EB病毒潜伏膜蛋白1羧基端活化区域2蛋白特异性结合短肽的筛选

Screening of EB virus latent membrane protein 1 C-terminal activating region 2 binding oligopeptides

目的 利用噬菌体12肽库筛选能与EB病毒潜伏膜蛋白1（LMPl）羧基端活化区域（CTAR）2蛋白特异性结合的短肽.方法 用纯化的CTAR2蛋白筛选噬菌体12肽库.通过夹心ELISA方法分析噬菌体克隆与CTAR2蛋白的亲和力.利用硝酸纤维膜斑点印迹法进行阳性克隆鉴定,并对阳性克隆进行测序及序列分析.结果 CTAR2蛋白对噬菌体12肽库进行3轮筛选,第3轮的产率是第1轮的143倍[（3.0×10^-6）/（2.1×10^-8）],噬菌体克隆得到较好的富集.ELISA方法提示筛选出来的噬菌体克隆能与CTAR2蛋白高效结合.硝酸纤维膜斑点印迹法证实阳性率达到100％.测序结果显示噬菌体上的短肽有共同序列：GLKHHSPGLLLY.结论 筛选出与CTAR2蛋白特异性结合的短肽序列GLKHHSPGLLLY,为研究LMP1致瘤机制和开发拮抗LMP1蛋白的小分子短肽类药物提供实验依据.

Objective To screen the oligopeptide with specific affinity to C-terminal activating region 2 （CTAR2） of EB virus latent membrane protein 1 （LMP1） by using the Ph D-12 Phage Display Peptide Library kit.Methods Purified CTAR2 protein was used to screen oligopeptide from Ph D -12Phage Display Peptide Library.Affinity of phage clones to CTAR2 was identified by sandwich ELISA.Dot blot assay was applied for identifying positive clones.DNA sequencing and analysis for these positive clones were performed.Results Yield of oligopeptide screened by CTAR2 protein in Round Ⅲ was 143 times of Round I [ （3.0× 10^-6）/（2.1 × 10^-8） ],with satisfactory enrichment of phage clones.All the screened clones efficiently bound to CTAR2 as shown by sandwich ELISA,and the positive rate of binding was 100% as confirmed by Dot blot assay.A common sequence of the oligopeptide on phages （GLKHHSPGLLLY） was detected by DNA sequencing.Conclusion A oligopeptide sequence GLKHHSPGLLLY binding to CTAR2 of EBV LMP1 is screened,which provides a laboratory evidence for further research on the role of LMP1 in oncogenesis and for development of small molecule vaccines against LMP 1.