抗黄曲霉毒素B1单链抗体(scFv)库的建立和筛选

Construction of an Anti-aflatoxin B1 scFv Library and Screening for Specific Antibodies Against AFB1

从抗黄曲霉毒素B1单克隆抗体细胞系中扩增到重链可变区(VH)和轻链可变区(VL),经PCR将VH、Linker和VL连接成单链抗体(scFv),并克隆到载体pCANTAB-5E,经电转化大肠杆菌TG1,构建了库容量为1×108的单链抗体库。通过辅助噬菌体M13KO7感染后,使单链抗体展示在噬菌体衣壳蛋白pIII的N端,得到客容量为1×108的噬菌体展示的抗体库。将抗体库用于"淘洗-富集-扩增"4轮以后,得到针对AFB1特异性的噬菌体抗体。进一步以噬菌体感染大肠杆菌HB2151,在细菌细胞周质中可溶性大量表达,最终经竞争ELISA分析表明获得了3株可以稳定分泌抗黄曲霉毒素B1 scFv菌株,分别命名为3C3、3B3和4A2。

ScFv antibodies can be used not only in detection,but also in neutralization of toxins.The VH and VL gene fragments were first amplified from cDNA of hybridoma cell line specific to aflatoxin B1,integrated as scFv with a linker,cloned into phagemid vector pCANTAB-5E,and then transformed into Escherichia coli TG1 by electroporation.A scFv antibody library as large as 1×108 clones was obtained.To produce phage-display scFv,the helper phage M13KO7 was added to the culture when bacteria were grown up to the log...