鸡TMEM9B同源基因的克隆及Akirin2基因的免疫功能研究
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摘要
近年来，随着新型传染性疾病的不断出现，使得疫苗防治和免疫机制等领域研究再次成为人们关注的热点之一。而TMEM9B和AKIRIN2是近来发现的，在免疫信号通路和炎症反应中起着重要的作用的基因，深入研究这些免疫相关新基因的功能特性，对于深入理解免疫防御机制和开发新型生物制剂用于疾病防治具有重要的理论和实践参考价值。鸡以其独特的生物学特性成为免疫学研究的重要模式动物之一，本研究以鸡为实验动物，克隆了Tmem9B基因并对Akirin2基因的免疫学功能特性进行了初步探索，主要研究方法和实验结果如下：
Tmem9B是TNF在NF-κB和MAPK途径的活化作用中不可缺少的重要因子，而且参与调控细胞膜脂筏的形成，与细胞信号传导和NF-κB调控有关。为了在鸡中克隆获得该新基因，本实验采用电子克隆技术预测得到海兰褐鸡Tmem9B基因全长编码contig序列，根据该预测序列设计特异性引物，利用RT-PCR技术克隆获得该基因序列，将该基因序列克隆到pMD18-T载体中进行测序；利用生物信息学方法分别对Tmem9B同源基因的核苷酸和氨基酸序列进行元件预测和结构分析，并对不同物种Tmem9B同源基因进行了序列同源性比较和进化性分析；提取不同发育阶段鸡的多个组织脏器，利用半定量RT-PCR技术对Tmem9B同源基因进行了组织表达谱分析和鉴定；将Tmem9B同源基因亚克隆到表达载体pcDNA3.1中，构建成pcDNA3.1-tmem9B为后续研究奠定物质基础。Tmem9B研究结果表明：成功获得了海兰褐鸡Tmem9B同源基因的全长编码序列570bp，与预测结果一致。生物信息分析表明Tmem9B氨基酸序列上存在3种修饰位点（豆蒄酰化位点、糖基化位点和蛋白激酶C磷酸化位点），具有信号肽，而且Tmem9B可能是定位于溶酶体和早期内涵体上的跨膜糖蛋白。Tmem9B基因在雏鸡和成鸡的不同组织间和不同发育阶段的表达活性存在差异，表明鸡TMEM9B可能是功能多样的免疫相关蛋白。
Akirin2是NF-κB重要的严格的核内调控子之一，在NF-κB依赖基因的转录调控中起关键作用。本研究首先通过对鸡Akirin2基因全长编码序列进行亚克隆，构建成重组真核表达载体pcDNA3.1-akirin2，然后大量提取和纯化载体质粒pcDNA3.1-akirin2；选取未免疫的海兰褐雏鸡进行新城疫LaSota疫苗免疫的同时，进行胸肌注射接种质粒pcDNA3.1-akirin2，对照组接种pcDNA3.1-EGFP质粒，在接种后的连续6周对实验雏鸡进行随机采血，利用血凝抑制实验（HI）对血清抗体效价进行检测分析。实验结果表明：pcDNA3.1-akirin2能够显著增强LaSota疫苗免疫后的新城疫抗体水平，而对照组注射pcDNA3.1-EGFP质粒未见对抗体水平的影响。本研究初步为重组载体质粒pcDNA3.1-akirin2作为基因工程免疫增强剂在兽医临床上的可能应用奠定了实验基础。
通过以上对鸡Tmem9B同源基因的克隆和表达特性鉴定，以及对Akirin2基因的免疫增强功能研究，这些基础性研究成果将为深入研究这些基因的功能作用奠定基础，同时也为人类相关同源基因功能特性的深入研究提供参考。
In recent years, with the continuous emergence of new infectious diseases, the studyfields of vaccine prevention and immune mechanisms become one of the focuses again.Also, TMEM9B and AKIRIN2were recently found, which play important roles in theimmune signaling pathways and inflammatory responses. Further studying the functionsand characters of these immune-related new genes, it has important theoretical andpractical reference value for deeply understanding of the immune defense mechanismsand developing new biological agents for disease control. Chicken with its uniquebiological characteristics, becomes one of important model animals for immunologyresearch. In this study, chicken is used as experimental animal, and we cloned Tmem9Bgene and preliminarily studied the immune functions of akirin2gene. The main researchmethods and the experimental results are as follows:
Tmem9B is an important and indispensable factor of Tumor Necrosis Factor (TNF)in the activation of nuclear factor-kappaB (NF-κB) and Mitogen-activated protein kinase(MAPK) pathways, and is involved in the formation of membrane lipid rafts, and cellsignaling and the regulation of NF-κB. In order to clone this gene in chicken, wepredicted the full length of Hi-Line Brown chicken Tmem9B cDNA contig sequenceusing in silico cloning. Specific primers were designed basis on this predicted sequence,so the sequence was cloned using RT-PCR technique. Then the sequence was cloned intothe pMD18-T vector for sequencing. We did elements