大黃魚TLR5與NF-κB家族部分基因的克隆與功能研究
發(fā)布時間：2018-08-01 10:41
【摘要】：本研究在實驗室前期獲得大黃魚(Larimichthys crocea)兩種類型TLR5(LcTLR5M,LcTLR5S)cDNA序列的基礎(chǔ)上,分別克隆了它們的啟動子序列,通過片段缺失,定點突變等技術(shù),篩選并驗證了其啟動子結(jié)構(gòu)及轉(zhuǎn)錄結(jié)合位點;構(gòu)建了LcTLR5M和LcTLR5S的亞細胞定位質(zhì)粒,研究了其在細胞內(nèi)的表達特征;構(gòu)建了它們的過表達質(zhì)粒,將其分別以及共同與NF-κB家族p65基因啟動子共轉(zhuǎn)染細胞系,檢測了LcTLR5M和LcTLR5S過表達對NF-κB-p65啟動活性的影響�？寺×舜簏S魚NF-κB家族RelB基因的全長cDNA序列,分析了它的分子結(jié)構(gòu)及組織表達譜,檢測了病原相關(guān)分子模式(PAMP)刺激后,其在細胞系內(nèi)的時空表達特征;研究了RelB亞細胞定位并分析了其過表達后對TNF-α等部分細胞因子的影響及IκB家族IκB-α對其調(diào)控機制。擴增了NF-κB家族成員p65基因的啟動子序列,檢測了其活性,以及病原刺激后對其啟動活性的影響;研究了p65基因亞細胞定位及分析了其過表達對下游細胞因子IL-1β、TNF-α和IFN-I基因的啟動子活性的影響,及IκB-α對其調(diào)控機制。結(jié)果如下:(1)大黃魚TLR5M啟動子區(qū)域在-1829bp至+189bp之間,無CpG島區(qū)域,轉(zhuǎn)錄結(jié)合位點含有CEBP、Oct-1、GATA-1、AP1等元件,系列片段缺失分析基本啟動子活性所需的順式作用元件可能位于-417至+189之間,負調(diào)控元件可能位于-799至-417之間。大黃魚TLR5S啟動子區(qū)域在-1932bp至+106bp之間,無CpG島區(qū)域,轉(zhuǎn)錄結(jié)合位點含有CEBP、SP1、Oct-1、AP1、NF-B等元件,片段缺失表明TLR5S基因順式作用元件可能位于-808至+106之間,Oct-1和NF-κB結(jié)合位點對TLR5S啟動子活性有正調(diào)控作用且大黃魚NF-κB家族亞基p65過表達可顯著上調(diào)TLR5S啟動子活性,表明預測正確。亞細胞定位分析表明,大黃魚TLR5M蛋白主要定位在細胞膜上,而TLR5S蛋白主要定位在細胞漿中。TLR5M過表達對NF-κB-p65啟動子的活性無顯著影響,而TLR5S過表達可以顯著上調(diào)NF-κB-p65啟動活性。(2)大黃魚RelB(LcRelB)基因全長為1946bp,ORF為1788bp,編碼595個氨基酸,包含RHD-DNA-bind結(jié)構(gòu)域和IPT結(jié)構(gòu)域,等電點p I=5.6,分子量為66.5KDa。進化分析表明,大黃魚RelB基因與紅鰭東方渶親緣關(guān)系最近;LcRelB廣泛分布于多種組織,其中在血細胞中含量最高,其次是脾臟和頭腎,皮膚中含量最低;LPS和poly I:C刺激后,大黃魚LCK細胞系中LcRelB上調(diào)表達顯著,而PGN免疫刺激后表達下調(diào)(p0.05)。LcRelB蛋白定位于細胞核中。LcRelB過表達后,其下游細胞因子IL-1β啟動子活性顯著下調(diào),而TNF-α和IFN-I啟動子活性顯著上調(diào)(p0.05),而當NF-κB家族抑制因子Lc IκB-α與其共轉(zhuǎn)染后,可以顯著抑制TNF-α啟動子活性(p0.01)。(3)Lcp65啟動子區(qū)域為-1695bp至+111bp,預測其轉(zhuǎn)錄結(jié)合位點含有USF、AP1、SRF、IRF-1、NF-κB等元件,含有CpG島以及(GT)n重復區(qū)域;LPS,PGN,Flagellin刺激后均能夠顯著誘導p65啟動活性上調(diào),其中Flagellin誘導能力最為顯著(p0.01);亞細胞定位表明,Lcp65蛋白主要定位于細胞核中;Lcp65過表達后能顯著上調(diào)其下游細胞因子IL-1β、TNF-α和IFN-I啟動子的活性,其中,TNF-α的啟動活性最強。抑制因子Lc IκB-α與其共轉(zhuǎn)染后,可以顯著抑制其對TNF-α啟動子活性的誘導(p0.01)。
[Abstract]:In this study, on the basis of two types of TLR5 (LcTLR5M, LcTLR5S) cDNA sequence of Larimichthys crocea in the laboratory, the promoter sequences were cloned respectively. The promoter structure and transcriptional binding sites were screened and verified by the technique of fragment deletion and fixed-point mutation, and the subcellular binding of LcTLR5M and LcTLR5S was constructed. The expression of the plasmid was studied in the cell, and their overexpressed plasmids were constructed to co transfect the cell lines with the NF- kappa B family p65 promoter, respectively. The effect of LcTLR5M and LcTLR5S overexpression on the activation of NF- kappa B-p65 was detected. The full-length cDNA sequence of the NF- kappa B family RelB gene of the rhubarb fish was cloned, and the analysis was made. Its molecular structure and tissue expression profiles were used to detect the spatio-temporal expression characteristics of the pathogen associated molecular model (PAMP), and to study the location of RelB subcellular localization and to analyze the effect of its overexpression on TNF- alpha and the regulation mechanism of I kappa B- alpha of the I kappa B