【作者】 徐忠伟；

【导师】 徐瑞成；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:从噬菌体7肽库中筛选与哇巴因特异性结合的短肽,并分析其生物学活性,为研究哇巴因与钠泵的相互作用及防治内源性哇巴因相关的原发性高血压奠定基础。方法:(1)哇巴因特异性结合短肽的筛选。①亲和筛选用哇巴因和卵清蛋白复合物包被酶标板,牛血清白蛋白封闭,快速洗板,加入稀释噬菌体文库,弃去未结合噬菌体,洗脱,收集洗脱液。将洗脱液加入大肠杆菌ER2738增殖培养。重复以上操作3次,获得高度富集的噬菌体。②吸附实验用卵清蛋白包被酶标板,加入第3轮淘筛的噬菌体,收集未结合的噬菌体,测定滴度。重复3次后保存洗脱液。③滴度测定将3轮淘选产物与ER2738混合接种于IPTG-Xgal培养板上。计数培养板上的蓝斑。④ELISA法鉴定用哇巴因与卵清蛋白复合物包被ELISA板的各孔,依次加入第3轮扩增后噬菌体、抗M13抗体和底物溶液H2O2,酶标仪测A450值。⑤噬菌体阳性克隆ssDNA的提取及测序分析,并推导出短肽序列,合成多肽。⑥同源性分析:应用NCBI/BLAST数据库,比较推导出的氨基酸序列同已知蛋白质的同源性。(2)哇巴因结合肽生物学活性的检测。①采用放射性核素标记法测定3H-哇巴因与哇巴因结合肽的结合活性。②MTT比色法检测哇巴因结合肽对哇巴因所致血管内皮细胞EAhy926生长抑制的影响。③形态学观察:倒置光显微镜观察细胞形态结构改变;Hoechst33342/PI双荧光染色,荧光显微镜观察细胞死亡特征。④半定量RT-PCR法检测哇巴因结合肽对哇巴因所致钠泵α1亚单位、β1亚单位、VE-cadherin和Snail mRNA表达改变的影响。结果:(1)从噬菌体7肽库中筛选出与卵清蛋白-哇巴因藕联物特异性结合肽的效率:第1轮: 2.0×10-3 %,第2轮: 2.1×10-3%,第3轮: 3.8%。使与卵清蛋白-哇巴因藕联物特异性结合肽得到高度富集。2×1013 pfu/ml的噬菌体库经过3轮卵清蛋白包板吸附实验,特异地与哇巴因结合的噬菌体库为1.8×102pfu/ml,经过3轮淘选并经ELISA鉴定,从噬菌体随机7肽库中筛选得到14个阳性克隆,对其ssDNA进行测序分析。筛选出3种多肽:肽A的筛选一致率达到64.3% (9/14),共有碱基序列:CAACGTACTGGACGTCCACT,蛋白序列:RCMTSRS,同源蛋白:一种多蛋白复合体,位于其643-648位置,主要参与细胞的多种信号传递;肽B的筛选一致率为28.6% (4/14),共有碱基序列:GAGCGTTGGTGCATGGTGT G,蛋白序列:LATTVPH,同源蛋白:氨甲酰天冬氨酸脱水酶,位于其26-32位置;肽C的筛选一致率为7.14% (1/14),共有碱基序列:TGCGTGTGTATGGATGG,蛋白序列:TATTIPT,同源蛋白:一种假象蛋白,位于其248-252位置。(2)放射配基实验结果:哇巴因结合肽能与3H-哇巴因结合,其回归方程:B/F = -0.743B + 94.5276(r =- 0.768),Kd= 0.836 nmol/L,Bmax = 127.7 fmol/mg蛋白。结果表明,3H-哇巴因与哇巴因结合肽(OCP)结合的平衡解离常数为0.836 nmol/L,受体密度为127.7 fmol/mg。(3)哇巴因可抑制EAhy926细胞生长,且呈浓度-时间依赖性;高浓度哇巴因(10μmol/L)对细胞有剧烈抑制作用,而低浓度(0.001μmol/L)对细胞增殖的抑制作用明显降低。经直线回归计算24 h、48 h、72 h IC50值分别为0.507、0.126、0.038μmol/L。(4)哇巴因结合肽拮抗作用:哇巴因结合肽可拮抗哇巴因对EAhy926细胞抑制生长作用,且呈浓度-时间依赖性;高浓度哇巴因结合肽(100μmol/L)对细胞有较强保护作用,而低浓度(0.001μmol/L)对细胞的保护作用则明显降低。经直线回归计算24 h、48 h、72 h IC50值分别为0.612、0.364、0.174μmol/ L。(5)细胞形态学改变:EAhy926为贴壁生长型细胞,哇巴因作用后细胞出现明显的形态学变化。5~10μmol/L哇巴因作用,12 h后即引起细胞死亡,给予0.1μmol/L哇巴因干预24 h,细胞由多角形变为圆形,细胞间缝隙增大,细胞折光性减弱,随着作用时间的延长,细胞出现肿胀、破碎、死亡。而10μmol/L哇巴因结合肽与0.1μmol/L哇巴因处理后,细胞死亡明显减少。荧光显微镜下观察证明,10μmol/L哇巴因处理细胞12h后有90%细胞剧烈坏死,被PI染成红色;0.1μmol/L哇巴因组细胞核染色质浓缩,呈亮珠状,并形成凋亡小体等典型的凋亡形态学改变,而联合组则凋亡细胞数量减少。(6)哇巴因结合肽与0.1μmol/L哇巴因联合对EAhy926细胞钠泵α1和β1亚单位mRNA表达的影响:联合实验组与单独使用0.1μmol/L哇巴因组作比较,联合实验组能下调EAhy926细胞钠泵α1亚单位的表达,上调β1亚单位表达,且两者均呈浓度依赖性。随着哇巴因结合肽浓度的增加,钠泵α1亚单位mRNA表达明显减弱;相反,β1亚单位mRNA表达增高。(7)哇巴因结合肽与0.1μmol/L哇巴因联合对EAhy926细胞VE-cadherin和Snail基因mRNA表达的影响:联合实验组与0.1μmol/L哇巴因对照组比较,哇巴因结合肽能浓度依赖性地上调VE-cadherin表达、下调Snail表达。且二者的表达存在逆反关系,Snail在转录水平抑制VE-cadherin的表达。结论:(1)从噬菌体7肽库中得到筛选一致率较高的哇巴因特异性结合短肽,其碱基序列为:CAACGTACTGGA C GTCCACT,蛋白序列为:RCMTSRS。(2)哇巴因结合肽可以阻抑哇巴因对血管内皮细胞的抑制增殖和诱导死亡作用,其作用呈浓度-时间依赖关系。(3)哇巴因结合肽可以阻抑哇巴因所致的血管内皮细胞钠泵α1亚单位和Snail基因的表达上调及钠泵β1亚单位和VE-cadherin的表达下调。这些分子的表达变化在血管内皮细胞的信号转导和细胞黏附中起重要作用。更多还原

