 ‘PELLT’ 以及位于可变区 FR4的 ‘FGGG’。将欧鳗IgL 恒定区序列与其它23种脊椎动物的相应序列进行比较并构建系统发育树,结果显示欧鳗轻链与虹鳟和大西洋鲑轻链 L1 型群聚为一簇,据此克隆的欧鳗轻链属于轻链L1型。根据已经克隆的欧鳗 IgL 全长cDNA序列,成功构建了具有双酶切位点欧鳗IgL成熟肽重组表达质粒,为 IgL 在大肠杆菌中的表达奠定基础。 6. 利用荧光定量PCR技术研究了经山羊 IgG 免疫注射欧鳗前后 Ig 在肾脏、脾脏、肝脏、鳃、肌肉以及心脏的分布和表达量的变化。之后研究了山羊IgG 免疫注射欧鳗后 28 d Ig在肾脏、脾脏和鳃中表达的时空变化。结果表明免疫接种前后欧鳗肾脏、脾脏和鳃 Ig 基因表达水平均高于肝脏、肌肉和心脏,且免疫后的肾脏、脾脏以及鳃 Ig 基因表达水平较免疫前均有显著提高。因此欧鳗肾脏、脾脏和鳃应为其主要免疫应答器官。经背部肌肉注射山羊IgG后,欧鳗系统免疫相关器官脾脏和肾脏免疫应答反应分别在第 7 d 和 第 14 d较为明显,而粘膜免疫相关组织鳃在第21 d 较为明显,说明系统免疫应答反应较粘膜免疫应答反应快。 7. 根据欧鳗 IgH和IgL基因序列,分别设计了IgH 恒定区1、恒定区2、恒定区3 以及 IgL恒定区特异性探针引物,以欧鳗cDNA为模板进行克隆与测序,进而通过地高辛(DIG)对以上基因片段分别进行标记,制备 IgH 恒定区1、恒定区2、恒定区3 以及 IgL恒定区探针。探针标记效率的检测结果显示:IgH 恒定区2 和恒定区 3 所标记的探针与对照探针的浓度达0.1 pg/ µL时均可见到斑点,而对IgH恒定区1和 IgL恒定区所标记的探针浓度低至0.03 pg/ µL尚可见到斑点,表明探针标记均达到预期的效果,其中IgH恒定区1与 IgL 恒定区探针有更高的标记效率。
英文摘要: European eel (Anguilla anguilla) is an important species in the aquaculture industry of China. The understanding of its immune system is great significant for the theory and application of aquaculture. The present study will be concerned with the following aspects: the purification of European eel serum immunoglobulin, distribution of immunoglobulin positive cells in different tissues, the molecular cloning, prokaryotic expression and the temporal and spatial expression analysis of the genes of IgH and IgL chains. The completion of the work of the European eel will provide the molecular basis of the preparation of engineering antibodies and transferring antibody gene breeding. The main contents and results of this study are as follows: 1. Purified immunoglobulin (Ig) was isolated from the serum of European eel using Protein A Sepharose and Sepharose-Goat IgG affinity chromatography. The corresponding polyclonal rabbit anti-eel Ig anti-sera were prepared by the two different purified Ig. Results of SDS-PAGE electrophoresis showed that the Ig purified by Protein A Sepharose was consisted of two singe bands at 68 ku and 26 ku, compatible with heavy chain and light chain. The Ig purified by Sepharose-Goat IgG affinity chromatography revealed one heavy chain with molecular weights of approximately 68 ku and two light chains with molecular weights of approximately 26 ku and 28 ku. The titers of two anti-eel Ig anti-sera were 1:51200. Western blotting analyses revealed that the rabbit anti-sera which were developed against the purified Ig, reacted more strongly with the IgH than with the IgL chain and the protein band with molecular weights of 56 ku was also found to react with the rabbit anti-sera. 2. The polyclonal rabbit anti-eel Ig antiserum prepared by the purified Ig was then used to detect the localization and distribution of the immunoglobulin positive ( Ig+ ) cells in the kidney, spleen and liver of the experimental (infected by Aeromonas hydrophila ) and the control (not infected) European eels by the method of immunohistochemistry. In the control group, the numbers of Ig+ cells were found very low in the spleen, relatively abundant in the kidney and non in the liver. In the experimental group, the number of Ig+ cells obviously increased in the spleen, but not obviously in the kidney, while no Ig+ cells were present in the liver. Both in the experimental and control groups, the melano-macrophage centres (MMCs), formed by the aggregated macrophages and the lymphoid cells, were identified in the spleen and the kidney. In the spleen, plenty of Ig+ cells which most distributed within or around the MMCs and generally close to blood vessels were found in the experimental group, and the similar distribution of Ig+ cells was found in the control group. In the kidney, Ig+ cells were distributed within or around the MMCs in the control group, but not found in the experimental group. Compared with the control group, the number and the volume of the melano-macrophages both in the spleen and the kidney were more and larger in the experimental group. The results obtained in this study revealed that spleen and kidney were primary immune organs which contained