人类巨细胞病毒编码US28受体拮抗性多肽的筛选鉴定及体外活性研究

Biological Functions, Screen and Identify Research on Chemokine Receptor Antagonist Encoded by US28 of Human Cytomegalovirus

立足US28的广谱趋化因子结合活性,寻找拮抗剂多肽的先导物.通过预测US28的结合模拟位设计多肽序列,合成相关多肽,再利用自建的25种趋化因子随机构成的噬菌体12肽库筛选结合模拟位,竞争性ELISA方法验证,从而获得30个阳性克隆,测序并推导氨基酸序列,得到7种序列:GSESLNAHCALW、EIDGFNAHCALL、VIARLNAHCALR、ATECLNAHCALW、VIESLNAHCALW、DNGSINAHCALL及VKKTLNAHCALR.所有序列富含疏水氨基酸,其中8个克隆有LNAHCAL保守序列.采用趋化抑制实验检测多肽对生理性趋化因子引起的细胞迁移活性的影响,流式细胞仪检测细胞内钙离子浓度变化.结果表明,利用人源趋化因子序列构建肽库并筛选有效序列的方法取得成功,筛选出的阳性克隆保守序列LNAHCAL可以模拟HumanMIP-1β与US28N端的结合位,从而与合成多肽H22结合,多肽H22可以阻断受体结合生理性趋化因子形成的趋化作用,本身不引起趋化运动,不影响胞内信号转导和细胞自然活性,具有广谱趋化因子拮抗剂的可能性.

Based on a large spectrum of US28, planning to find out new antagon. Membrane spanning domain and epitopes were used to predict a binding mimotope that US28 bound with CC chemokines, the result of which were used to design and synthesize the peptide. The 12 peptide phage library was built sourcing from 25 different chemokines random phages including four families. The binding mimotope of synthesized peptide was screened by phage display technique, and assessed by ELISA assay. PL＋S method was used to screen the 12 phage library with the target as biotinylated soluble form peptide H22. Biological activity of H22 were measured with cellular chemotaxis assays and calcium mobilization. Amino acid sequences of the displayed peptides in 10 phage clones were deduced from DNA sequences. They are GSESLNAHCALW, EIDGFNAHCALL, VIARLNAHCALR, ATECLNAHCALW, VIESLNAHCALW, DNGSINAHCALL and VKKTLNAHCALR. Every peptide sequence contains at least 2 hydrophobic residues. Eight of the 10 clones have a conservative sequence LNAHCAL. Chemotaxis assays showed that H22 induced migration of peripheral blood mononuclear, and H22 suppressed PBMCs＇ migration induced by hMIP-1β and EC50=30.9 μg/L. H22 itself was not remarkably associated with the normal, rapid mobilization of calcium from intracellular stores. Instead, it blocked calcium mobilization induced by endogenous chemokines. It proves that using chemokine sequence of human source to build a peptide library and screen effective sequence is available. The conservative sequence could simulate the binding mimotope of Human MIP-1β interacting with HCMV US28 N-terminus to bind with synthesized peptide H22. The study has demonstrated that peptide H22 cannot stop the biological function of hMIP-1β and block the signal transduction from hMIP-1β by the way of calcium pathway, but itself did not affect cells activity.