摘要
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.
出处
《江苏农业学报》
CSCD
北大核心
2010年第5期1093-1097,共5页
Jiangsu Journal of Agricultural Sciences
基金
国家公益性行业科研专项(3-15)
中国博士后科学基金面上资助项目(20080441061)
江苏省博士后科研资助计划项目(0901067C)
农产品安全检测实验室开放课题(BM2006611)