摘要
目的:揭示人趋化因子受体5(human C-C chemokine receptor 5,hCCR5)中与人趋化因子配体5(hu⁃man C-C motif chemokine ligand 5,hCCL5)结合并影响hCCL5趋化作用的关键氨基酸。方法:根据生物信息学分析提示的信息,采用定点突变技术,对CCR5受体中Phe112、Trp86和Tyr108分别构建了CCR5-F112A、CCR5-W86A和CCR5-Y108A突变体质粒,并通过慢病毒感染技术感染人胚胎肾细胞HEK293T,分别获得了CCR5-F112A、CCR5-W86A和CCR5-Y108A突变体细胞株。通过激光共聚焦显微镜技术,进一步检测了这些突变对CCR5受体与趋化因子CCL5结合的影响。此外,通过CCK8细胞增殖和Transwell侵袭实验,检测了这些突变对趋化因子CCL5对CCR5受体所带来的肿瘤细胞增殖和侵袭的影响。结果:实验结果显示,CCR5中的Phe112或Trp86位点发生突变后,CCR5受体与CCL5的结合能力以及CCL5的趋化作用均显著减弱,但Tyr108并不显著。结论:CCR5受体中Phe112和Trp86是影响CCR5与CCL5结合的关键氨基酸。此外,这些构建的突变体,也可为进一步揭示CCL5/CCR5生物轴对其在肿瘤疾病中重要生物学功能的研究提供实验研究工具。
Objective:To reveal the key amino acids of human C-C chemokine receptor 5(hCCR5)for binding to human C-C motif chemokine ligand 5(hCCL5)and the related chemotaxis effects.Methods:According to the in⁃formation suggested by bioinformatics analysis,site-directed mutagenesis technique was used to construct mutant plasmids CCR5-F112A,CCR5-W86A and CCR5-Y108A for Phe112,Trp86 and Tyr108 in CCR5,and human embryonic kidney cells HEK293T were infected with such mutants by lentivirus infection to obtain CCR5-F112A,CCR5-W86A and CCR5-Y108A mutants.The binding effects of CCR5-F112A,CCR5-W86A and CCR5-Y108A mutants with CCL5 were further detected by laser scanning confocal microscopy.In addition,the effects of these mutations on tumor proliferation and invasion were also evaluated by CCK8 and Transwell invasion experiments.Results:The experimental results showed that after the mutation of Phe112 or Trp86 in CCR5,the binding ability of CCR5 receptor to CCL5 and the chemotaxis of CCL5 were significantly reduced,but Tyr108 was not significant.Con⁃clusion:Phe112 and Trp86 in CCR5 are the key amino acids for the binding of CCR5 to CCL5,and the construction of these mutants also provides an experimental tool for further elucidating the biological roles of the CCL5/CCR5 axis on tumor progression.
作者
宋晓旭
陈玲
于晶
SONG Xiaoxu;CHEN Ling;YU Jing(School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)
出处
《现代肿瘤医学》
CAS
北大核心
2023年第4期591-597,共7页
Journal of Modern Oncology
基金
国家自然科学基金面上项目(编号:32071298)。
