摘要
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34kD处出现特异性条带,其表达量约占细胞总蛋白的3%。Westernblotting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。
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 hi, 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.
出处
《昆虫学报》
CAS
CSCD
北大核心
2006年第1期16-21,共6页
Acta Entomologica Sinica
基金
上海交通大学院青年基金(AE150054)
国家自然科学基金(30271008)~~
关键词
中华蜜蜂
溶血肽
表达
杆状病毒系统
粉纹夜蛾细胞系
Apis cerana cerana
melittin
expression
baculovirus system
TrichopUusia ni cell line
