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题名: 中华鳖免疫相关基因的筛选、克隆及表达分析
作者: 周秀霞
答辩日期: 2009-06-05
导师: 戴和平 ; 郭琼林
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: 中华鳖 ; 抑制性差减杂交 ; 免疫相关基因 ; 嗜水气单胞菌 ; 荧光定量分析 ; rIL-8 ; 急性期反应
其他题名: Screening, cloning and expression analysis of immune-relevant genes in Chinese soft-shelled turtle Trionyx sinensis
摘要: 摘 要 本研究在最为薄弱的爬行动物免疫学研究领域进行了有意义的探索。应用抑制性差减杂交技术,首次构建了一个与嗜水气单胞菌 (Aeromonas hydrophila) 感染有关的中华鳖 (Trionyx sinensis) 主要内脏器官的差减cDNA文库。从200多个克隆中筛选到42个差异或上调表达的基因,其中16个与免疫相关基因系爬行类首次鉴定。经虚拟Northern 杂交和RT-PCR验证,其中的6个基因(IL-8、SAA、CD9、ATF4、CL和毒素样蛋白)在中华鳖感染组织均上调表达。 通过RACE方法得到中华鳖IL-8、SAA、毒素样蛋白、CD9和Fg基因的全长cDNA序列并进行了分子结构和进化分析;通过Genome Walking方法得到前3个基因的基因组及启动子序列。中华鳖IL-8基因组全长4924 bp,由三个内含子和四个外显子组成;cDNA全长为1188 bp,开放阅读框包含312 bp,编码蛋白为104个氨基酸,含有保守的CXC结构域、4个半胱氨酸和与功能相关的ELR基序。SAA基因组全长3153 bp,由两个内含子和三个外显子组成;cDNA全长为554 bp,开放阅读框包含381 bp,编码蛋白为127个氨基酸,C-端氨基酸十分保守,N-端由疏水氨基酸组成。毒素样蛋白基因组全长1995 bp,由两个内含子和三个外显子组成;cDNA全长为580 bp,开放阅读框包含267bp,编码蛋白为89个氨基酸,含有8个保守的半胱氨酸和三指蛋白家族的标志序列C-末端CCXXXCN基序。CD9 cDNA全长为1146 bp,开放阅读框包含672bp,编码蛋白为224个氨基酸,包含四个跨膜结构域(TM)和大小两个胞外环(LEL和SEL),LEL内含有保守的CCG基序和4个半胱氨酸,SEL内含一个N-端糖基化位点。Fg cDNA全长为1605 bp,开放阅读框包含1320bp,编码蛋白为440个氨基酸,含有FReD结构域、N-末端球形结构域C-gamma、Ca2+结合位点和聚合口袋结构等。系统进化分析显示,除毒素样蛋白与一种美洲大毒蛇的毒素样蛋白聚为一支外,其余四个蛋白均与鸟类的同源分子聚为一支。 RT-PCR分析显示在中华鳖相关组织中均检测到IL-8、CD9和毒素样蛋白基因的转录产物。其中IL-8基因为组成型表达;CD9基因主要在肝和脾组织表达;而毒素样蛋白基因主要在肝和肾组织中表达。RT-PCR结果表明,除SAA外,其余4种急性期蛋白(APP)Fg、C3、ALB和 CL基因均在肝组织表达。 通过RQ-PCR,发现在嗜水气单胞菌感染条件下,IL-8、CD9和毒素样蛋白在中华鳖相关组织中呈现动态表达变化。其中IL-8基因于感染后第4天以肝、脾和肾组织上调表达最明显;而CD9基因在2-4天以肝、脾和血液上调表达最明显;毒素样蛋白基因于感染后第1–2天以肝、脾、心脏和血液上调表达最明显;除ALB基因下调表达外,其它4种APP基因在肝组织均有不同程度的诱导表达,其中SAA于感染后第2天在肝组织上调至2000倍;表明这些基因的表达变化与爬行类抗细菌免疫密切相关。 完成了中华鳖IL-8基因的原核表达和蛋白纯化。趋化实验表明,rIL-8蛋白浓度为1.0―20ng/ml时对中华鳖嗜中性粒细胞有趋化作用,对淋巴细胞趋化作用不明显。首次证实了爬行动物rIL-8对嗜中性粒细胞的趋化作用。
英文摘要: The valuable explore was performed in the reptile immunology, which was the least studied. By using the suppressive subtractive hybridization technique, a cDNA library was first constructed from the main internal organs of Aeromonas hydrophila infected Chinese soft-shelled turtles. 42 genes were identified from more than 200 clones, and 16 of them were immune-relevant genes and firstly identified in reptile. Then, IL-8, SAA, CD9, Toxin-like protein, ATF4 and CL genes were further observed to be up-regulated in the infected tissues of turtles by virtual Northern hybridization and RT-PCR assays. By using RACE-PCR, the full-length cDNA and turtle IL-8, SAA, Toxin-like protein, CD9 and Fg were isolated, and the molecular and phylogenetic characteristics were analyzed. The genome DNA and promoter sequences of the first three genes were amplified by genome-working. The turtle IL-8 gene spans 4924 kb, contains three introns and four exons. The full-length cDNA was 1188 bp contained an open reading frame (ORF) of 312 bp, which coding a protein of 104 amino acids (aa). The CXC domain, four cysteines and the ELR motif (involved in IL-8 founction) were well conserved in turtle IL-8. The genome sequence of turtle SAA was 3153 bp, contained two introns