【Abstract】 Objective: To screen the combination peptide of ouabain from Ph.D.-7 phage display peptide library, and analyze its biological activity, and study the interaction between the ouabain and sodium pump and it’s role in essential hypertension which is correlated with endogenous ouabain.Methods: (1) Ouabain combination peptides were screened from Ph.D.-7 peptide library by biopanning.①Affinity screening: Ouabain-ovalbumin compounds were adsorbed in enzyme-labeled plates. After incubation overnight, the plates were blocked with 5% BSA, and incubated with peptide library samples diluted 1:10 in blocking buffer. Non-combination peptides were abandoned and special combination peptides were added into ER2738 enriched culture, the enriched phages were obtained.②Adsorption experiment: Ovalbumin was adsorbed in enzyme-labeled plates and then the plates were cultured with the third round phages, non-combination peptides were collected.③Tite measurement: Each round’s infected phages were spreaded on IPTG-Xgal cultivate plates, and cultured 12h and the locus cinereus were counted.④ELISA identification: Enzyme- labeled plates were adsorbed by ouabain-ovalbumin comp- ounds,then the third round’s phages, anti-M13 antibody and H2O2 were added by turns. The phages’activity were valuated by A450.⑤The ssDNA of phage positive clone were extracted, then identified by electrophoresis. The DNA sequences of each selected phage was determined,the sequences of amino acid were deduced and analyzed through internet NCBI/BLAST.⑥Homology analysis: The sequences and the homology of the deduced amino acid were analyzed with gene bank.(2)Biological activity of ouabain combination peptide.①Combin- ant activity of 3H-ouabain and OCP was detected by radioligand binding assay.②Growth inhibition effect of ouabain and OCP on endothelial cell line EAhy926 was analyzed by MTT assay.③Microscopic structure changes. The feature of cell death was studied by Hoechst 33342/PI staining, nuclear morphological assessment of dead cells was observed by fluorescence micro- scopy.④The mRNA expression of Na+-K+-ATPaseα1-subunit,β1-subunit, VE-cadherin and Snail was studied by reverse transcription PCR(RT-PCR).Results: (1) Efficiency of screening phage display peptide library for ouabain combination peptides with OVA-OUA combination was 2.0×10-3 % in the first round, 2.1×10-3% in the second round, and 3.8% in the third round. Through three times’biopanning, the specific combination peptide was highly enriched with OVA-OUA combination. The phage peptide library(2×1013 pfu/ml) was absorbed by OVA for three times, and more specific peptide library(1.8×102 pfu/ml) was obtained. Three kinds of peptide were selected. Peptide A(Arg-Cys-Met -Thr-Ser-Arg-Ser) was