prediction and structures analysisabout Tmem9B homologous nucleotide and amino acid sequences using bioinformaticsmethods, as well as did sequence homology comparison analysis and evolutionaryanalysis about the different species Tmem9B homologous genes.Various tissues andinternal organs at different developmental stages of chicken were used for RNAextraction, and tissue expression profile and identification of tmem9B homologous genewere analyzed using semi-quantitative RT-PCR technique. Tmem9B homologous genewas subcloned into the expression vector pcDNA3.1to construct the recombinantplasmid pcDNA3.1-tmem9B for following research. The results show that: We succeedto obtain Hi-Line Brown chicken Tmem9B homologous gene full-length coding sequenceof570bp, and these results are consistent with the predictions. Bioinformatics analysis shows that there are three kinds of modification sites (myristoylation sites, glycosylationsites and protein kinase C phosphorylation sites), a signal peptide in Tmem9B amino acidsequence, and Tmem9B may be a glycoprotein which is located in the lysosomal and theearly endosomal transmembrane. Tmem9B gene expression activity has differences indifferent tissues and different developmental stages of chickens, which shows that thechicken Tmem9B may be an immune-related protein that has variety of functions.
Akirin2is one of the important strict nuclear regulator, which plays key roles in thetranscriptional regulation of NF-κB dependent genes. In this study, the chicken akirin2gene full-length coding sequence was subcloned to construct the recombinant eukaryoticexpression vector pcDNA3.1-akirin2, then, the plasmid pcDNA3.1-akirin2was largelyextracted and purified. The non-vaccinated Hi-Line Brown chickens were vaccinatedwith Newcastle disease LaSota vaccine. At the same time, the pectoral muscles of thesechickens were injected with the plasmid pcDNA3.1-akirin2, and the control groupchicken were injected with pcDNA3.1-EGFP plasmid. After inoculation, we drewchicken blood serum randomly every week, which were used for detection and analysisserum antibody titers using Hemagglutination Inhibition (HI) Test. The results show that:pcDNA3.1-akirin2can significantly enhance the level of antibodies against the LaSotavaccine after immunization, whereas the control groups that injected withpcDNA3.1-EGFP have no impact on the antibody levels. This study will establishpreliminary experimental foundation for the possible application of the recombinantplasmid pcDNA3.1-akirin2used as genetic engineering immunostimulants in veterinaryclinical.
Through the above of the chicken Tmem9B homologous gene cloning andexpression characteristic identification, and akirin2gene immune function study, thesebasic researches will not only establish the foundation for further studying these genesfunctions, but also provide the references for the function study of human relatedhomologous genes.
