NF-κB p65亚基DNA结合域相互作用多肽的筛选及其功能鉴定
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摘要
核因子-κB(NF-κB)是一种普遍存在的转录因子，是多种信号转导途径的汇聚点，不仅参与介导了免疫应答、病毒复制、细胞凋亡和增殖的多种基因的表达调控，而且在调节炎症反应的基因中起关键作用。NF-κB可高效诱导多种细胞因子、粘附分子、趋化因子和急性期反应蛋白的基因表达，同时对参与炎症反应放大与延续的多种酶的基因表达也具有重要的调控作用。许多急慢性炎性相关疾病的发生或致病过程，均与NF-κB过度激活密切相关。因此，多数学者最近提出NF-κB是极具潜力的新型抗炎靶点。从理论上讲，如能有效拮抗NF-κB的活化，则可达到一定程度地缓解或／和治疗炎性相关疾病的目的。
为获得具有拮抗NF-κB活性的拮抗剂，本实验利用酵母双杂交系统筛选与NF-κB发生相互作用的多肽，并通过凝胶阻滞实验初步鉴定多肽的拮抗功能，以期获得具有拮抗NF-κB活性的拮抗多肽。本研究进行了以下实验工作：1．GAL4 DNA-BD／p65BD融合蛋白酵母表达质粒构建及其表达鉴定。
(1) 采用引物二聚体搭桥PCR扩增法，克隆p65亚基DNA结合域的cDNA。后将其构建到带有GAL4 DNA-BD基因的克隆载体pGBKT_7 DNA-BD上，与GAL4 DNA-BD基因构成融合基因，获得pGBKT_7 DNA-BD／p65BD重组质粒，并保持靶基因的阅读框与GAL4 DNA-BD基因的阅读框一致。DNA测序结果表明，重组质粒中p65 DNA结合域的cDNA碱基序列和阅读框架均正确。
(2)将阳性重组质粒转化入酵母菌AH109，培养诱导重组融合蛋白的表达。抽提酵母总蛋白，经SDS-PAGE电泳，Western-blot鉴定，结果显
示：在约26kD的位置有重组融合蛋白的表达。凝胶扫描软件分析结果显
示，表达的重组蛋白约占酵母总蛋白的5％。
2．GAL4 AD／肽库的扩增、自主报告基因活化、酵母细胞毒性及＊ 遗漏表
达检测。
＊）按肽库操作说明扩增肽库，采用碱裂解法抽提肽库质粒，聚乙二醇
沉淀法纯化肽库质粒，最终获得大量高纯度的肽库质粒。随机挑选10个扩
增的肽库质粒进行序列测定，结果表明10个肽碱基序列备不相同，说明扩
增没有发生偏差。
o）将重组质粒pGBKT，DNAID／p65BD转化入AH109酵母菌中，在
SD人Tp选择培养基上培养，观察细胞生长状况，并与对照 pGBKT，
DNA．BD比较，结果表明细胞生长良好，菌落大小与对照一致，说明融合
蛋白对AH 09酵母菌无毒性。检测AH109酵母质粒转化株p一半乳糖昔酶
表达，结果显示，pGBKT，DNAED／p65BD重组质粒转化株不表达p一半乳
糖着酶，提示无自主报告基因激活活性。AH 09酵母质粒转化菌株不能在
SD／－HIS平板上生长，无m 遗漏表达。
3．NF－KB P65亚基 DNA结合域相互作用多肽筛选及其功能鉴定
＊）用电穿孔法依次将重组质粒 pGBKT7 DNA－BD／p65BD和肽库质粒
共转化AH 09酵母菌，在SD人Tp人工eu人His选择培养基上培养，结果筛选
出64个阳性候选克隆。经SD人Trp人Leu／－His人Ade选择培养基进一步筛选，
12个阳性候选克隆被淘汰。分析剩余阳性候选克隆的e一半乳糖苦酶，共
获得32个表达6一半乳糖昔酶的克隆。
o）采用酵母交配法，将阳性候选克隆分别与pGBKT7 DNA－BD、
PGBKT7 DNA－BD／P65BD和 pGBKT广＊am质粒共转化入 Y187酵母菌，对
32个候选阳性候选克隆进行假阳性鉴定。结果发现，仅有5个阳性候选克
隆能特异地识别p65靶蛋白，为真阳性克隆。
o）抽提阳性克隆质粒，并进行测序，结果获得5个碱基序列各不相同
的多肽。在Genebank进行氨基酸同源序列比对，没有发现与之有高度同
源的蛋白。
·Vlll·
O）选择5个多肽中相互作用最强的2个多肽，合成并纯化。通过凝胶
阻滞实验验证多肽对NF－。B探针的竞争抑制作用，结果表明，其中一个多
肽具有竞争抑制作用，并随多肽的浓度增加，竞争抑制效果越明显。
NF-KB is a ubiquitous transcription factor, and one of cross-talk points of multiple signal transduction pathway. It plays a key role in basic processes such as regulation of the immune and inflammatory responses, virus replication, cell proliferation and apoptosis. It can regulate transcription and expression of genes such as cytokines, adhesion molecules, chemotaxins, acute phase response proteins and enzymes, which take part in inflammatory responses. Over expression of NF-KB is associated with pathogenesis and processes of various acute and chronic inflammatory diseases. Therefore, many experts suggested recently that NF-KB can be a potential novel anti-inflammatory target. Theoretically, effectively antagonizing NF-KB activity is one of effective ways to relieve and/or treat those inflammatory diseases.
