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当前位置: 首页 > 文章 > 小鼠抑制素α亚基在杆状病毒表达系统中的表达和纯化 中国兽医科学 2015,45 (12) 1295-1299
Position: Home > Articles > Expression and purification of mouse inhibin-αin baculovirus expression system Chinese Veterinary Science 2015,45 (12) 1295-1299

小鼠抑制素α亚基在杆状病毒表达系统中的表达和纯化

作  者：
李悦;陈伟贞;陈建国;胡长敏;刘东明
单  位：
华中农业大学动物医学院;华中农业大学农业微生物学国家重点实验室华中农业大学动物医学院
关键词：
抑制素α亚基;杆状病毒表达系统;表达;纯化
摘  要：
为利用杆状病毒表达系统表达小鼠的抑制素α亚基基因(INHα),将模板质粒pcDNA3.1(+)-INHα中的INHα基因克隆至载体质粒pFastBac1中,构建出重组转座质粒pFastBac1-INHα,转化至感受态DH10Bac细胞,形成重组穿梭质粒Bacmid-INHα,转染至Sf9细胞,获得第一代重组杆状病毒(P1)。然后病毒连续传代2次,用RT-PCR鉴定重组的杆状病毒,用SDS-PAGE验证纯化后蛋白质的表达,并使用BCA蛋白质浓度检测试剂盒检测纯化后的蛋白质样品的质量浓度。结果表明,感染重组杆状病毒后的Sf9细胞能够明显看到细胞皱缩、碎裂等感染症状;RT-PCR能够扩增出1 272bp的条带,证明重组的杆状病毒构建正确;SDS-PAGE结果表明重组的杆状病毒能够表达出45ku的条带,与理论大小一致;利用BCA蛋白质浓度检测试剂盒测得纯化样品中蛋白质的质量浓度为(216.05±10.02)μg/mL。小鼠抑制素α亚基成功地在杆状病毒表达系统中被表达,为今后抑制素的广泛应用奠定了基础。
译  名：
Expression and purification of mouse inhibin-αin baculovirus expression system
作  者：
LI Yue;CHEN Wei-zhen;CHEN Jian-guo;HU Chang-min;LIU Dong-ming;DING Ming-xing;HAN Li;State Key Laboratory of Agricultural Microbiology/Huazhong Agricultural University;College of Veterinary Medicine,Huazhong Agricultural University;
关键词：
INHα;;baculovirus expression system;;expression;;purification
摘  要：
To express mouse inhibinαsubunit by using baculovirus expression system,the INHαgene in template plasmid pcDNA3.1(+)-INHα was inserted into vector pFastBac1 to construct recombinant transposition plasmid pFastBac1-INHα.Then,it was transformed into competent Escherichia coil DH10 Bac for transposition.After the recombinant Bacmid-INHαobtained and transfected into Sf9 insect cells,the prepared recombinant baculovirus of passage 1was obtained.The passage 1was subcultured for 2passages.The prepared recombinant Bacmid-INHαwas identified by RT-PCR and the expression of purified protein was determined by SDS-PAGE.The BCA Protein Assay Kit was used to quantify the production of protein.The result showed that the Sf9 cells infected with recombinant Bacmid-INHαwere obviously seen cell shrinkage,fragmentation and other symptoms of infection.The gene fragment of 1 272 bp was amplified by RT-PCR which showed that the recombinant Bacmid-INHαwas constructed correctly.The SDS-PAGE result showed that the recombinant Bacmid-INHαexpressed in insect cells with 45 ku band which was consistent with the actual mass of INHα.The concentration of the INHαprotein was(216.05±10.02)μg/mL by using BCA Protein Assay Kit.The mouse INHα was successfully expressed in baculovirus expression system,which lay the foundation for the wide application of INHαin the future.

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