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

目的　探索恶性疟原虫感染红细胞(PRBCs)与细胞粘附因子1(ICAM1)之间的结合位点,研制治疗脑型疟的抗粘附药物。　方法　以抗ICAM1(I区)的单抗15.2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、dotELISA及Westernblotting鉴定获得的噬菌体短肽与单抗15.2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ICAM1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26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ICAM1与15.2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ICAM1的64～73位aa有50%的同源性。　结论　阳性噬菌体表达的短肽是15.2单抗所识别的模拟表位,K··L···GSV几个氨基酸可能对ICAM1与PRBCs的结合起重要作用

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

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.