To obtain antagonist of NF-KB, the following experiments were performed in this paper.
1. Construction of pGBKT7 DNA-BD/p65BD(DNA-BD/p65 binding domain) and identification of its expression in the yeast.
(1) By means of primer dimer bridging, the fragment of DNA binding domain of NF-KB p65 subunit was amplified, then inserted into pGBKT7 DNA-BD vector, and pGBKT7 DNA-BD/p65BD was constructed. Sequencing result indicated that the fragment sequence of DNA binding domain of NF-KB p65 subunit and its open reading frame were completely correct.
(2) The recombinant plasmid was transformed into yeast AH 109, and the expression of GAL4 DNA-BD/p65BD fusion protein in the yeast cells was
identified by means of SDS-PAGE and Western-blot. The results indicated that expression of fusion protein showed on the lane of 26kD could be found in the yeast AH109. Gel scans analysis showed that the expressed fusion protein accounted for 5 percent of the total yeast proteins.
2. Amplification of GAL4 AD/peptide library, identication of autonomous activation, measurement of cell toxicity and His3 leaky expression in the yeast.
(1) Peptide library plasmids were amplified according to the operating manual, extracted by means of alkaline lysis, purificated by means of polyethylene glycol precipitation, and then a lot of pure library plasmids were obtained. Ten clones were selected at random and an automated sequencer analyzed each sequence of their inserted peptides. Sequencing results showed that there was no bias towards one particular random sequence (each of 10 clones contained a different sequence).
(2) The recombinant plasmid was transformed into yeast AH 109. The growth condition of the transformant in the selected medium SD/Trp was observed. The results showed that colony size of transformant was the same as that of the controls, p-galactosidase activity of positive clones was tested by using both liquid assay and plate assay. The experimental results suggested that DNA binding domain of NF-KB p65 subunit have neither ability of autonomous reporter gene activation nor yeast cell toxicity. The recombinant plasmid transformant could not grow in the SD/-His medium, and did not have His leaky expression.
3. Screening of polypeptides interacted with DNA binding domain of NF-KB p65 subunit.
(1) By means of electroperforation, the pGBKT7 DNA-BD/p65BD plasmids and the peptide library plasmids were sequentially transformed into yeast AH 109. Transformants were spreaded on the plates containing
SD/-Trp/-Leu/-His medium, 64 positive candidate clones expressing His3 were obtained. These positive candidate clones were cultured in the SD/"Ade/Trp/"Leu/"His medium, and 12 false positive clones were eliminated. -galactosidase activity of remaining positive candidate clones was detected, and 32 clones expressing p-galactosidase were finally obtained.
