一种新型家蚕核多角体病毒Bac to Bac系统的构建

CONSTRUCTION OF A NOVEL Bm NPV Bac to Bac SYSTEM

用家蚕核型多角体病毒的全基因组DNA与AcBacmidDNA共转染家蚕BmN细胞,构建转座一穿梭载体BmBacmid。另一供体质粒以转座方式将乙肝病毒e抗原基因HBeAg整合到BmBacmid的attTn7位点上成为重组rBmHBe。结果表明BmBacmid既能在大肠杆菌中以质粒的形式复制,又能在家蚕BmN细胞和草地夜蛾Sf9细胞中复制,形成感染性病毒粒子。Southernblotting证实重组病毒的构建是正确的。BmN细胞能正确识别与切割HBeAg信号肽序列,SDSPAGE表明HBeAg基因在家蚕细胞中高效表达,ELISA测定培养上清中HBeAg效价达1∶32000,细胞内HBeAg效价为1∶2000,培养液及细胞内的HBcAg含量极低(〈1∶160)。

A Bi Shuttle vector Bm Bacmid was constructed by co infecting Bm N cells with wild type genomic DNA from BmNPV and Ac Bacmid DNA. It could not only replicate in \%E. coli\% cells as a large plasmid and but also remain infectious when induced into Bm N or Sf9 cells. Recombinant virus rBmHBe was obtained after transposition of a donor plasmid carrying Hapatitis Be antigen gene (HBeAg) into att Tn7, and was demonstrated by Southern blotting. SDS PAGE analysis showed that HBeAg gene were highly expressed in Bm N cells. By ELISA testing, the highest antigenecity titer of HBeAg protein in cell cultural medium was up to a dilution of 1∶32?000. Although HBeAg protein also presented in the Bm N cells the titer was only 1∶?2000. The HBcAg protein was much fewer than HBeAg (<1∶160) whatever in culture medium and in cells. The results showed that Bm N cells was able to recognize the signal peptide sequence and cut it correctly for HBeAg protein\'s excreting production.