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题名: 鲤T细胞标志基因的克隆及表达分析
作者: 尚娜
答辩日期: 2009-05-25
导师: 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: 硕士
关键词: T细胞标志基因 ; ; 原位杂交 ; 病毒感染 ; 寄生虫感染 ; 荧光定量分析
其他题名: Cloning and expression analysis of several T cell marker genes of carp
摘要: 鱼类T淋巴细胞标志基因是鱼类免疫学研究中一个备受关注的问题。通过同源克隆与RACE相结合的方法,获得了鲤(Cyprinus carpio L.)CD8α基因的全长cDNA序列。根据本实验室已获得的CD3γ/δ、CD4L-1、CD8α和CD8β的基因序列设计反义RNA探针,应用原位杂交技术检测了CD3γ/δ、CD8α、CD8β和CD4L-1表达细胞在鲤胸腺、头肾、体肾和脾脏中的分布特征。CD3γ/δ、CD8α、CD8β和CD4L-1表达细胞在鲤胸腺的淋巴样外区呈点状密集分布,而在上皮样内区分布较少且分散;在头肾、体肾和脾脏组织中分散分布,在血管周围呈现不同程度的聚集分布。 通过同源克隆与RACE相结合的方法,获得鲤T-bet、GATA-3、HCK和LCK基因的全长cDNA序列;通过反向PCR的方法,获得鲤T-bet和GATA-3基因的启动子序列。 RT-PCR分析显示在6、18月龄鲤相关组织中均检测到TCRγ、CD3γ/δ、LCK和HCK基因的转录产物,其中以胸腺、鳃和脾脏表达较强;在18月龄鲤相关组织中均检测到T-bet和GATA-3基因的转录产物,其中以胸腺和脾脏表达较强。T-bet、GATA-3和HCK基因主要在转GH基因鲤胸腺、脾脏和头肾组织上调表达,而LCK和CD8α基因主要在转GH基因鲤胸腺组织上调表达,进一步表明转GH基因对鲤T细胞发育的促进作用。 通过RQ-PCR检测,发现在鲤春病毒(SVCV)感染条件下,LCK、HCK、T-bet、GATA-3和CD8α基因在鲤胸腺、头肾、脾脏、鳃、肠和皮肤中呈现动态表达变化。其中在胸腺以表达下降为主,头肾和脾脏在感染早期以上调表达为主;在3-7天,鳃和肠以上调表达为主;表明T细胞系统在鱼类抗病毒免疫中发挥非常活跃的作用。而在小瓜虫(Ichthyophthirius multifiliis)感染条件下,LCK、HCK、T-bet、GATA-3和CD8α基因在1-3天鲤胸腺和头肾以上调表达为主;HCK、GATA-3基因在3-4天皮肤以上调表达为主;提示T细胞系统与鱼类抗寄生虫免疫密切相关。
英文摘要: The study of fish T cell marker genes is a question attracted much attention in fish immunology. We cloned the full length cDNA of carp CD8α using degenerate primers combined with RACE-PCR. Based on the sequences of CD3γ/δ, CD4L, CD8α, CD8β, we examined the distribution of CD3γ/δ, CD4L, CD8α and CD8β positive cells in carp thymus, headkidney, tunk kidney and spleen. In thymus, CD3γ/δ, CD4L, CD8α, CD8β positive cells were mainly distributed in lymphoid outer zone; in lymphoid inner zone, the distribution of these cells was rather dispersedly. In headkidney, tunk kidney and spleen, CD3γ/δ, CD4L, CD8α, CD8β positive cells were mainly scattered, also distributed in groups near the blood vessels. We cloned the full length cDNA of carp T-bet, GATA-3, HCK and LCK using degenerate primers combined with RACE-PCR, and cloned the promoter sequences of carp T-bet and GATA-3. RT-PCR analysis showed that TCRγ, CD3γ/δ, LCK and HCK appeared in all the tissues examined in 6-month and 18-month carp, and the expression of these genes in thymus, gill and spleen was stronger than in other tissues. T-bet and GATA-3 appeared in all the tissues examined in 18-month carp, and the expression of T-bet and GATA-3 in thymus and spleen was stronger than in other tissues. The expression of T-bet, GATA-3 and HCK in thymus, spleen and headkidney of trans-GH carp was stronger than in common carp; and the expression of LCK and CD8α in thymus, spleen and headkidney of trans-GH carp was stronger than in common carp. RQ-PCR analysis showed that after SVCV infection, the expression of T-bet, GATA-3, HCK, LCK and CD8α in thymus, headkidney, spleen, gill, intestine and skin changed dynamically. The expression of these genes was mainly down regulated in thymus after infection, and up regulated in headkidney and spleen in early days after infection; and mainly up regulated in gill and intestine in the later days of infection. This indicates that T cells play very active roles in anti-virus infection. After Ichthyophthirius multifiliis infection, the expression of T-bet, GATA-3, HCK, LCK and CD8α in thymus and headkidney was mainly upregulated in 1-3d after infection, the expression of GATA-3 and HCK in skin was mainly up-regulated in 3-4d, and indicates that T cell immunity is related to anti-parasite infection.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12470
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:
鲤T细胞标志基因的克隆及表达分析.尚娜[d].中国科学院水生生物研究所,2009.20-25
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