利用Bac-to-Bac杆状病毒载体表达系统将真菌细胞色素P450nor基因克隆至转移载体pFastBac1中, 得到重组质粒pFastBac-P450nor, 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P450nor基因的重组穿梭载体rBacmid pAc-P450nor.分离提取重组Bacmid DNA, 并转染培养的昆虫细胞Sf9, 得到重组病毒rAc-p450nor.经酶切和PCR 鉴定, 细胞色素P450nor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43kD左右.Western blotting分析结果表明:有一条特定的杂交带存在, 且分子量相同(约43kD).进一步证明了含有真菌细胞色素P450nor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P450nor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 Bam HⅠ/Xba Ⅰ 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 transpositionrn 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 werrne 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 hrnighly 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.
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