(2) By means of yeast mating, plasmids of positive candidate clones were cotransformed into Y187 respectively with pGBKT7 DNA-BD, pGBKT7 DNA-BD/p65BD and pGBKT7 Lam. p-galactosidase activity of transformants was detected and false positive clones identified. 5 clones were found to possess capability of specially interacting with DN
为获得具有拮抗NF-κB活性的拮抗剂，本实验利用酵母双杂交系统筛选与NF-κB发生相互作用的多肽，并通过凝胶阻滞实验初步鉴定多肽的拮抗功能，以期获得具有拮抗NF-κB活性的拮抗多肽。本研究进行了以下实验工作：1．GAL4 DNA-BD／p65BD融合蛋白酵母表达质粒构建及其表达鉴定。
(1) 采用引物二聚体搭桥PCR扩增法，克隆p65亚基DNA结合域的cDNA。后将其构建到带有GAL4 DNA-BD基因的克隆载体pGBKT_7 DNA-BD上，与GAL4 DNA-BD基因构成融合基因，获得pGBKT_7 DNA-BD／p65BD重组质粒，并保持靶基因的阅读框与GAL4 DNA-BD基因的阅读框一致。DNA测序结果表明，重组质粒中p65 DNA结合域的cDNA碱基序列和阅读框架均正确。
(2)将阳性重组质粒转化入酵母菌AH109，培养诱导重组融合蛋白的表达。抽提酵母总蛋白，经SDS-PAGE电泳，Western-blot鉴定，结果显
示：在约26kD的位置有重组融合蛋白的表达。凝胶扫描软件分析结果显
示，表达的重组蛋白约占酵母总蛋白的5％。
2．GAL4 AD／肽库的扩增、自主报告基因活化、酵母细胞毒性及＊ 遗漏表
达检测。
＊）按肽库操作说明扩增肽库，采用碱裂解法抽提肽库质粒，聚乙二醇
沉淀法纯化肽库质粒，最终获得大量高纯度的肽库质粒。随机挑选10个扩
增的肽库质粒进行序列测定，结果表明10个肽碱基序列备不相同，说明扩
增没有发生偏差。
o）将重组质粒pGBKT，DNAID／p65BD转化入AH109酵母菌中，在
SD人Tp选择培养基上培养，观察细胞生长状况，并与对照 pGBKT，
DNA．BD比较，结果表明细胞生长良好，菌落大小与对照一致，说明融合
蛋白对AH 09酵母菌无毒性。检测AH109酵母质粒转化株p一半乳糖昔酶
表达，结果显示，pGBKT，DNAED／p65BD重组质粒转化株不表达p一半乳
糖着酶，提示无自主报告基因激活活性。AH 09酵母质粒转化菌株不能在
SD／－HIS平板上生长，无m 遗漏表达。
3．NF－KB P65亚基 DNA结合域相互作用多肽筛选及其功能鉴定
＊）用电穿孔法依次将重组质粒 pGBKT7 DNA－BD／p65BD和肽库质粒
共转化AH 09酵母菌，在SD人Tp人工eu人His选择培养基上培养，结果筛选
出64个阳性候选克隆。经SD人Trp人Leu／－His人Ade选择培养基进一步筛选，
12个阳性候选克隆被淘汰。分析剩余阳性候选克隆的e一半乳糖苦酶，共
获得32个表达6一半乳糖昔酶的克隆。
o）采用酵母交配法，将阳性候选克隆分别与pGBKT7 DNA－BD、
PGBKT7 DNA－BD／P65BD和 pGBKT广＊am质粒共转化入 Y187酵母菌，对
32个候选阳性候选克隆进行假阳性鉴定。结果发现，仅有5个阳性候选克
隆能特异地识别p65靶蛋白，为真阳性克隆。
o）抽提阳性克隆质粒，并进行测序，结果获得5个碱基序列各不相同
的多肽。在Genebank进行氨基酸同源序列比对，没有发现与之有高度同
源的蛋白。
·Vlll·
O）选择5个多肽中相互作用最强的2个多肽，合成并纯化。通过凝胶
阻滞实验验证多肽对NF－。B探针的竞争抑制作用，结果表明，其中一个多
肽具有竞争抑制作用，并随多肽的浓度增加，竞争抑制效果越明显。
NF-KB is a ubiquitous transcription factor, and one of cross-talk points of multiple signal transduction pathway. It plays a key role in basic processes such as regulation of the immune and inflammatory responses, virus replication, cell proliferation and apoptosis. It can regulate transcription and expression of genes such as cytokines, adhesion molecules, chemotaxins, acute phase response proteins and enzymes, which take part in inflammatory responses. Over expression of NF-KB is associated with pathogenesis and processes of various acute and chronic inflammatory diseases. Therefore, many experts suggested recently that NF-KB can be a potential novel anti-inflammatory target. Theoretically, effectively antagonizing NF-KB activity is one of effective ways to relieve and/or treat those inflammatory diseases.
To obtain antagonist of NF-KB, the following experiments were performed in this paper.
1. Construction of pGBKT7 DNA-BD/p65BD(DNA-BD/p65 binding domain) and identification of its expression in the yeast.
(1) By means of primer dimer bridging, the fragment of DNA binding domain of NF-KB p65 subunit was amplified, then inserted into pGBKT7 DNA-BD vector, and pGBKT7 DNA-BD/p65BD was constructed. Sequencing result indicated that the fragment sequence of DNA binding domain of NF-KB p65 subunit and its open reading frame were completely correct.
