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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。
In this study, Mycoplasma hominis P37 protein as a screening molecule, phage display random peptide library affinity panning. The positive phage clones with high affinity were identified by ELISA and sequenced. The amino acid sequence of the random peptide was deduced, and then the recombinant protein of GST-polypeptide was constructed and expressed. The GST- Binding force. After 4 rounds of screening and monoclonal assays, 18 positive clones with strong specific responses were obtained. Eighteen clones included five different small peptide sequences, which were ACAPKPPWLC (12/18), RPLSIDP-WSPHL (3 / 18), RPLSNDPWSPHL (1/18), QNMMSPIEGVRI (1/18) and WAPEKDYMQLMK (1/18). GST-pull down experiments using ACAPK-PPWLC, RPLSIDPWSPHL and GST recombinant fusion protein showed that RPLSIDPWSPHL polypeptide could interact with P37. This study provides experimental and structural basis for further screening of proteins that interact with P37.