Tmem9B是TNF在NF-κB和MAPK途径的活化作用中不可缺少的重要因子，而且参与调控细胞膜脂筏的形成，与细胞信号传导和NF-κB调控有关。为了在鸡中克隆获得该新基因，本实验采用电子克隆技术预测得到海兰褐鸡Tmem9B基因全长编码contig序列，根据该预测序列设计特异性引物，利用RT-PCR技术克隆获得该基因序列，将该基因序列克隆到pMD18-T载体中进行测序；利用生物信息学方法分别对Tmem9B同源基因的核苷酸和氨基酸序列进行元件预测和结构分析，并对不同物种Tmem9B同源基因进行了序列同源性比较和进化性分析；提取不同发育阶段鸡的多个组织脏器，利用半定量RT-PCR技术对Tmem9B同源基因进行了组织表达谱分析和鉴定；将Tmem9B同源基因亚克隆到表达载体pcDNA3.1中，构建成pcDNA3.1-tmem9B为后续研究奠定物质基础。Tmem9B研究结果表明：成功获得了海兰褐鸡Tmem9B同源基因的全长编码序列570bp，与预测结果一致。生物信息分析表明Tmem9B氨基酸序列上存在3种修饰位点（豆蒄酰化位点、糖基化位点和蛋白激酶C磷酸化位点），具有信号肽，而且Tmem9B可能是定位于溶酶体和早期内涵体上的跨膜糖蛋白。Tmem9B基因在雏鸡和成鸡的不同组织间和不同发育阶段的表达活性存在差异，表明鸡TMEM9B可能是功能多样的免疫相关蛋白。
Akirin2是NF-κB重要的严格的核内调控子之一，在NF-κB依赖基因的转录调控中起关键作用。本研究首先通过对鸡Akirin2基因全长编码序列进行亚克隆，构建成重组真核表达载体pcDNA3.1-akirin2，然后大量提取和纯化载体质粒pcDNA3.1-akirin2；选取未免疫的海兰褐雏鸡进行新城疫LaSota疫苗免疫的同时，进行胸肌注射接种质粒pcDNA3.1-akirin2，对照组接种pcDNA3.1-EGFP质粒，在接种后的连续6周对实验雏鸡进行随机采血，利用血凝抑制实验（HI）对血清抗体效价进行检测分析。实验结果表明：pcDNA3.1-akirin2能够显著增强LaSota疫苗免疫后的新城疫抗体水平，而对照组注射pcDNA3.1-EGFP质粒未见对抗体水平的影响。本研究初步为重组载体质粒pcDNA3.1-akirin2作为基因工程免疫增强剂在兽医临床上的可能应用奠定了实验基础。
通过以上对鸡Tmem9B同源基因的克隆和表达特性鉴定，以及对Akirin2基因的免疫增强功能研究，这些基础性研究成果将为深入研究这些基因的功能作用奠定基础，同时也为人类相关同源基因功能特性的深入研究提供参考。
In recent years, with the continuous emergence of new infectious diseases, the studyfields of vaccine prevention and immune mechanisms become one of the focuses again.Also, TMEM9B and AKIRIN2were recently found, which play important roles in theimmune signaling pathways and inflammatory responses. Further studying the functionsand characters of these immune-related new genes, it has important theoretical andpractical reference value for deeply understanding of the immune defense mechanismsand developing new biological agents for disease control. Chicken with its uniquebiological characteristics, becomes one of important model animals for immunologyresearch. In this study, chicken is used as experimental animal, and we cloned Tmem9Bgene and preliminarily studied the immune functions of akirin2gene. The main researchmethods and the experimental results are as follows:
Tmem9B is an important and indispensable factor of Tumor Necrosis Factor (TNF)in the activation of nuclear factor-kappaB (NF-κB) and Mitogen-activated protein kinase(MAPK) pathways, and is involved in the formation of membrane lipid rafts, and cellsignaling and the regulation of NF-κB. In order to clone this gene in chicken, wepredicted the full length of Hi-Line Brown chicken Tmem9B cDNA contig sequenceusing in silico cloning. Specific primers were designed basis on this predicted sequence,so the sequence was cloned using RT-PCR technique. Then the sequence was cloned intothe pMD18-T vector for sequencing. We did elements prediction and structures analysisabout Tmem9B homologous nucleotide and amino acid sequences using bioinformaticsmethods, as well as did sequence homology comparison analysis and evolutionaryanalysis about the different species Tmem9B homologous genes.Various tissues andinternal organs at different developmental stages of chicken were used for RNAextraction, and tissue expression profile and identification of tmem9B homologous genewere analyzed using semi-quantitative RT-PCR technique. Tmem9B homologous genewas subcloned into the expression vector pcDNA3.1to construct the recombinantplasmid pcDNA3.1-tmem9B for following research. The results show that: We succeedto obtain Hi-Line Brown chicken Tmem9B homologous gene full-length coding sequenceof570bp, and these results are consistent with the predictions. Bioinformatics analysis shows that there are three kinds of modification sites (myristoylation sites, glycosylationsites and protein kinase C phosphorylation sites), a signal peptide in Tmem9B amino acidsequence, and Tmem9B may be a glycoprotein which is located in the lysosomal and theearly endosomal transmembrane. Tmem9B gene expression activity has differences indifferent tissues and different developmental stages of chickens, which shows that thechicken Tmem9B may be an immune-related protein that has variety of functions.