(2) The recombinant plasmid was transformed into yeast AH 109, and the expression of GAL4 DNA-BD/p65BD fusion protein in the yeast cells was
identified by means of SDS-PAGE and Western-blot. The results indicated that expression of fusion protein showed on the lane of 26kD could be found in the yeast AH109. Gel scans analysis showed that the expressed fusion protein accounted for 5 percent of the total yeast proteins.
2. Amplification of GAL4 AD/peptide library, identication of autonomous activation, measurement of cell toxicity and His3 leaky expression in the yeast.
(1) Peptide library plasmids were amplified according to the operating manual, extracted by means of alkaline lysis, purificated by means of polyethylene glycol precipitation, and then a lot of pure library plasmids were obtained. Ten clones were selected at random and an automated sequencer analyzed each sequence of their inserted peptides. Sequencing results showed that there was no bias towards one particular random sequence (each of 10 clones contained a different sequence).
(2) The recombinant plasmid was transformed into yeast AH 109. The growth condition of the transformant in the selected medium SD/Trp was observed. The results showed that colony size of transformant was the same as that of the controls, p-galactosidase activity of positive clones was tested by using both liquid assay and plate assay. The experimental results suggested that DNA binding domain of NF-KB p65 subunit have neither ability of autonomous reporter gene activation nor yeast cell toxicity. The recombinant plasmid transformant could not grow in the SD/-His medium, and did not have His leaky expression.
3. Screening of polypeptides interacted with DNA binding domain of NF-KB p65 subunit.
(1) By means of electroperforation, the pGBKT7 DNA-BD/p65BD plasmids and the peptide library plasmids were sequentially transformed into yeast AH 109. Transformants were spreaded on the plates containing
SD/-Trp/-Leu/-His medium, 64 positive candidate clones expressing His3 were obtained. These positive candidate clones were cultured in the SD/"Ade/Trp/"Leu/"His medium, and 12 false positive clones were eliminated. -galactosidase activity of remaining positive candidate clones was detected, and 32 clones expressing p-galactosidase were finally obtained.
(2) By means of yeast mating, plasmids of positive candidate clones were cotransformed into Y187 respectively with pGBKT7 DNA-BD, pGBKT7 DNA-BD/p65BD and pGBKT7 Lam. p-galactosidase activity of transformants was detected and false positive clones identified. 5 clones were found to possess capability of specially interacting with DN
引文
1. Sen R,Baltimore D.Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism. Cell, 1986;46:705-716.
2. Siebenlist U,Franzoso G,Brown K et al. Structure, regulation and function of NF-κB.Annu Rev Cell Biol, 1994; 10:405-455.
3. Yamamoto Y, Gaynor RB. Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer. J Clin Invest, 2001, 107(2):135-142.
4. Mattson MP, Camandola S. NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest. 2001; 107(3):247-254.
5. Tomita N,Morishita R,Lan HY, et al. In vivo administration of a nuclear transcription factor-kappaB decoy suppresses experimental crescentic glomerulonephritis. J Am Soc Nephrol, 2000; 11(7):1244-1251.
6. Baldwin AS. Series Introduction: The transcription factor NF-kappaB and human disease. J Clin Invest. 2001; 107(1):3-10.
7. Neurath MF, et al.Role of NF-kappaB in immune and inflammatory responses in the gut. Gut 1998 Dec; 43(6):856.
8. Chen FE, et al. Crystal structure of P50/P65 heterodimer of transcription factor NF-kB bound to DNA. Nature, 1998;391(22):413.
9. Wialliam C, et al. Targeted disruption of the P50/P65 subunits of NF-κB leads to multifocal defects in immune responses. Cell, 1995;80(27):321.
10. Marumo T, et al. Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells. Diabetes, 1999;48(5): 1131.
11. Chung KC, et al. Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels, NF-kappaB, and MAP kinases. J
Neurosci Res, 2000;59(1): 117.
12. Parry GC, Backman N. A set of inducible genes expressed by activated human monocytic and endothelial cells contain kappa B-like sites that specifically bind c-Rel-p65 heterodimers. J Biol Chem, 1994; 269(33):20823-20825.
13. Ganchi PA, Sun SC,Greene WC et al. I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding.Mol Cell Biol,1992;3(12):1339-1352.
14. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol,2000; 18:621-663.
