利用噬菌体随机肽库筛选抗呼吸道合胞病毒多肽的实验研究  

Anti-RSV polypeptides selected from a phage displayed random peptide library

作　　者：赖克方[1] 黄海鹭[1] 孙宝清[1] 钟南山[1] 

机构地区：[1]广州呼吸疾病研究所,510120

出　　处：《中华微生物学和免疫学杂志》2002年第6期670-674,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的　采用完整的呼吸道合胞病毒 (respiratorysyncytialvirus ,RSV)颗粒筛选噬菌体肽库 ,以获得能高亲和力结合并能抑制该病毒复制的特异性多肽。方法　Hep 2细胞培养RSV ,采用蔗糖密度梯度 (30 %、4 5 %、6 0 % )和超滤离心纯化病毒。病毒用生物素标记后 ,应用M13噬菌体PⅢ呈现随机 7肽库进行筛选。经过 3轮淘筛后 ,ELISA进一步鉴定噬菌体克隆与RSV的亲和力 ,通过TCID50检测其抗病毒复制能力。选取与RSV具有高亲和力的阳性克隆进行DNA测序 ,据此推导多肽的氨基酸序列。结果　经鉴定得到 13个阳性噬菌体克隆能与RSV呈高亲和力结合 ,其中 3个克隆体外能明显抑制RSV的复制 ,使TCID50 由 10 - 8.1 SFU ml分别降至 10 - 4.1 、10 - 4.3、10 - 3.8SFU ml。DNA测序发现各个克隆之间缺乏明显的同源序列 ,但普遍含有较多的脯氨酸。结论　通过噬菌体肽库能够筛选到抗病毒作用的阳性克隆 。Objective To find specific polypeptides which bind RSV with high affinity and inhibit replication of RSV, we screened ligands on intact RSV virion by display libraries of phage. Methods Human RSV was harvested in Hep 2 cells. The PEG concentrated virus was layered over sucrose density gradients (30%, 45%, 60%), followed by ultrafiltration. The purified RSV was biotinylated in vitro with NSH LC biotin and then to react with random peptide library displaying 7 amino acids fused on protein Ⅲ of M13 phage. The selected peptides for target binding were assayed by ELISA after 3 rounds of biopanning and measured by 50 percent tissue culture infection dose (TCID 50 ). The positive clones with high affinity were used for automated sequencing with dye labeled dideoxynucleotides, and the amino acid sequence of polypeptide displayed on phage was deduced. Results The enrichment was shown by ELISA after 3 rounds of biopanning and 13 positive clones bound to coat protein of RSV with high affinity. Three positive clones were identified to inhibit the replication of RSV in Hep2 cells in vitro that decreased RSV TCID 50 from 10 -8.1 SFU/ml to 10 -4.1 , 10 -4.3 , 10 -3.8 SFU/ml respectively. Sequencing of the genes encoding these peptides in 13 positive clones showed absence of any conserved motifs, although most peptides had high proportion of argenines. Conclusion Positive clones against RSV can be selected from phage display peptide library and so provide a potential tool for highly sensitive diagnostic kits and novel antiviral agents.

关 键 词：呼吸道合胞病毒 噬菌体呈现肽库 DNA测序 RSV 呼吸道感染 儿童 

分 类 号：R346[医药卫生—基础医学]