鸡堆型艾美耳球虫单链抗体基因的构建及表达  

Construction and expression of gene of ScFv antibody for detection of Eimeria acervulina surface antigens

作　　者：康桂英[1] 赵月兰[2] 秦建华[1] 郭红斌[2] 包永占[1] 张宁[1] 郭莉[1] 

机构地区：[1]河北农业大学动物科技学院 [2]河北农业大学中兽医学院,河北定洲073000

出　　处：《中国兽医学报》2008年第9期1028-1031,共4页Chinese Journal of Veterinary Science

基　　金：河北省自然科学基金资助项目(303226)

摘　　要：轻链可变区和重链可变区基因产物等摩尔浓度混合后,用重叠延伸拼接法扩增出单链抗体基因,再用带限制性内切酶位点的引物(NcoⅠ和HindⅢ)扩增出带有特定酶切位点的单链抗体基因片段。NcoⅠ和HindⅢ双酶切单链抗体与表达载体pET-22b(+),用回收试剂盒回收酶切后的单链抗体与表达载体pET-22b(+)基因片段,用连接试剂盒将单链抗体与表达载体pET-22b(+)基因片段连接,并将连接产物转化感受态细胞JM109。氨苄青霉素筛选阳性重组子,以T7promoter/T7terminator primer进行菌落PCR鉴定,测定正确后,37℃摇菌扩增,测菌浓度至D600为0.6时,加入IPTG诱导表达,对表达产物进行SDS-PAGE初步检测。SDS-PAGE检测显示其相对分子质量是31 000,与预期结果一致,证明单链抗体基因在大肠杆菌中得到表达。Same quality of purified VH and VL genes were joined together with a linker DNA by splicing overlap extension PCR and a desired fragment(scfv) was generated.After scfv and expression vector were digested with the corresponding restriction enzymes NcoⅠand HindⅢ and purified on an agarose gel,the scfv gene was ligated into expression vector pET-22b(+)and transformed into competent E.coli JM109 cells.the recombinants was grown on the ampr plate.The aimed product was identified with T7 promoter primer and T7 te...

关 键 词：鸡 堆型艾美耳球虫 单链抗体 可变区 

分 类 号：S852.723[农业科学—基础兽医学]