15. Thanos D,Maniatis T. NF-kappa B: a lesson in family values. Cell,1995;80(4):529-532.
16. Goebeler M, et al. Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells. Blood. 2001;97(1):46.
17. Jacobs MD, Harrison SC. Structure of an IκBα/NF-κB Complex[J]. Cell, 1998, 95(11):749-758.
18. Fields S, Song O.A novel genetic system to detect protein-protein interactions.Nature, 1989;340(6230):245-246.
19．伍新尧，罗超权，马涧泉．分子遗传学与基因工程．郑州：河南医科大学出版社，1997．14～19．
20．颜子颖，王海林 译．精编分子生物学实验指南。第一版，北京：科学出版社，1998．
21．金冬雁，黎孟枫等译，分子克隆实验指南．第三版．北京，科学出版社，2000．
22．田丁，张基增等译．PCR技术-DNA扩增的原理及应用．第一版，北京：北京医科大学和协和医科大学联合出版社，1991．
23．徐祥，刘昕，梁华平等．引物二聚体在基因克隆中的初探．临床检验杂志，2001；19(3)：3-5．
24. Clontech,In "Matchmaker Gal4 two-hybrid system3 user manual" (PT3247-1).
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26. Schmitz ML, Baeuerle PA.The p65 subunt is responsible for the strong transcription activating potential of NF-κB.EMBO J, 1991; 10(12):3805-3817.
27. Ma J, Ptashne M.A new class of yeast transcriptional activators. Cell, 1987;51(1): 113-119.
28. Yang M, Wu Z, Fields S.Protein-peptide interactions analyzed with the yeast two-hybrid system. Nucleic Acids Res. 1995;23(7):1152-1156.
29. Wu Q, Li-Y, Liu-R, et al. Modulation of retinoic acid sensitivity in lung cancer cells through dynamic balance of orphan receptors nur77 and COUP-TF and their heterodimerization. EMBO J, 1997; 16(7):1656-1669.
30. Gietz RD, Triggs-Raine B, Robbins-A, et al. Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system. Mol Cell Biochem, 1997; 172(1-2):69-79.
2. Siebenlist U,Franzoso G,Brown K et al. Structure, regulation and function of NF-κB.Annu Rev Cell Biol, 1994; 10:405-455.
3. Yamamoto Y, Gaynor RB. Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer. J Clin Invest, 2001, 107(2):135-142.
4. Mattson MP, Camandola S. NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest. 2001; 107(3):247-254.
5. Tomita N,Morishita R,Lan HY, et al. In vivo administration of a nuclear transcription factor-kappaB decoy suppresses experimental crescentic glomerulonephritis. J Am Soc Nephrol, 2000; 11(7):1244-1251.
6. Baldwin AS. Series Introduction: The transcription factor NF-kappaB and human disease. J Clin Invest. 2001; 107(1):3-10.
7. Neurath MF, et al.Role of NF-kappaB in immune and inflammatory responses in the gut. Gut 1998 Dec; 43(6):856.
8. Chen FE, et al. Crystal structure of P50/P65 heterodimer of transcription factor NF-kB bound to DNA. Nature, 1998;391(22):413.
9. Wialliam C, et al. Targeted disruption of the P50/P65 subunits of NF-κB leads to multifocal defects in immune responses. Cell, 1995;80(27):321.
10. Marumo T, et al. Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells. Diabetes, 1999;48(5): 1131.
11. Chung KC, et al. Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels, NF-kappaB, and MAP kinases. J
Neurosci Res, 2000;59(1): 117.
12. Parry GC, Backman N. A set of inducible genes expressed by activated human monocytic and endothelial cells contain kappa B-like sites that specifically bind c-Rel-p65 heterodimers. J Biol Chem, 1994; 269(33):20823-20825.
13. Ganchi PA, Sun SC,Greene WC et al. I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding.Mol Cell Biol,1992;3(12):1339-1352.
14. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol,2000; 18:621-663.
15. Thanos D,Maniatis T. NF-kappa B: a lesson in family values. Cell,1995;80(4):529-532.
16. Goebeler M, et al. Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells. Blood. 2001;97(1):46.
17. Jacobs MD, Harrison SC. Structure of an IκBα/NF-κB Complex[J]. Cell, 1998, 95(11):749-758.
18. Fields S, Song O.A novel genetic system to detect protein-protein interactions.Nature, 1989;340(6230):245-246.
19．伍新尧，罗超权，马涧泉．分子遗传学与基因工程．郑州：河南医科大学出版社，1997．14～19．
20．颜子颖，王海林 译．精编分子生物学实验指南。第一版，北京：科学出版社，1998．
21．金冬雁，黎孟枫等译，分子克隆实验指南．第三版．北京，科学出版社，2000．
22．田丁，张基增等译．PCR技术-DNA扩增的原理及应用．第一版，北京：北京医科大学和协和医科大学联合出版社，1991．
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