Ig+ cells, and the change of the numbers and the volume of the melano-macrophages was closely associated with the process of the immune response in the European eels. 3. The full-length cDNAs encoding the IgH of the European eel was for the first time cloned by RT-PCR and RACE. The full-length cDNA of IgH has 2,089 nucleotides encoding a putative protein of 581 amino acids; the IgH was composed of leader peptide (L), variable domain (VH), CH1, CH2, Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 up to the second cysteine forming the disulfide bridges. Furthermore, one conserved amino acid motifs His-X-Asp-X-X-X-Pro close to the second cysteine was found, which would be a binding motif for C1q. The deduced amino acid sequence of the European eel IgH constant region shared similarities to that of the Ladyfish (Elops saurus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus cario), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. 4. The recombinant expression plasmids carrying the cDNA sequences of IgH chain were then constructed, and subsequently expressed efficiently in Escherichia coli. Using Western blotting analyses, the products expressed by the inserted genes were recognized by the rabbit anti-purified-European eel Ig sera, confirming that the expressed protein shared the similar immunogenicity with the nature IgH chain. 5. The full-length cDNAs encoding the IgL of the European eel was for the first time cloned by RT-PCR and RACE. The full-length cDNA of IgL has 1016 nucleotides encoding a putative protein of 238 amino acids; the IgL was composed of leader peptide (L), variable domain (VL) and constant domain (CL). The conserved amino acid motif ‘PELLT’ in FR2 and ‘FGGG’ in FR4 were found in VL, which were necessary for dimerization of the variable heavy- and light-chain domains. The comparison of amino acid sequence of the European eel CL domain with those of other vertebrates and the phylogenetic tree was constructed. The result showed the CL of European eel was clustered with the rainbow trout(O. mykiss) and Atlantic salmon(S. salar) IgL isotype L1, which suggested that the cloned European eel IgL belong to the IgL isotype L1. The recombinant expression plasmids carrying the cDNA sequences of IgL chain mature peptide with a pair of restriction sites were then successfully constructed, which would be the foundation of gene expression in E. coli. 6. IgH gene expression in the kidney, liver, spleen, heart, gills, and muscle was quantified at both day 0 and day 21 post intramuscular injection with goat IgG by quantitative real-time PCR. The kinetics of Ig gene expression in the kidney, spleen, and gills were also analyzed over 28 days after immunization. The highest level of Ig gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the un-immunized European eels, and the similar situation also appeared at day 21 post immunization except that the level of liver of IgH gene expression decreased significantly, which indicated that that the kidney, spleen, and the gills were the primary tissues for immune response in European eels. The highest level of Ig gene in the spleen, kidney and gills appeared respectively at day 14, day 21 and day 21 post immunization, which suggested that the systemic immune response could be induced faster that the mucosal immune response. 7. According to the European eel IgH and IgL gene sequences, specific primers were designed to clone the specific DNA fragment of CH1, CH2, CH3, and CL which were then labeled by DIG. The result showed the 0.1 pg/µL dilution spots of CH2 and CH3 were visible and the 0.03 pg/µL dilution spots CH1 and CL were visible. All the labeled probes reached the expected labeling efficiency and the probes of CL and CH1 had a higher labeled efficiency.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/11930
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
欧鳗免疫球蛋白基因克隆与表达的研究.冯建军[d].中国科学院水生生物研究所,2008.20-25
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