and three extrons. The 554 bp cDNA sequence is obtained with an ORF of 381 bp encoding 127aa. The N-terminus of turtle SAA was composed of hydrophobic residues; the C-terminus amino acids were well conserved. The full-length genome DNA of Toxin-like protein was 1995 bp and occupied two introns and three extrons. The full-length cDNA was 580 bp and contained an ORF of 267 bp coding for a protein of 89 aa. There were 8 conserve cycteines and the consensus sequence motif -CCXXXCN- at the C-terminal end, which was a marker sequence of the “three-finger” protein family. The full-length cDNA of turtle CD9 was 1146 bp and contained a 672 bp ORF coding for a protein of 224 amino acids (aa). Turtle CD9 contained four TM domains, large and small extracellular loops (LEL and SEL). The conserved CCG motif and four cycteines were found within the LEL, and a potential N-glycosylation site was found in the SEL of turtle CD9. The full-length cDNA of turtle Fg was 1605 bp and contained a ORF of 1320 bp, which coding a protein of 440 aa. It contained the FReD, C terminal globular domain, Ca2+ binding site and a polymerization pocket. Phylogenetic analysis showed that, except Toxin-like protein, the rest four proteins of soft-shelled turtle were clustered with their birds’ homologues. RT-PCR analysis identified the transcripts of IL-8, CD9 and Toxin-like protein in the tissues of control turtles. IL-8 was constitutively expressed; CD9 was mainly expressed in liver and spleen, and Toxin-like protein was mainly expressed in liver and kidney. RT-PCR also showed that, except SAA, the rest four APP genes (Fg, C3, CL and ALB) were all constitutively expressed in liver. After infected with A. hydrophila, RQ-PCR analysis showed that there were dynamic changes of IL-8, CD9 and Toxin-like protein mRNA expression in related tissues of soft-shelled turtles. The most induction of turtle IL-8 gene was found in liver, spleen and kidney 4 d after infection. CD9 gene was significantly up-regulated in liver, spleen and blood during 2-4 d after infection. So was the Toxin-like protein gene during 1-2 d after infection. Except the ALB gene was observably suppressed, the rest four APP genes were induced at different levels in liver after infection, and SAA was induced and reached to about 2000-fold at 2 d. These results suggested that the dynamic changes of these genes expression were closely related to the anti-bacteria immune responses of reptiles. The recombinant turtle IL-8 protein was expressed in E. coli and purified. Chemotaxis experiments showed that the turtle rIL-8 induced a migration of turtle neutrophils. There attraction of lymphocytes was not obvious. This was the fist identification of the chemotaxis effects of reptile rIL-8 to neutrophils.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12492
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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中华鳖免疫相关基因的筛选、克隆及表达分析.周秀霞[d].中国科学院水生生物研究所,2009.20-25
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