occupied in 64.3% (9/14) , its homologous protein was a kind of multiprotein complex which participates in cell signal transduction, located in 643-648 site. Peptide B(Leu-Ala-Thr-Thr-Val-Pro-His) was occupied in 16.7% (4/14), homologous protein: carbamyl aspartate, located in 26-32 site. Peptide C(Thr-Ala-Thr-Thr-Llr-Pro-Thr) was occupied in 7.14% (1/14),homologous protein: imagination protein, located in 248-252 site. (2) There was some bonding ability between 3H-ouabain and Ouabain combination peptide (OCP). Regression Equation: B/F=-0.743B+94.5276 (r=-0.768). K=0.836nmol/L, Bmax=127.7fmol/mg protein. Equilibrium constant: 0.836 nmol/L. Receptor density: 127.7 fmol/mg. (3) 10μmol/L ouabain treating for 24h could stimulate the necrosis of EAhy926 cells. The median inhibitive concentration (IC50) of ouabain for 24, 48, 72h on EAhy926 cells was 0.507, 0.126, 0.038μmol/L respectively. Ouabain could inhibit EAhy926 cells growth in a dose and time-dependment manner. (4) OCP could antegia the inhibition action of ouabain on EAhy926 cells. 1μmol/L OCP has stronger protection action on EAhy926 cells. The median inhibitive concentration (IC50) of ouabain for 24, 48, 72h on EAhy926 cells was 0.612, 0.364, 0.174μmol/L respectively. OCP could antegia ouabain in a dose and time-dependent manner. (5) When EAhy926 cells were treated with 0.1μmol/L ouabain for 24～48 hours, the cells became sphericalin shape, detached, obviously defluxion. Apoptotic cells shew nuclear chromatin condensation, chromatin margination and the loss of cell-cell adhesion. When EAhy926 cells were treated with 0.1μmol/L ouabain combined with 0.1 and 1μmol/L OCP respectively for 24～48 hours, more cells had better shape and adhesiveness than ouabain group, and the number of apoptotic cells had decreased. (6) OCP could inhibit the up-regulated expression of Na+,K+-ATPaseα1-Subunit and down-regulated expression of Na+,K+-ATPaseβ1-Subunit induced by ouabain in EAhy926 cells. (7) OCP could inhibit the up-regulated expression of Snail and down-regulated expression of VE-cadherin induced by ouabain in EAhy926 cells.Conclusion: (1) A specific ouabain combination peptide was obtained, it’s base sequence was CAACGTACTGGACG T CCACT, and it’s amino acid sequence was RCMTSRS. (2) OCP could antegia the growth inhibition and death induction of ouabain in EAhy926 cells in a dose and time-dependent maner. (3) OCP could inhibit the up-regulated expression of Na+, K+-ATPaseα1-Subunit and Snail, and down-regulated expres- sion of Na+,K+-ATPaseβ1-Subunit and VE-cadherin induced by ouabain in EAhy926 cells. These molecular changes may play important role in signal transduction and cell-cell adhesion.更多还原