p15在杆状病毒-昆虫细胞表达系统中的表达  

Expression and identification of p15 in BAC-TO-BAC baculovirus expression sys-tems

作　　者：艾永兴[1] 肖昌[2] 郝军元[1] 胡薇[3] 潘风光[1] 郑梅竹[1] 张玉静[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]西南大学生命科学院,重庆400715 [3]吉林农业大学生物技术学院,吉林长春130118

出　　处：《中国兽医学报》2009年第5期632-636,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30471284);吉林大学农学部博士启动基金资助项目

摘　　要：为探讨鸡p15蛋白的特性及其在细胞周期抑制通路中与其他蛋白的作用,本研究选用昆虫细胞表达系统来表达有活性的p15蛋白。为后期纯化的需要,对pFAST供体质粒的多克隆位点进行了重新改造,并成功构建带有TAP纯化标签的供体质粒pFAST-NMCS-N-TAP-p15和pFAST-NMCS-C-TAP-p15。将2个供体质粒转化到DH10Bac感受态菌中,对阳性转化子Bacmid-N-TAP-p15和Bacmid-C-TAP-p15进行反复涂板的去阴性单克隆纯化,提取大质粒Bacmid-N-TAP-p15和Bacmid-C-TAP-p15并进行转座鉴定。将纯化的大质粒Bacmid-N-TAP-p15和Bacmid-C-TAP-p15转染入sf9昆虫细胞中,72h后收集培养上清中的杆状病毒,并进行病毒的扩增和滴度测定,结果所获2株重组杆状病毒N-TAP-p15和C-TAP-p15的滴度分别为3.6×107,5.4×107pfu/mL。将2株高滴度的重组杆状病毒再感染新培养的sf9昆虫细胞进行重组p15蛋白的表达,结果与对照组相比,重组p15融合蛋白得到了有效的表达,表达量分别为4.32%和4.7%。Western blotting结果表明,所表达的重组p15融合蛋白只特异地与p15单克隆抗体和兔的IgG反应而未与其他细胞蛋白反应;说明带有TAP标签的重组p15融合蛋白在昆虫细胞表达系统中得到了有效表达。To characterize the properties and functions of chicken pl 5 protein, BAC TO-BAC baculovirus expression systems was used to express active p15 protein. The multiple clone site of donor plasmid in this system was reconstructed. The donor plasmids of p15 fused TAP-tag for purification, pFAST-NMCS-N-TAP-pl5 and pFAST- NMCS-C-TAP-pl5 ,were constructed and transformed into DH10Bac competent cells, respectively. From two trans- formants, Bacmid-N-TAP-pl5 and Bacmid-C-TAP plS, were isolated after three times monoclonal plating. Two recombinant bacmid were extracted and transfected into sf9 cells and then the recombinant virus was harvested from cell culture supernatant at 72 hours after transfection. The titer assay was performed after amplifying the recombinant baculoviral stock, sf9 cells were infected with two recombinant baculovirus as high titer respectively for the ex- pression of p15 fusion proteins. The results showed that the titers of N-TAP-pl5 and C-TAP-pl5 recombinant baculovirus were 3.6× 10^7 pfu/mL and 5.4 × 10^7 pfu/mI., respectively. Two p15 fusion proteins were expressed in sf9 cells and amounting to 4. 32% and 4.7% ,respectively. Western blotting showed that both of them specifically bound to McAb against p15 and rabbit IgG,respectively.

关 键 词：P15 TAP sf9昆虫细胞 杆状病毒 

分 类 号：S852.2[农业科学—基础兽医学] Q78[农业科学—兽医学]