中华蜜蜂溶血肽基因在杆状病毒系统中的表达

构建含中华蜜蜂溶血肽基因的重组转移载体pBacHT-GFPTAccM，转化受体菌DH10Bac，得重组穿梭载体Bacmid-GFPTAccM, Lipofectin介导其基因组DNA转染粉纹夜蛾细胞系Tn-5B1-4。SDS-PAGE分析表明，感染重组杆状病毒Bacmid-GFPTAccM的细胞表达产物在约为34 kD处出现特异性条带，其表达量约占细胞总蛋白的3%。Western blotting和细胞表达时相动态分析证明中华蜜蜂溶血肽基因已在粉纹夜蛾细胞系Tn-5B1-4中进行了成功的表达。

Expression of the melittin gene from Apis cerana cerana in baculovirus system

A cDNA fragment encoding melittin from Apis cerana cerana was obtained by restriction enzyme digestion from the recombinant plasmid pGEM-AccMT, and inserted into the multiple cloning site of the pBacHTeGFPT to construct the recombinant donor plasmid pBacHT-GFPTAccM, which was transposed to the target Bacmid in E. coli (DH10) by Tn7 transposition function. Then Bacmid-GFPTAccM recombinant genome DNA was used to transfect Tn-5B1-4 cell of the cabbage looper, Trichoplusia ni, mediated by lipofectin. The expressed protein band of about 34 kD was determined by SDS-PAGE, and the thin layer scanning showed that the expression amount of GFPT-AccM fusion protein was about 3% of the total cell protein. Western blotting and the cytopathic effect of Tn cell after infection of Bacmid-GFPTAccM at different time proved that the fusion protein GFPT-AccM had been successfully expressed in Tn-5B1-4 cell.