基于模拟酶切vMIP-II的CCR1受体拮抗多肽的设计与生物活性研究

发布时间：2018-06-02 09:16

本文选题：vMIP-II + CCR1　；参考：《暨南大学》2014年硕士论文

【摘要】：目的：以广谱趋化因子拮抗剂vMIP-II为研究基础，基于vMIP-II与趋化因子家族的结构相似性，利用生物信息学方法设计和筛选来源于vMIP-II的CCR1受体拮抗性多肽，并对其进行生物学活性研究。方法：运用基于结构的多重序列比对分析和蛋白酶模拟酶切等生物信息学方法，寻找vMIP-II结合CCR1的保守区和包含保守区的多肽片段，根据理化性质分析结果筛选合适的多肽片段进行化学合成。采用脂质体转染法，将重组质粒pcDNA4/TO-CCR1转染HEK293细胞，构建表达CCR1受体的HEK293/CCR1细胞系，作为多肽C18P的研究平台。通过[35S]GTPγS结合实验验证CCR1的受体功能以及检测多肽C18P激活受体的能力，趋化实验证明多肽C18P的趋化及趋化抑制作用，钙流实验评价多肽C18P对受体引发的细胞信号转导的阻断作用，配体受体结合实验测定多肽C18P对受体的亲和力，利用ELISA和荧光定量PCR从分子水平探究多肽C18P对内毒素（LPS）炎症细胞模型的影响。结果：（1）建立的HEK293/CCR1细胞系能高效表达CCR1，，而且CCR1具有正常的受体功能，MIP-1α对CCR1的EC50为5.57ng/ml。（2）多肽C18P是利用生物信息学筛选到的来源于vMIP-II的CCR1的特异性拮抗剂，本身不具有趋化性，其抑制HCC-1、MIP-1α和RANTES引起的趋化运动的IC50分别为19.08ng/ml、21.27ng/ml和10.56ng/ml，并可基本阻断由HCC-1、MIP-1α和RANTES引起的钙离子内流。（3）配体受体实验结果表明，多肽C18P能完全抑制125I-MIP-1α与CCR1受体结合，其IC50为14.63ng/ml。当配体浓度达到100ng/ml时，其对125I-MIP-1α结合受体的抑制率均达到80%，对配体受体结合的IC50为15.09ng/ml。[35S]GTPγS结合实验进一步证明C18P是CCR1的拮抗剂，而非激动剂。（4）成功建立内毒素细胞炎症模型。ELISA法检测结果显示：与LPS组相比，低剂量（1.5ng/ml）的多肽C18P和地塞米松（DM）对细胞模型TNF-α、IL-6和IL-8的蛋白分泌没有明显的抑制作用；而中、高剂量（5ng/ml和15ng/ml）的多肽C18P和DM均对TNF-α、IL-6和IL-8的蛋白分泌具有明显的抑制作用。荧光定量PCR检测结果显示：多肽C18P组（15ng/ml）和DM对照组（15ng/ml）对TNF-α、IL-6和IL-8mRNA的表达具有显著的抑制作用。NO含量检测结果显示：低剂量（1.5ng/ml）的多肽C18P和DM对细胞模型NO的分泌没有明显的抑制作用，而中、高剂量（5ng/ml和15ng/ml）的多肽C18P和DM均对NO的分泌具有明显的抑制作用。结论：本研究证明来源于vMIP-II的多肽C18P是CCR1受体的特异性拮抗剂，能抑制细胞的趋化运动和钙离子内流，与受体有较好的亲和力并能阻碍配体激活受体G蛋白，且能抑制内毒素炎症细胞模型分泌炎性细胞因子，具有作为抗炎药物的研究前景。
[Abstract]:Objective: Based on the broad-spectrum chemokine antagonist vMIP-II, based on the structural similarity between vMIP-II and chemokine family, the bioinformatics method was used to design and screen the antagonistic peptides of CCR1 receptor from vMIP-II, and to study their biological activities. Methods: Bioinformatics methods such as structure-based multiple sequence alignment analysis and protease mimic enzyme digestion were used to search for conserved regions and polypeptide fragments containing conserved regions of vMIP-II combined with CCR1. According to the results of physicochemical analysis, suitable polypeptide fragments were selected for chemical synthesis. The recombinant plasmid pcDNA4/TO-CCR1 was transfected into HEK293 cells by liposome transfection, and the HEK293/CCR1 cell line expressing CCR1 receptor was constructed, which was used as the research platform of the peptide C18P. The [35s] GTP 纬 S binding assay was used to verify the receptor function of CCR1 and to detect the ability of the peptide C18P to activate the receptor. The chemotactic experiment demonstrated the chemotaxis and chemotaxis inhibition of the peptide C18P, and the calcium flow assay evaluated the blocking effect of the peptide C18P on the receptor-induced cell signal transduction. Ligand receptor binding assay was used to determine the affinity of peptide C18P to the receptor. ELISA and fluorescence quantitative PCR were used to explore the effect of C18P on the inflammatory cell model of lipopolysaccharide (LPS). Results: (1) the established HEK293/CCR1 cell line was able to express CCR1 efficiently, and CCR1 had normal receptor function and the EC50 of CCR1 was 5.57 ng / ml for MIP-1 伪. The peptide C18P is a specific antagonist from vMIP-II CCR1 screened by bioinformatics and is not chemoattractant by itself. The inhibitory effects of IC50 on chemotaxis induced by HCC-1 and RANTES were 19.08 ng / ml and 10.56 ng / ml, respectively, and calcium influx induced by HCC-1 / MIP-1 伪 and RANTES was basically blocked. The ligand receptor assay showed that the peptide C18P could completely inhibit the binding of 125I-MIP-1 伪 to CCR1 receptor, and its IC50 was 14.63 ng / ml. When the concentration of ligand reached 100ng/ml, the inhibition rate of 125I-MIP-1 伪 -binding receptor was 80%, and the IC50 binding to ligand receptor was 15.09 ng / ml. [35s] GTP 纬 S binding assay further proved that C18P was an antagonist of CCR1, not an agonist. The results of Elisa showed that compared with LPS group, the low dose of C18P and Dexamethasone (Dexamethasone) did not inhibit the secretion of TNF- 伪, IL-6 and IL-8. Both C18P and DM at high doses of 5 ng / ml and 15 ng / ml inhibited the secretion of TNF- 伪 -IL-6 and IL-8 protein. Fluorescence quantitative PCR assay showed that the expression of TNF- 伪 -IL-6 and IL-8mRNA was significantly inhibited by the peptide C18P group (15 ng / ml) and DM control group (15 ng / ml). The results showed that the low dose peptide C18P and DM had no effect on no secretion in the cell model. The obvious inhibitory effect, However, the high dose of C18P and DM of 5ng / ml and 15ng / ml respectively inhibited the secretion of no. Conclusion: This study demonstrated that C18P, a peptide derived from vMIP-II, is a specific antagonist of CCR1 receptor, which can inhibit chemotaxis and calcium influx of cells, have good affinity with receptor and block ligand activated receptor G protein. It can inhibit the secretion of inflammatory cytokines in endotoxin inflammatory cell model, so it has the research prospect as an anti-inflammatory drug.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91;R96

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