利用杆状病毒Bac-to-Bac系统在Sf9细胞中表达真菌细胞色素P450nor(英文)  

Over-expression of Fungal Cytochrome P450nor Gene in Sf9 Cells by Using Bac-to-Bac Baculovirus Expression System

作　　者：周建刚[1] 江月[1] 段媛媛[1] 彭建新[1] 刘德立[1] 

机构地区：[1]华中师范大学生命科学学院,武汉430079

出　　处：《微生物学报》2004年第2期177-181,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金 ( 3 0 170 0 11)~~

摘　　要：利用Bac to Bac杆状病毒载体表达系统将真菌细胞色素P4 5 0nor基因克隆至转移载体pFastBac1中 ,得到重组质粒pFastBac P4 5 0nor,再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中发生转座作用 ,得到含P4 5 0nor基因的重组穿梭载体rBacmidpAc P4 5 0nor。分离提取重组BacmidDNA ,并转染培养的昆虫细胞Sf9,得到重组病毒rAc p4 5 0nor。经酶切和PCR鉴定 ,细胞色素P4 5 0nor基因正确地插入到病毒基因组的多角体蛋白基因启动子下 ,SDS PAGE分析证明 :表达蛋白的分子量为 4 3kD左右。Westernblotting分析结果表明 :有一条特定的杂交带存在 ,且分子量相同 (约 4 3kD)。进一步证明了含有真菌细胞色素P4 5 0nor基因的重组表达载体和重组病毒构建成功 ,并在昆虫细胞Sf9中实现了高效表达 ,经MTT法测定表达的细胞色素P4 5 0nor具有还原NO的生物学活性。The Bac-to-Bac baculovirus expression system is a novel gene expression system that allows the rapid and efficient generation of recombinant baculovirus DNA by site-specific transposition in Escherichia coli, rather than homologous recombination in insect cells. The recombinant vector, pFast-P450nor, was constructed by inserting the cytochrome P450nor gene from Cylindrocarpon tonkinense into the BamHⅠ/XbaⅠ sites of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. After transformation, pFast-P450nor was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid. Recombinant bacmid pAc-P450nor was constructed by transposing a mini-Tn7 element from the donor plasmid, pFast-P450nor, to the mini-attTn7 attachment site on the bacmid, where the Tn7 transposition functions were provided in trans by a helper plasmid. The recombinant bacmid DNA was isolated and transfected into the insect cells (Spodoptera frugiperda, Sf9) to produce the recombinant virus, rAc-P450nor. Fresh insect Sf9 cells were infected with the recombinant virus containing cytochrome P450nor to express the target protein. Result showed that the recombinant bacmid expression vector, pAc-P450nor, was constructed successfully and the cytochrome P450nor gene was highly expressed in Sf9 insect cells. A specific band (about 43kD) was detected by SDS-PAGE and confirmed by Western blot analysis. Availability of this expression system should facilitate characterization of the role of cytochrome P450nor in the metabolism of NO.

关 键 词：杆状病毒 BAC-TO-BAC系统 SF9细胞 表达 真菌 细胞色素P450nor 基因 生物学活性 

分 类 号：S852.65[农业科学—基础兽医学]