重组人ADAM15去整合素结构域蛋白的制备及其作用机理

发布时间：2018-07-07 17:15

  本文选题：rhddADAM15 + 小鼠黑色素瘤细胞(B16)　； 参考：《江南大学》2008年博士论文

【摘要】： ADAM (A Disintegrin And Metalloproteinase)是一类含有七个功能结构域并在细胞间相互识别和相互作用中发挥重要作用的跨膜糖蛋白家族。ADAM15作为该家族唯一在去整合素结构域(disintegrin)含有RGD(Arg-Gly-Asp)基序的成员,在细胞外基质降解、细胞粘附、细胞内信号转导和肿瘤的病理变化等过程中发挥重要作用,但是其抗肿瘤机理需进一步阐释。本文在成功获取高表达水平的重组人ADAM15去整合素结构域蛋白片段(rhddADAM15)的基础上,以小鼠黑色素瘤细胞(B16)为研究模型,研究rhddADAM15对B16细胞体外增殖、粘附和迁移等细胞行为的影响,通过筛选rhddADAM15特异结合的12肽以及rhddADAM15在B16细胞上相互作用的靶点蛋白,探讨rhddADAM15干预肿瘤细胞行为的生理机制。主要研究结果如下: 1)为获得大量具有生物活性的rhddADAM15蛋白,在详尽分析目标蛋白的cDNA序列的基础上,(1)选择能为大肠杆菌稀有密码子提供额外tRNA的大肠杆菌Escherichia coli Rosetta(DE3)作为宿主菌,将目标蛋白以GST(Glutathione-S-transferase)融合蛋白形式表达,在最佳诱导表达条件下融合蛋白GST-ADAM15浓度为298 mg/L,最佳凝血酶酶切条件下,rhddADAM15浓度为42 mg/L; (2)采用PCR体外定点突变技术将凝血酶识别序列附近稀有密码子GGA(Gly)替换为GGC,同时消除Pro-Glu-Phe残基,构建突变表达质粒,提高凝血酶酶切效率。在相同的表达和酶切条件下获得GST-△-ADAM15和rhddADAM15蛋白浓度分别为326 mg/L和68mg/L,比野生型分别提高9.4%和61.9%。这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达; 2)采用MTT法测定rhddADAM15对B16、MCF-7/W以及HMEC-1细胞体外增殖的作用,采用创伤愈合实验、Matrigel基质胶以及Transwell侵袭小室法观察rhddADAM15对B16细胞体外迁移、粘附和侵袭的影响;采用台盼蓝染色法分析细胞活力、流式细胞仪检测rhddADAM15对B16细胞周期的影响。结果表明rhddADAM15对肿瘤细胞和正常内皮细胞生长均有一定的抑制作用(其IC50分别为9.5、11.8和14.9μg/mL),且明显抑制B16细胞的体外增殖、迁移、粘附和侵袭。台盼蓝染色法表明低剂量的rhddADAM15并不能导致B16细胞出现坏死,且rhddADAM15并不导致B16细胞出现典型的凋亡峰,而是将细胞阻滞于G2/ M期,并且这种作用呈剂量依赖关系; 3)采用亲和甄别磁珠技术筛选rhddADAM15在B16细胞上的结合蛋白。将rhddADAM15采用蛋白生物素化方法标记生物素,借助生物素-亲和素系统,将生物素化的rhddADAM15固定于亲和素标记的免疫磁珠。从B16细胞裂解液中钓出与之相互作用的蛋白,经浓缩富集后,采用双向电泳分离技术分离获得主要的结合蛋白点,然后对这些蛋白点进行电喷雾串联质谱技术(ESI-MS)分析,在蛋白数据库中检索,共鉴定了8个蛋白质,按功能可分为如下三类:(1)临床应用的肿瘤标志物(苹果酸脱氢酶、磷酸甘油醛脱氢酶和谷氨酸脱氢酶);(2)能量代谢相关蛋白(烯醇酶、磷酸丙糖异构酶、原肌球蛋白和血红蛋白);(3)信号转导通路成员(p38MAPK激酶)等。在此基础上通过分子对接软件molsoft ICM模拟计算获得rhddADAM15与p38激酶相互作用的最佳模式和重要的结合位点; 4)采用噬菌体展示技术筛选与rhddADAM15特异性相互作用的多肽序列。采用噬菌体随机十二肽库对固定有rhddADAM15的亲和磁珠系统进行生物淘洗,获得4条与rhddADAM15特异结合的12肽。将获得的12肽与蛋白质库进行序列比对,发现细胞周期相关蛋白如Cdc25和p53,信号通路相关蛋白/受体,如整合素αvβ3的胞外区,丝裂原活化蛋白激酶p38α等与所获得的4条12肽具有高度同源性; 5)基于上述研究所获得的rhddADAM15在B16细胞上潜在的靶点蛋白,进一步研究rhddADAM15与p38MAPK激酶的体外相互作用,结合rhddADAM15对B16细胞行为的抑制作用,采用蛋白点杂交和激酶活性测定方法分析rhddADAM15在体外与重组p38激酶蛋白的结合情况,同时研究不同剂量rhddADAM15对B16细胞中p38MAPK激酶磷酸化水平的影响,并观察rhddADAM15对p38激酶活性受到部分抑制的B16细胞的增殖和细胞周期抑制作用的变化。发现:(1)rhddADAM15在离体低温条件下能够和重组p38激酶蛋白结合,但rhddADAM15(0-10μg/mL)在体外对p38MAPK激酶活性没有影响;(2)同样剂量的rhddADAM15作用于B16细胞24 h后,细胞中p38MAPK激酶的磷酸化水平提高(被激活);(3)p38激酶的特异性抑制剂SB203580(0.5 mmol/L)预处理B16细胞30 min后,rhddADAM15对细胞增殖的抑制作用减弱(IC50由未经处理的9.5μg/mL增加到11.4μg/mL),对细胞周期的阻滞作用也减弱(G2/M期细胞比率从未经处理的的28.77%降到16.59%)。上述结果表明,p38MAPK信号转导通路参与了rhddADAM15对B16细胞增殖和细胞周期的抑制作用,同时也表明生理条件下rhddADAM15与p38MAPK激酶相互作用的复杂性。
[Abstract]:ADAM (A Disintegrin And Metalloproteinase) is a class of transmembrane glycoprotein family.ADAM15 that contains seven functional domains and plays an important role in intercellular recognition and interaction as the only member of the family that contains RGD (Arg-Gly-Asp) motif in the domain of the integrin domain (disintegrin), and is degraded in the extracellular matrix. Adhesion, intracellular signal transduction and tumor pathological changes play an important role, but their anti-tumor mechanisms need to be further explained. On the basis of successful acquisition of recombinant human ADAM15 domain protein fragment (rhddADAM15), a rat melanoma cell (B16) was used as a model to study rhddADA. The effects of M15 on the proliferation, adhesion and migration of B16 cells in vitro, and to explore the physiological mechanism of rhddADAM15 intervention in tumor cell behavior by screening the specific binding 12 peptide of rhddADAM15 and the target protein of rhddADAM15 interaction on B16 cells. The main results are as follows:
