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目的:寻找基孔肯雅病毒中和表位,并将其表达为重组融合蛋白。方法:对噬菌体展示12肽库进行了生物淘洗,随机挑取噬菌体克隆,使用ELISA方法筛选并鉴定阳性克隆,分析阳性克隆的DNA序列,将其翻译成氨基酸序列进行同源性比较。将其中一个表位连同M13噬菌体PⅢ蛋白结构域Ⅰ~Ⅱ的编码序列用PCR技术扩增后插入原核表达载体pQE30,用IPTG诱导融合蛋白表达。结果与结论:从经过3轮淘洗的噬菌体中随机挑选123个克隆,从中筛选到20个阳性克隆,其中包含7种不同的氨基酸序列,这些序列缺乏同源性,表明该单抗表位应为构象型表位。在室温培养条件下成功地在大肠杆菌周质腔表达了一种可溶性的表位融合蛋白,经亲和层析得到了纯化的表位融合蛋白。
OBJECTIVE: To search for the chikungunya virus neutralizing epitope and to express it as a recombinant fusion protein. Methods: The phage display 12 peptide library was biopanned, the phage clones were randomly selected, and the positive clones were screened and identified by ELISA. The DNA sequences of the positive clones were analyzed. The amino acid sequences were compared for homology. One of the epitopes was amplified by PCR with the coding sequence of domain I ~ II of the PIII protein of M13 phage and inserted into the prokaryotic expression vector pQE30. The expression of the fusion protein was induced by IPTG. RESULTS AND CONCLUSION: 123 clones were randomly selected from the 3 rounds of eluted phage, and 20 positive clones were screened out and contained 7 different amino acid sequences. These sequences lacked homology, indicating that the McAb epitope should be Conformational epitopes. A soluble epitope fusion protein was successfully expressed in the periplasm of Escherichia coli at room temperature. The purified epitope fusion protein was obtained by affinity chromatography.