利用噬菌体随机肽库筛选可结合猪繁殖与呼吸综合症病毒的多肽序列

Screening of polypeptides binding to porcine reproductive and respiratory syndrome virus by phage display library

【目的】采用完整的猪繁殖与呼吸综合症病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)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】用纯化的病毒粒子包被ELISA板,再用M13噬菌体随机12肽库进行筛选。经过3轮淘筛,ELISA鉴定噬菌体单克隆与PRRSV的亲和力,选取与PRRSV具有高亲和力的噬菌体单克隆进行DNA测序,据此推导多肽的氨基酸序列。通过TCID50检测其抗病毒复制能力,同时人工合成FITC标记的展示肽用于PRRSV的检测。【结果】经筛选和鉴定得到17个阳性噬菌体克隆能与PRRSV呈高亲和力结合,DNA测序发现各克隆之间有部分共有基序,其中2个克隆体外能明显抑制PRRSV的复制,使TCID50由10-7.3/0.1mL分别降至10-3.2、10-3.6/0.1mL,而FITC标记该展示肽能够在5mg/L工作浓度检测PRRSV。【结论】通过噬菌体肽库能够筛选到具有抗病毒作用的阳性噬菌体克隆,为进一步开发高效PRRSV的诊断和治疗试剂奠定基础。

[Objective ]To find specific polypeptides that bind porcine reproductive and respiratory syndrome virus （PRRSV） with high affinity and inhibit replication of PRRSV,we screened ligands on intact PRRSV virion by phage display library.[Methods]The purified PRRSV was coated and then reacted with random peptide library displaying 12 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% tissue culture infection dose.The positive clones with high affinity were used for automated sequencing,and the amino acid sequence of polypeptide displayed on phage was deduced.Synthesis of fluorescein isothiocyanate labelled polypeptide and establish a method for detection of PRRSV.[Results]The enrichment was shown by ELISA after 3 rounds of biopanning and 17 positive clones bound to PRRSV with high affinity.Sequencing of the genes encoding these peptides in positive clones show some of conserved motifs.Two positive clones inhibited the replication of PRRSV in Marc-145 cells in vitro and decreased PRRSV TCID50 from 10-7.3 /0.1 mL to 10-3.2,10-3.6 /0.1 mL respectively.The fluorescein isothiocyanate labelled peptide was able to detect PRRSV at the concentration of 5 mg /L.[Conclusion]Positive clones against PRRSV can be selected from phage display peptide library and so provide a potential tool for highly sensitive diagnostic kits and novel antiviral agents.