1) in order to obtain a large number of bioactive rhddADAM15 proteins, on the basis of detailed analysis of the cDNA sequence of the target protein, (1) select the Escherichia coli Escherichia coli Rosetta (DE3) which can provide extra tRNA for the rare codon of Escherichia coli as the host bacteria, and the target egg white is expressed in the form of GST (Glutathione-S-transferase) fusion protein. Under the optimal induction condition, the concentration of fusion protein GST-ADAM15 was 298 mg/L, and the concentration of rhddADAM15 was 42 mg/L under the optimum thrombin enzyme digestion condition. (2) the rare codon GGA (Gly) near the thrombin recognition sequence was replaced with GGC in vitro by PCR, and the Pro-Glu-Phe residue was eliminated and the mutant expression plasmid was constructed to improve the coagulation. The concentration of GST- Delta -ADAM15 and rhddADAM15 protein was 326 mg/L and 68mg/L respectively under the same expression and enzyme cutting conditions, respectively, which was 9.4% and 61.9%. respectively higher than that of wild type. The results showed that the selective expression of the host and the expression plasmid on the basis of fully understanding the characteristics of the target protein could achieve the high water content of the exogenous protein. Flat expression;
2) the effect of rhddADAM15 on the proliferation of B16, MCF-7/W and HMEC-1 cells in vitro was measured by MTT, and the effects of rhddADAM15 on the migration, adhesion and invasion of B16 cells in vitro were observed by the wound healing experiment, Matrigel matrix gel and Transwell invasion, and the cell viability was analyzed by trypan blue staining, and the flow cytometry was used to detect rhddADAM15 pairs. The effect of B16 cell cycle shows that rhddADAM15 inhibits the growth of tumor cells and normal endothelial cells (IC50 is 9.5,11.8 and 14.9 mu g/mL respectively), and obviously inhibits the proliferation, migration, adhesion and invasion of B16 cells in vitro. Trypan blue staining shows that low dose rhddADAM15 does not cause B16 cells to appear bad. RhddADAM15 did not cause typical apoptotic peak in B16 cells, but blocked G2/ M phase.
3) the binding protein of rhddADAM15 on B16 cells was screened by affinity screening magnetic beads. Biotin was used to mark biotin by protein biotin, and biotin avidin system was used to immobilization of biotinylated rhddADAM15 on avidin labeled immunomagnetic beads. The proteins interacting with them from the B16 cell lysate were found. After concentration and enrichment, the main binding protein points were obtained by two-dimensional electrophoresis separation technology, and then the protein spots