细胞粘附因子-1与恶性疟原虫感染红细胞结合位点的鉴定

Identification of the Binding Site on ICAM-1 for Red Blood Cells Infected by Plasmodium falciparum

目的 探索恶性疟原虫感染红细胞 (PRBCs)与细胞粘附因子 1 (ICAM 1 )之间的结合位点 ,研制治疗脑型疟的抗粘附药物。 方法 以抗ICAM 1 (I区 )的单抗 1 5 .2为靶 ,采用亲和筛选法对噬菌体随机十二肽库进行 3轮筛选 ,通过ELISA、竞争抑制试验、dot ELISA及Westernblotting鉴定获得的噬菌体短肽与单抗 1 5 .2之间的结合特性。对阳性克隆进行DNA序列测定 ,推导其十二肽的氨基酸序列并与ICAM 1氨基酸全序列进行同源性比较。 结果 经 3轮亲和筛选后 ,结合噬菌体得到良好富集。从第 3轮洗脱液铺制的琼脂板中随机挑取 30个噬菌体单克隆 ,ELISA检测有 2 6个为阳性 ,阳性率达 86 .7%。竞争性ELISA显示多数阳性噬菌体能竞争抑制ICAM 1与 1 5 .2单抗结合。DNA及氨基酸序列分析表明半数以上的噬菌体克隆表达十二肽KLYLIAEGSVAA ,该短肽中K(XX)L(XXX)GSV与ICAM 1的 64～ 73位aa有 50 %的同源性。 结论 阳性噬菌体表达的短肽是 1 5 .2单抗所识别的模拟表位 ,K··L···GSV几个氨基酸可能对ICAM

Objective To identify the binding site on ICAM 1 to PRBCs in order to explore anti adhesive agent against cerebral malaria. Methods Monoclonal antibody 15.2 against ICAM 1 domain 1 was chosen as target molecule to screen mimetic peptides of ICAM 1 from a 12 mer random peptide library. Three rounds of biopanning were carried out and then ELISA, competitive ELISA, dot ELISA and Western blotting were used to evaluate the binding character between phage borne peptides and McAb 15.2. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. Results Thirty clones from the third round were randomly selected, and 26 of them were found positive by sandwich ELISA. The competitive ELISA test proved that most phage borne peptides could competitively inhibit the binding of antibody (15.2 McAb) with ICAM 1. Analysis of DNA and amino acid sequences indicated that over a half positive phage clones expressed 12 mer peptide KLYLIAEGSVAA. Comparison of peptide K(XX)L(XXX)GSV with the 64-73 aa of primary sequence of ICAM 1 showed a 50% homogeneity. Conclusion These peptides displayed by phage may be analogs of ICAM 1, K··L···GSV probably plays a significant role on the binding reaction of ICAM 1 and PRBCs.