【摘要】 本研究以猪鼻支原体P37蛋白作为筛选分子,对噬菌体展示随机肽库进行亲和淘选。利用ELISA法鉴定亲和力高的阳性噬菌体克隆,对其进行DNA测序和分析,据此推导随机多肽的氨基酸序列,然后构建、表达GST-多肽重组蛋白,经GST-pull down进一步鉴定该多肽与P37的结合力。经过4轮筛选和单克隆检测,获得18个有较强特异反应的阳性克隆,18个克隆包括了5种不同的小肽序列,它们分别为ACAPKPPWLC(12/18)、RPLSIDP-WSPHL(3/18)、RPLSNDPWSPHL(1/18)、QNMMSPIEGVRI(1/18)和WAPEKDYMQLMK(1/18)。用ACAPK-PPWLC、RPLSIDPWSPHL与GST重组的融合蛋白进行GST-pull down实验表明,RPLSIDPWSPHL多肽能与P37相互作用。本研究为进一步筛选与P37相互作用的蛋白提供了实验依据和结构基础。更多还原

【Abstract】 Phage display random heptapeptide library was screened with recombinant P37 in this study.The positive phage clones were identified by ELISA and were sequenced,and the amino acid sequences of the polypeptides displayed on phage were deduced.After GST-polypeptides fusion protein was constructed and expressed,its binding to P37 was determined by GST-pull down and Western blot.After 4 rounds of bio-panning,the enriched positive phage clones were identified by ELISA.Eighteen positive phage clones were sequenced and the peptide sequences were as follows: ACAPKPPWLC(12/18),RPLSIDPWSPHL(3/18),RPLSNDPWSPHL(1/18),QNMMSPIEGVRI(1/18) and WAPEKDYMQLMK(1/18).The results from GST-pull down and Western blot showed that peptide RPLSIDPWSPHL could interact with P37.The study will be helpful for identifying the protein reacting with P37.更多还原