摘要
使用同源重组方法,在昆虫细胞内将多角体启动子驱动的EGFP表达盒插入杆状病毒穿梭载体Bacmid的p74位相,经5轮空斑纯化获得重组穿梭载体Bacmid-egfp。然后将Bacmid-egfp转化含转座助手质粒的E.coliDH10B,获得受体菌E.coliDH10Bac-egfp,由于Bacmid-egfp保留了完整的转座结构和α互补功能,因此该菌株和原始E.coliDH10Bac一样能有效的利用各种pFastBac系列的载体进行转座并构建出能指示病毒繁殖和目的基因表达的重组病毒。使用红色荧光蛋白DsRed对系统进行了验证,结果表明重组病毒Bac-egfp-DsRed感染的细胞中绿色荧光蛋白和红色荧光蛋白均得到了高效表达。进一步使用该系统在昆虫细胞中高效表达并纯化了IL-6蛋白,为研究和应用该细胞因子提供物质基础,同时也进一步证明所改造的杆状病毒表达系统的可靠性和实用性。
Based on site-specific transposition of an expression cassette into a baculovirus shuttle vector （Bacmid） which propagated in Escherichia coli, the Bac-to-Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. And the efficiency of recombinant baculovirus infecting cells plays an important role on the protein expression. In this study, we introduced an EGFP expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp was then transformed into E, coli DH10B containing the transposition helper plasmid to gain a new transposition receipt strain E. coli DHlOBac-egfp. Because of the intact attTn7 sites and lacZ＇, target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracing of the target protein expression and the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac- egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coliDHlOBac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6 × his tag was expressed and purified sucessfully from Sf9 cells infected with recombinant virus Bac-egfp-6 × his-IL-6 constructed by the improved Bac-to-Bac system.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第4期572-580,共9页
Chinese Journal of Biotechnology