family. The p65 gene of NF- kappa B family members was amplified. The promoter sequence was used to detect its activity and the effect of pathogen stimulation on its activity. The effect of over expression of p65 gene on the promoter activity of the downstream cytokine IL-1 beta, TNF- A and IFN-I genes and the regulation mechanism of I kappa B- a were studied. The results were as follows: (1) the TLR5M promoter region of the yellow croaker was in -1829 Between BP and +189bp, there is no CpG island region, and the transcription binding site contains CEBP, Oct-1, GATA-1, AP1 and other components. The sequence of sequence deletion analysis of the basic promoter activity may be located between -417 to +189, and the negative regulatory element may be between -799 to -417. The transcriptional binding site contains CEBP, SP1, Oct-1, AP1, NF-B and other components. The deletion of fragment indicates that the cis acting element of the TLR5S gene may be located between -808 and +106. Oct-1 and NF- kappa B binding sites have positive regulation on the activity of the TLR5S promoter. The subcellular localization analysis showed that the TLR5M protein was mainly located on the cell membrane, while the TLR5S protein was mainly located in the cytoplasm, and the.TLR5M overexpression had no significant effect on the activity of NF- kappa B-p65 promoter, while TLR5S overexpression could significantly increase the activation activity of NF- kappa B-p65. (2) the RelB (LcRelB) gene of large yellow croaker was 1946bp, ORF was 1788bp, which encodes 595 amino acids, contains the RHD-DNA-bind domain and IPT domain, isoelectric point P I=5.6. The molecular weight of 66.5KDa. evolution analysis shows that the RelB gene is closest to the red fin Orient; LcRelB is widely distributed in a variety of tissues, with the highest content in the blood cells, followed by the spleen and the head kidney, and the lowest in the skin. After the stimulation of LPS and poly I:C, the expression of LcRelB in the LCK cell line of the large yellow croaker was significantly up-regulated, while the expression of P0.05.LcRelB protein was down regulated after.LcRelB overexpression in the nucleus after PGN immunization, and the activity of the downstream cytokine IL-1 beta promoter was down significantly down, while TNF- alpha and IFN-I promoter activity were significantly up-regulated. Factor Lc I kappa B- alpha and co transfection can significantly inhibit the activity of TNF- alpha promoter (P0.01). (3) Lcp65 promoter region is -1695bp to +111bp, and predicts its transcriptional binding sites contain USF, AP1, SRF, IRF-1, and repeating regions. The Flagellin induction ability was most significant (P0.01); subcellular localization showed that Lcp65 protein was mainly located in the nucleus; Lcp65 overexpression could significantly increase the activity of IL-1 beta, TNF- A and IFN-I promoter, among which TNF- a was the strongest. The inhibitory factor Lc I kappa B- alpha and co transfection could significantly inhibit it. Induction of TNF- alpha promoter activity (P0.01).
