抗脱氧雪腐镰刀菌烯醇噬菌体单链抗体库的构建与鉴定

Construction and Identification of Phage ScFv Library against Deoxynivalenol

从抗脱氧雪腐镰刀菌烯醇(DON毒素)杂交瘤细胞株中扩增出VH和VL基因片段,再经重叠延伸PCR拼接扩增得到单链抗体基因(ScFv),克隆到pCANTAB-5E噬菌粒载体上。然后转化感受态大肠杆菌TG1,经辅助噬菌体超感染,构建成抗DON毒素噬菌体单链抗体库,库容约为1.1×105,滴度为1.3×108pfu。随机选取10个克隆进行双酶切鉴定,结果显示均有ScFv基因片段插入。抗DON毒素单链抗体库的构建,为进一步富集筛选并表达抗DON毒素单链抗体奠定了基础。

The VH and VL gene fragments were first amplified from cDNA of hybridoma cell line specific to deoxynivalenol,integrated as ScFv with a pair of linker primers,cloned into phagemid vector pCANTAB-5E,and then transformed into Escherichia coli TG1.To produce phage-display ScFv,the helper phage M13KO7 was added to the cultures when bacteria were grown up to the log-phase,in which ScFv antibody was displayed in N-terminus of the bacteriophage coat protein pⅢ.The phage antibody contained 1.1×10^5 independent clones,and the titer of which was about 1.3×10^8 pfu.Enzymatic digestions of 10 random clones showed that the recombinant plasmids contained an intact ScFv gene.These results indicated that the phage antibody library was constructed successfully.