抗Bt(Cry1B)毒素单链抗体在sf9昆虫细胞中的表达及活性测定  

Expression and characterization of anti-Bt( Cry1B) toxin scFv antibody in sf9 insect cells

作　　者：徐重新[1,2] 张霄[1] 张存政[1] 刘媛[1] 谢雅晶[1] 黄鹰[2] 刘贤金[1] 

机构地区：[1]江苏省农业科学院食品质量安全与检测研究所江苏省食品质量安全重点实验室--省部共建国家重点实验室培育基地/农业部农产品质量安全控制技术与标准重点实验室,江苏南京210014 [2]南京师范大学生命科学学院/江苏省微生物与功能基因组学重点实验室,江苏南京210023

出　　处：《江苏农业学报》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]

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.

关 键 词：Cry1B毒素 单链抗体 真核表达系统 酶联免疫分析 

分 类 号：Q786[生物学—分子生物学]