【學位授予單位】：集美大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4;Q78
本文編號：2157308
[Abstract]:In this study, on the basis of two types of TLR5 (LcTLR5M, LcTLR5S) cDNA sequence of Larimichthys crocea in the laboratory, the promoter sequences were cloned respectively. The promoter structure and transcriptional binding sites were screened and verified by the technique of fragment deletion and fixed-point mutation, and the subcellular binding of LcTLR5M and LcTLR5S was constructed. The expression of the plasmid was studied in the cell, and their overexpressed plasmids were constructed to co transfect the cell lines with the NF- kappa B family p65 promoter, respectively. The effect of LcTLR5M and LcTLR5S overexpression on the activation of NF- kappa B-p65 was detected. The full-length cDNA sequence of the NF- kappa B family RelB gene of the rhubarb fish was cloned, and the analysis was made. Its molecular structure and tissue expression profiles were used to detect the spatio-temporal expression characteristics of the pathogen associated molecular model (PAMP), and to study the location of RelB subcellular localization and to analyze the effect of its overexpression on TNF- alpha and the regulation mechanism of I kappa B- alpha of the I kappa B family. The p65 gene of NF- kappa B family members was amplified. The promoter sequence was used to detect its activity and the effect of pathogen stimulation on its activity. The effect of over expression of p65 gene on the promoter activity of the downstream cytokine IL-1 beta, TNF- A and IFN-I genes and the regulation mechanism of I kappa B- a were studied. The results were as follows: (1) the TLR5M promoter region of the yellow croaker was in -1829 Between BP and +189bp, there is no CpG island region, and the transcription binding site contains CEBP, Oct-1, GATA-1, AP1 and other components. The sequence of sequence deletion analysis of the basic promoter activity may be located between -417 to +189, and the negative regulatory element may be between -799 to -417. The transcriptional binding site contains CEBP, SP1, Oct-1, AP1, NF-B and other components. The deletion of fragment indicates that the cis acting element of the TLR5S gene may be located between -808 and +106. Oct-1 and NF- kappa B binding sites have positive regulation on the activity of the TLR5S promoter. The subcellular localization analysis showed that the TLR5M protein was mainly located on the cell membrane, while the TLR5S protein was mainly located in the cytoplasm, and the.TLR5M overexpression had no significant effect on the activity of NF- kappa B-p65 promoter, while TLR5S overexpression could significantly increase the activation activity of NF- kappa B-p65. (2) the RelB (LcRelB) gene of large yellow croaker was 1946bp, ORF was 1788bp, which encodes 595 amino acids, contains the RHD-DNA-bind domain and IPT domain, isoelectric point P I=5.6. The molecular weight of 66.5KDa. evolution analysis shows that the RelB gene is closest to the red fin Orient; LcRelB is widely distributed in a variety of tissues, with the highest content in the blood cells, followed by the spleen and the head kidney, and the lowest in the skin. After the stimulation of LPS and poly I:C, the expression of LcRelB in the LCK cell line of the large yellow croaker was significantly up-regulated, while the expression of P0.05.LcRelB protein was down regulated after.LcRelB overexpression in the nucleus after PGN immunization, and the activity of the downstream cytokine IL-1 beta promoter was down significantly down, while TNF- alpha and IFN-I promoter activity were significantly up-regulated. Factor Lc I kappa B- alpha and co transfection can significantly inhibit the activity of TNF- alpha promoter (P0.01). (3) Lcp65 promoter region is -1695bp to +111bp, and predicts its transcriptional binding sites contain USF, AP1, SRF, IRF-1, and repeating regions. The Flagellin induction ability was most significant (P0.01); subcellular localization showed that Lcp65 protein was mainly located in the nucleus; Lcp65 overexpression could significantly increase the activity of IL-1 beta, TNF- A and IFN-I promoter, among which TNF- a was the strongest. The inhibitory factor Lc I kappa B- alpha and co transfection could significantly inhibit it. Induction of TNF- alpha promoter activity (P0.01).
【學位授予單位】：集美大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4;Q78
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