Tayapiwatana Chatchai, Arooncharus Pramoon, Kasinrerk Watchara
Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
J Immunol Methods. 2003 Oct 1;281(1-2):177-85. doi: 10.1016/s0022-1759(03)00270-9.
The external domain of a human leukocyte surface molecule, CD147 was displayed on the surface of phage. Two Escherichia coli laboratory strains, XL-1 Blue and TG-1, were chosen to separately propagate the recombinant phages. By sandwich enzyme linked immunosorbent assay (ELISA), CD147 on phage particles were individually captured by six CD147 mAbs and subsequently detected by anti-M13 conjugated HRP. All mAbs specifically bound the CD147 on phage particles derived from TG-1. On the contrary, only four of them could recognize the CD147 on phages produced by XL-1 Blue. The results indicate that the environment in the TG-1 periplasm is more appropriate than that of XL-1 Blue for promoting the suitable folding of CD147. This finding emphasizes the importance of selecting the appropriate E. coli host for display of a complex protein. The epitopes of CD147 displayed on the phage were further mapped by competitive inhibition ELISA, which is a reliable and economical method. Certain clusters of mAb recognition areas were identified and will provide valuable information for the discovery of the ligand for CD147.
人白细胞表面分子CD147的胞外结构域展示在噬菌体表面。选择两种大肠杆菌实验室菌株XL-1 Blue和TG-1分别繁殖重组噬菌体。通过夹心酶联免疫吸附测定（ELISA），六种CD147单克隆抗体分别捕获噬菌体颗粒上的CD147，随后用抗M13共轭辣根过氧化物酶进行检测。所有单克隆抗体均特异性结合源自TG-1的噬菌体颗粒上的CD147。相反，其中只有四种能够识别由XL-1 Blue产生的噬菌体上的CD147。结果表明，TG-1周质中的环境比XL-1 Blue的环境更适合促进CD147的正确折叠。这一发现强调了选择合适的大肠杆菌宿主用于展示复杂蛋白质的重要性。通过竞争抑制ELISA进一步绘制了噬菌体上展示的CD147的表位，这是一种可靠且经济的方法。确定了某些单克隆抗体识别区域的簇，这将为发现CD147的配体提供有价值的信息。
