摘要
通过构建pFastBacTM HT-A-scFv重组质粒,并将重组质粒化转DH10BacTM Escherichia coli感受态细胞,进行蓝白斑筛选,获得阳性克隆.PCR和基因测序检测证实挑取的白斑阳性单克隆存在完整、正确的单链抗体基因.提取阳性克隆中的重组杆状病毒穿梭载体（Bacmid-scFv）侵染sf9昆虫细胞,细胞发生明显病变特征.通过SDS-PAGE、Western blotting和间接竞争性ELISA等方法检测,发现单链抗体在sf9昆虫细胞中表达成功,测得纯化后蛋白浓度为103.000 0 μg/ml,Cry1B毒素对单链抗体的抑制中浓度（IC50）为1.010 0 μg/ml,最低检测线IC10为0.011 3μg/ml,线性检测范围为0.500 0～10.000 0 μg/ml.可溶性scFv对抗原类似物无交叉反应.
The single-chain fragment variable （scFv）gene was cloned into pFastBacTMHT-A to construct a recombi- nant plasmid pFastBacTM HT-A-scFv . The recombinant plasmid was then transformed into competent cell of DH10BaeTM Escherichia coli and blue-white selection was performed for positive clones. The positive clones with correct and inextenso scFv sequence were confirmed by polymerase chain reaction （PCR） and sequencing. Adenovirus shuttle vector （Bacmid-scFv） plasmids from positive clones were extracted to infect sf9 insect ceils. The sf9 cells presented significant pathological changes after 72-h transfection. SDS-PAGE, Western blotting and indirect competitive ELISA, con- firmed that the scFv proteins were successfully expressed in sf9 insect cells, and the concentration of purified scFv reached 103. 000 0 μg/m. In the concentrations of CrylBranging from 0. 500 0 μg/m to 10.000 0 μg/m, the IC5o and the detection limit （IC10） of CrylB toxin against scFv were 1. 010 0 μg/m and 0. 011 3 μg/m. The cross-reaction between antigen analogues and soluble scFv was not detected, in- dicating the sensitivity and specificity of the antibody.
出处
《江苏农业学报》
CSCD
北大核心
2013年第5期985-991,共7页
Jiangsu Journal of Agricultural Sciences
基金
国家“973”计划项目(2012CB722505)
国家自然科学基金项目(31272109、31201535)
江苏省农业自主创新基金项目[CX(12)5042]
关键词
Cry1B毒素
单链抗体
真核表达系统
酶联免疫分析
Cryl B toxin
single-chain fragment variable（scFv） antibody
eukaryotic expression system
ELISA
