摘要
目的获得靶向人CD147的纳米抗体。方法以胞外区全长CD147蛋白为靶标,从噬菌体展示文库中筛选CD147的纳米抗体,将筛选获得的单域抗体可变区(VHH)目的基因克隆至p ET-28a表达载体上,转化大肠杆菌(E.coli)BL21(DE3),异丙基硫代半乳糖苷(IPTG)诱导表达纳米抗体,使用AKTA start+GE Ni-NTA预装柱对带有组氨酸(His)标签的重组蛋白进行纯化并用酶联免疫吸附实验、生物膜层干涉技术、蛋白质免疫印迹法及激光共聚焦显微镜成像技术检测纳米抗体的特异性和亲和力。结果经过3轮筛选获得了1个特异性结合CD147抗原的纳米抗体12-3,其亲和力达到9.46×10-9mol/L,且可特异性地识别肿瘤细胞表面的CD147抗原。结论从新疆双峰驼单域抗体噬菌体展示文库中筛选获得了1个高亲和力抗CD147的纳米抗体,有望用于肿瘤诊断。
Objective To obtain nanobody targeting CD147 protein. Methods CD147 nanobody was screened from the phage display library by the target for ectodomain protein of CD147（CD147 ect）. The target gene of single domain antibody variable region（VHH）was inserted into a p ET-28 a（＋）expression vector,and then used to transform E. coli BL21（DE3）. After isopropyl-β-Dthiogalactoside（IPTG）induction,the expressed histone-tagged recombinant protein was purified using AKTA start＋GE Ni-NTA preloaded column. The specificity and affinity of nanobodies were detected by ELISA,biolayer interferometry（BLI）,Western blotting and confocal laser scanning microscopy. Results A nanobody 12-3 that could specifically bind with the CD147 antigen was obtained via 3 rounds of screening,and its affinity reached 9.46×10^-9 mol/L. Meanwhile,the nanobody 12-3 could specifically recognize CD147 antigens on the surface of tumor cells. Conclusion An anti-CD147 nanobody with a high affinity was obtained from the phage display library,which could be used for the diagnosis of tumors.
作者
李日飞
周鹏
王婷婷
李日勇
殷惠
王子豪
张文远
左儒楠
李引乾
LI Ri-fei;ZHOU Peng;WANG Ting-ting;LI Ri-yong;YIN Hui;WANG Zi-hao;ZHANG Wen-yuan;ZUO Ru-nan;LI Yin-qian(College of Veterinary Medicine,Northwest Agriculture and Forest University,Yangling 712100,China;Synthetic and Functional Biomolecules Center,College of Chemistry and Molecular Engineering,Peking University,Beijing 100871,China;Clinical College,Hospital 307 of Chinese People ＇s Liberation Army,Anhui Medical University,Beijing 100071,China;Peking University Health Sciences Center,Beijing 100191,China;Institute of Pharmacology＆ Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《国际药学研究杂志》
CAS
CSCD
北大核心
2018年第3期211-218,共8页
Journal of International Pharmaceutical Research
