摘要
目的:利用昆虫杆状病毒表达系统表达早期快速反应基因5(IER5)蛋白,为后续蛋白质结晶及探索蛋白质三级结构提供线索。方法:以HeLa细胞cDNA为模板扩增出IER5基因片段,构建重组转移质粒pFastBac1-IER5;重组转移质粒经双酶切及测序鉴定后转化至感受态细胞DH10Bac,以获得重组的穿梭质粒rBacmid-IER5;将重组穿梭质粒感染Sf9细胞,待细胞出现明显病变时收集重组杆状病毒;用间接免疫荧光、SDS-PAGE及Western blot以及对表达产物进行分析鉴定。结果:重组转移质粒pFastBac1-IER5经双酶切后得到与预期相同的2条条带;重组穿梭质粒rBacmid-IER5经PCR鉴定在3300 bp左右出现一条特异性条带;间接免疫荧光结果提示IER5蛋白在Sf9细胞中得到表达;SDS-PAGE结果显示,表达产物的相对分子质量约为48 k,Western blot结果表明表达产物能与IER5抗体特异性结合。结论:利用昆虫-杆状病毒表达系统成功表达了人IER5蛋白,获得了体外表达重组IER5蛋白的相对分子质量、溶解性等物理化学特性。
OBJECTIVE:The insect baculovirus expression system was used to express the early rapid response gene 5 protein,and to provide clues for subsequent protein crystallization and exploration of tertiary protein structure.METHODS:The recombinant transfer vector pFastBac1-IER5 was constructed via amplification of the IER5 gene fragment with HeLa cell cDNA as template.After confirmation by restriction enzyme digestion and DNA sequencing,the recombinant transfer vector was transformed into E.coli DH10Bac^TM competent cells to obtain the recombinant shuttle vector rBacmid-IER5.The recombinant shuttle vector was transfected into Sf9 cells and the recombinant baculovirus was collected when the cells showed obvious lesions.The expression products were analyzed and identified using indirect immunofluorescence,SDS-PAGE and Western blot.RESULTS:The expected two bands were observed after the recombinant transfer vector pFastBac1-IER5 was digested by restriction enzymes;a band with the excepted size of about 3300 bp was obtained from the recombinant shuttle vector rBacmid-IER5 by PCR amplification;the results from indirect immunofluorescence indicate that IER5 protein was expressed correctly in the Sf9 cells.The SDS-PAGE analyses reveal that the molecular weight of the expressed product was about 48k.The Western blot analyses show that the expressed product specifically reacted with the IER5 antibody.CONCLUSION:The recombinant IER5 protein from human was expressed successfully using the insect baculovirus expression system,and revealed the physical and chemical properties of the recombinant IER5 protein,like the molecular weight and dissolubility.
作者
熊强
姜晓燕
丁立新
刘晓丹
周平坤
丁库克
XIONG Qiang;JIANG Xiaoyan;DING Lixin;LIU Xiaodan;ZHOU Pingkun;DING Kuke(National Institute for Radiological Protection,China CDC,Beijing 100088;Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Science,Beijing Key Laboratory for Radiobiology,Beijing 100850,China)
出处
《癌变．畸变．突变》
CAS
2020年第1期57-61,66,共6页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金项目(31640022,31770907)
北京市自然科学基金项目(7172146)。
