桃拉综合征病毒CP2蛋白的克隆表达及其结合肽的噬菌体肽库筛选

Cloning,expression of the CP2 gene of Taura syndrome virus and screen ligands from phage display random dodecapeptide library for CP2 protein

目的：克隆表达出桃拉综合征病毒（TSV）主要衣壳蛋白CP2蛋白,并通过对噬菌体随机肽库的淘选,得到与CP2蛋白结合的相关多肽,以探讨CP2蛋白与配体作用的特异位点。方法：根据重组质粒pMD-CP2的核苷酸序列,设计并合成了一对引物,扩增TSV CP2基因高变区376~1 371 bp,扩增片段插入pET-32构建成原核表达载体pET-CP2。该重组载体在IPTG诱导下进行可溶性表达,SDS-PAGE电泳检测表达的CP2蛋白。以纯化的CP2蛋白作为筛选分子,对噬菌体展示12肽库进行亲和淘选。结果：原核表达出56 kD大小的CP2蛋白,对噬菌体12肽库4轮＂吸附-洗脱-扩增＂淘洗后,所得的噬菌体回收率逐步提高,由6.4×10-5上升到2.8×10-2,显示淘选过程对随机肽库有很好的富集效果。随机挑选第4轮淘筛后的36个单克隆噬菌体,ELISA鉴定有24个单克隆（66.6%）为强阳性。将这24个阳性克隆扩增、测序,推导随机多肽的氨基酸序列。分析发现,有7个克隆（克隆号2、6、7、9、18、20、33）完全一致,序列为HTSFCSTHLCLI,其它的几个克隆如克隆3及28序列为HCSNLFCSLDLP;克隆5为HCSHTLCALHVM;克隆26为HCNSWLCPLITD也与该7个克隆有相似的序列,经比对核心序列为HCS及LCL。结论：首次用重组的TSV CP2蛋白从噬菌体随机12肽库中筛选到特异结合的肽序列,其共有结构特征为明确CP2与结合蛋白的相互作用位点提供支持。

Objective： The major capsid protein CP2 of Taura syndrome virus（ TSV） was cloned and expressed,peptides binding to CP2 were successfully obtained from a phage display random dodecapeptide library. Methods： According to the nucleotide sequence of recombinant plasmid pMD-CP2,a pair of primer was designed to amplify the high variable region 376-1 371 bp of CP2 gene.The product was purified and cloned into the prokaryotic vector pET-32a to construct the recombinant plasmid,designated pET-CP2.The CP2 gene could be dissolubility expressed by the induction of IPTG,the fusion protein was detected by SDS-PAGE. Phage display random dodecapeptide library was screened with the purified CP2 protein. Results： The molecular weight of CP2 protein was about 56kD,after 4 rounds of panning（ absorption-elution-amplification） with dodecapeptide library,the ratio of output to input increased from6. 4 × 10- 5to 2. 8 × 10- 2,indicated well enrichment. ELISA test revealed high affinity of the fourth panning phage peptides to CP2 protein. 36 clones random selected from the fourth panning phage library showed 66. 6% positive rate by ELISA test. The twenty-four positive clones were selected for automated sequencing and the amino acid sequence of polypeptide displayed on phage was deduced. Same peptide sequence HTSFCSTHLCLI was found in 7 clones,three similar sequences HCSNLFCSLDLP,HCSHTLCALHVM,HCNSWLCPLITD have homologous sequence compared with the above. Comparison of the amino acid sequences of foreign peptides of the clones,HCS and LCL motif were considered to be the necessary amino acid sequence for the affinity. Conclusion： Peptides binding to CP2 were successfully obtained from a phage display random dodecapeptide library. The results provide useful experimental and structure data for identification of the binding domain for CP2 with its ligand.