摘要
目的对SDF-1进行遗传改造,将其第2位氨基酸由脯氨酸(P)突变为甘氨酸(G),且缺失其C-端α螺旋结构,以获得一种CXCR4特异性拮抗剂SDF-1P2G54。方法将PCR扩增的SDF-1突变体SDF-1p2g54的基因插入表达载体pET-30a(+),并转化BL21(DE3)菌株。IPTG诱导表达的重组蛋白SDF-1P2G54在变性条件下采用镍柱亲和层析纯化,并通过梯度稀释和超滤方法得以复性。利用趋化实验鉴定SDF-1P2G54对Jurkat细胞的趋化活性及对SDF-1趋化活性的抑制效应,流式细胞仪检测SDF-1P2G54诱导MOLT4细胞钙内流及细胞表面CXCR4内在化的能力。结果 SDF-1P2G54完全丧失激活CXCR4、趋化Jurkat细胞跨膜迁移和诱导MOLT4细胞钙内流的能力,却保持了与CXCR4的高亲和性,能有效抑制野生型SDF-1对Jurkat的趋化效应、诱导MOLT4细胞表面CXCR4的快速内在化。结论 SDF-1P2G54是一种新型的CXCR4特异性拮抗剂,具有开发成抑制HIV-1感染和癌细胞转移等重大疾病特效药物的潜在应用价值。
Objective To obtain a specific antagonist of CXCR4,SDF-1P2G54 by mutating SDF-1 second proline（P） into glycin（G） and removing the α-helix of its C-terminal.Methods SDF-1p2g54 gene amplified by PCR was inserted into the vector pET-30a（＋） and transformed into Escherichia coli（E.coli） strain BL21.After IPTG induction of E.coli,the expressed recombinant protein was purified with nickel-affinity chromatography column under denaturing conditions and refolded with gradient dilution and ultra-filtration.The chemotactic effect of SDF-1P2G54 on Jurkat cells and its antagonistic effect against SDF-1 were determined by transwell assay;flow cytometry was used to assay the ability of SDF-1P2G54 to induce calcium influx and CXCR4 internalization in MOLT4 cells.Results The recombinant protein SDF-1P2G54 completely lost the functions to activate CXCR4 or to induce transmembrane migration of Jurkat cells and calcium influx in MOLT4 cells,but maintained a high affinity to CXCR4.SDF-1P2G54 effectively inhibited the chemotactic effect of wild-type SDF-1 to Jurkat cells,and induced rapid CXCR4 internalization in MOLT4 cells.Conclusion SDF-1P2G54 is a new antagonist of CXCR4 with a potential value as an effective inhibitor of HIV-1 infection,cancer metastasis or other major diseases.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2012年第1期55-60,共6页
Journal of Southern Medical University
基金
国家自然科学基金(81101732)
教育部新教师基金(20104433120013)
广东省医学科学技术研究基金(B2009139)~~
