脂多糖结合蛋白／CD14结合位点的初步定位  

Preliminary locating for CD14 binding site with lipopolysaccharide binding protein

作　　者：徐智[1] 李琦[1] 冯起甲[1] 钱桂生[1] 徐顺贵[1] 李昆霖[2] 王兴胜[2] 吴国明[1] 

机构地区：[1]第三军医大学新桥医院全军呼吸内科研究所 全军呼吸病研究重点实验室,重庆400037 [2]第三军医大学大坪医院野战外科研究所呼吸内科,重庆400042

出　　处：《第三军医大学学报》2009年第7期565-568,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30500230)~~

摘　　要：目的应用噬菌体随机12肽库、DNAStar分析软件、亲和实验及竞争抑制实验初步定位脂多糖结合蛋刍（lipopolysaccharide binding protein，LBP）与CD14的结合位点。方法以CD14为筛选分子，联合应用噬菌体肽库展示技术、噬菌体ELISA鉴定、LBP竞争抑制实验及DNA测序推导LBP／CD14结合位点的展示肽序列，采用DNAStar分析软件：E对所获展示肽序列与LBP一级结构，初步定位LBP／CD14结合位点并人工合成其模拟肽。采用ELISA检测该模拟肽与CD14的亲和力及与LBP的竞争抑制活性。结果DNAStar比对结果表明，经噬菌体肽库筛选获得的8条展示肽氨基酸序列与LBP第252～263位氨基酸有一定相似性。LBP第252～263位氨基酸具有结合位点的特性，即有良好的亲水性、可及性、可塑性及抗原性，该部位可能是LBP／CD14的结合位点。体外活性实验表明，模拟肽FHRNHRsPVTLL可与CD14有臭好的亲和力，有较强的与LBP竞争结合CD14的活性。结论LBP第252—263位氨基酸的模拟肽FHRNHRSPVTLL与CD14有良好的亲和力及有较强的与LBP竞争结合CD14的活性，具有LBP／CD14结合位点的生物学特性，LBP／CD14结合位点初步定位在该区域。Objective To investigate CD14 binding site with lipopolysaccharide binding protein （LBP） by phage peptide library display, DNAStar software analyses, affinity test and LBP competitive inhibition experiments. Methods With CD14 as a target molecule, phage peptide library display, phage ELISA, LBP competitive inhibition experiments and DNA screening for testing sequences were jointly adopted to deduct the display peptide sequences of the binding site of LBP/CD14. The display peptide sequences were matched with primary structure of LBP by DNAStar analysis software, to initially locate CD14 binding site with LBP and synthesis its mimic peptide. The affinity activity and competitive inhibition activity of mimic peptide were detected by ELISA. Results DNAStar software analyses indicated that the sequences of 8 peptides obtained by phage peptide library were similar to 252 to 263 amino acids of LBP, and satisfactory hydrophilicity, accessibility, flexibility and antigenieity were seen in this amino acid fragment of LBP. The binding site of LBP/CD14 might locate in this fragment. In vitro activity test indicated that mimic peptide （FHRNHRSPVTLL） had satisfactory affinity activity to CD14 and competitive inhibition activity to LBP. Conclusion The mimic peptide （FHRNHRSPVTLL） of the 252 to 263 amino acid fragment of LBP has satisfactory affinity activity to CD14 and competitive inhibition activity to LBP. It displays the bioaetivity of the binding site of LBP/CD14 and indicates CD1.4 binding site with LBP is locate in this region preliminarily.

关 键 词：脂多糖结合蛋白 CD14 噬菌体肽库 模拟肽 内毒素 

分 类 号：Q503[生物学—生物化学] Q513