were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS), and 8 proteins were identified in the protein database. According to their functions, they can be divided into three categories: (1) the clinical application of the tumor markers (malate dehydrogenase, phosphoric acid) Glyceraldehyde dehydrogenase and glutamate dehydrogenase (2) energy metabolism related proteins (enolase, phosphosin isomerase, promyosin and hemoglobin); (3) signal transduction pathway members (p38MAPK kinase), and so on. On this basis, the best model of the interaction between rhddADAM15 and p38 kinase is obtained by molecular docking software molsoft ICM. Important binding sites;
4) using phage display technique to screen the polypeptide sequence with rhddADAM15 specific interaction. Using the phage random twelve peptide library, 4 rhddADAM15 affinity magnetic beads system was washed, and 4 12 peptides specifically binding to rhddADAM15 were obtained. The sequence alignment of the obtained 12 peptide and the protein pool was compared, and the cell cycle correlation was found. Proteins such as Cdc25 and p53, signaling pathway related proteins / receptors, such as the extracellular domain of integrin alpha v beta 3, and mitogen activated protein kinase p38 alpha are highly homologous to the 4 12 peptides obtained.
5) based on the potential target protein of rhddADAM15 on B16 cells obtained from the above study, the interaction of rhddADAM15 and p38MAPK kinase in vitro was further studied, and the inhibition action of rhddADAM15 on B16 cell behavior was combined, and the binding of protein point hybridization and kinase activity was used to analyze the binding of rhddADAM15 in vitro and the recombinant p38 kinase protein. At the same time, the effects of different doses of rhddADAM15 on the phosphorylation of p38MAPK kinase in B16 cells and the changes in the proliferation and cell cycle inhibition of B16 cells partially inhibited by p38 kinase activity were observed. (1) rhddADAM15 can be combined with the recombinant kinase protein under the isolated cryogenic strip, but rhddADAM, but rhddADAM 15 (0-10 g/mL) had no effect on the activity of p38MAPK kinase in vitro; (2) after the same dose of rhddADAM15 acted on B16 cells 24 h, the level of phosphorylation of p38MAPK kinase in the cells was increased (activated); (3) the inhibitory effect of rhddADAM15 on cell proliferation was weakened after the specific inhibitor SB203580 (0.5 mmol/L) of p38 kinase (0.5 mmol/L). C50 was increased to 11.4 UU g/mL from untreated 9.5 mu g/mL), and the cell cycle retardation was also reduced (the ratio of G2/M cell ratio never treated by 28.77% to 16.59%). The results showed that the p38MAPK signal transduction pathway was involved in the inhibition of rhddADAM15 on the proliferation and cell cycle of B16 cells, and also indicated rhddAD under physiological conditions. The complexity of the interaction between AM15 and p38MAPK kinase.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R341

【引证文献】

中国硕士学位论文全文数据库 前1条

1 金雄华;刚性陶瓷/琼脂糖复合蛋白质层析介质的制备及应用研究[D];江南大学;2012年

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