Akirin2is one of the important strict nuclear regulator, which plays key roles in thetranscriptional regulation of NF-κB dependent genes. In this study, the chicken akirin2gene full-length coding sequence was subcloned to construct the recombinant eukaryoticexpression vector pcDNA3.1-akirin2, then, the plasmid pcDNA3.1-akirin2was largelyextracted and purified. The non-vaccinated Hi-Line Brown chickens were vaccinatedwith Newcastle disease LaSota vaccine. At the same time, the pectoral muscles of thesechickens were injected with the plasmid pcDNA3.1-akirin2, and the control groupchicken were injected with pcDNA3.1-EGFP plasmid. After inoculation, we drewchicken blood serum randomly every week, which were used for detection and analysisserum antibody titers using Hemagglutination Inhibition (HI) Test. The results show that:pcDNA3.1-akirin2can significantly enhance the level of antibodies against the LaSotavaccine after immunization, whereas the control groups that injected withpcDNA3.1-EGFP have no impact on the antibody levels. This study will establishpreliminary experimental foundation for the possible application of the recombinantplasmid pcDNA3.1-akirin2used as genetic engineering immunostimulants in veterinaryclinical.
Through the above of the chicken Tmem9B homologous gene cloning andexpression characteristic identification, and akirin2gene immune function study, thesebasic researches will not only establish the foundation for further studying these genesfunctions, but also provide the references for the function study of human relatedhomologous genes.
引文
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2Tiersch T R, Wachtel S S.On the evolution of genome size inbirds. Journal of Heredity,1991,82(5):363~368
3满朝来，刘玉芬，李响：鸡胚胎在生命科学研究中的应用.生命的化学.2010,30(2):207~210
4Davison TF. The immunologists’ debt to the chicken. British Poultry Science,2003,44:6~21
5满朝来.鸡myostatin基因的研究进展.中国离收杂志2009,45(17):60~64
6Hughes AL, Hughes M K. Small genomes for better flyers. Nature,1995,377(6548):391~391
7Burt D W, Bumstead N, Bitgood J J, et al. Chicken genome mapping: A new era inavian genetics. Trends Genet,1995,11(5):190~194
8Hu J, Bumstead N, Burke D, et al.Genetic and physical mapping of the naturalresistanceassociated macrophage protein1(NRAMP1) in chicken. MammalianGenome.1995,6(11):809~15
9Marit K, Ellen T, Guri D, et al. Characterization of the novel human transmembraneprotein9(TMEM9) that localizes to lysosomes and late endosomes. Biochemical andBiophysical Research Communications.2002,297(4):912~917
10Francis D, Marie G, Dieter H, et al. The Lysosomal Transmembrane Protein9BRegulates the Activity of Inflammatory Signaling Pathways. The Journal ofBiological Chemistry.2008,283(31):21487~21494
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