| 亚非马蜂溶血肽基因与增强型绿色荧光蛋白基因融合在昆虫细胞中的表达 |
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| 引用本文： | 施婉君,杨章女,张传溪,程家安. 亚非马蜂溶血肽基因与增强型绿色荧光蛋白基因融合在昆虫细胞中的表达[J]. 农业生物技术学报, 2006, 14(1): 102-106,i0002 |
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| 作者姓名： | 施婉君 杨章女 张传溪 程家安 |
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| 作者单位： | 1. 浙江大学应用昆虫学研究所,杭州,310029;上海交通大学资源环境科学系,上海,201101
2. 浙江大学应用昆虫学研究所,杭州,310029 |
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| 基金项目： | 上海交通大学院青年基金(No.AE150054)，国家自然基金(No.30271008)资助 |
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| 摘 要： | 构建亚非马蜂(Polisteshebraeus)溶血肽基因重组转移载体pBacHT-GFPTPhMT,转化含穿梭载体Bacmid的受体菌Escherichiacoli(DH10Bac)中,得重组穿梭载体Bacmid-GFPTPhM,再用Lipofectin介导其DNA转染粉纹夜蛾(Trichoplusiani)细胞系Tn。SDS-PAGE分析表明,感染Bacmid-GFPTPhM的Tn-5B1-4细胞的表达产物在约为34kD处出现特异性条带,表达量约占细胞总蛋白的3%。Westernblot显示表达产物具有良好的免疫活性。感染Bacmid-GFPTPhM的细胞表达时相动态分析进一步表明,与增强型绿色荧光基因融合的亚非马蜂溶血肽基因的重组病毒已在昆虫细胞中进行了成功地表达,感染后72h表达量可达最高水平。
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| 关 键 词： | 亚非马蜂 蜂毒溶血肽 增强型绿色荧光蛋白基因 表达 |
| 文章编号： | 1006-1304（2006）01-0102-05 |
| 收稿时间： | 2005-01-26 |
| 修稿时间： | 2005-01-262005-04-03 |
Expression of the Melittin Gene of Polistes hebraeus Fused with Enhanced Green Fluorescent Protein Gene in Insect Cells |
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| SHI Wan-Jun,YANG Zhang-Nv,ZHANG Chuan-Xi,CHENG Jia-An. Expression of the Melittin Gene of Polistes hebraeus Fused with Enhanced Green Fluorescent Protein Gene in Insect Cells[J]. Journal of Agricultural Biotechnology, 2006, 14(1): 102-106,i0002 |
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| Authors: | SHI Wan-Jun YANG Zhang-Nv ZHANG Chuan-Xi CHENG Jia-An |
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| Affiliation: | 1. Institute of Applied Entomology, Zhejiang University, Hangzhou 310029, China; 2. School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 201101, China |
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| Abstract: | A cDNA fragment encoding melittin from the venom gland of female Polistes hebraeus was cloned into pBacHTeGFPT to construct the recombinant donor plasmid pBacHT-GFPTPhM. The donor plasmid was then transformed into Escherichia coli (DH10Bac) to form the recombinant, Bacmid-GFPTPhM. Bacmid-GFPTPhM DNA was further transfected into Tn-5B1-4 cells of the cabbage looper, Trichoplusia ni, mediated by Lipofectin. SDS-PAGE result showed the expressed protein was about 34 kD. The thin layer scanning showed that the expressed fusion protein GFPT-PhM was accumulated up to about 3 % of the total cell protein. The recombinant product was confirmed by Western blot using the His-Tag monoclonal antibody and a rabbit derived polyclonal antibody against melittin, respectively. The cytopathic effect of Tn-5B1-4 cells after infection of Bacmid-GFPTPhM at different times was investigated and the result indicated that the recombinant protein was expressed to the highest level in Tn-5B1-4 cells 72 h post infection. |
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| Keywords: | Polistes hebraeus melittin enhanced green fluorescent protein gene expression |
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