| 棉铃虫核型多角体病毒ORF27基因的表达和细胞内定位 |
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| 引用本文： | 刘艳荷,陈艳霞,章亦卿,徐海君,张传溪.棉铃虫核型多角体病毒ORF27基因的表达和细胞内定位[J].农业生物技术学报,2007,15(3):503-507. |
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| 作者姓名： | 刘艳荷 陈艳霞 章亦卿 徐海君 张传溪 |
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| 作者单位： | 1. 浙江大学昆虫科学研究所,杭州,310029;广东海洋大学农学院,湛江,524088
2. 浙江大学昆虫科学研究所,杭州,310029 |
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| 基金项目： | 教育部全国百篇优秀博士学位论文作者专项基金;国家自然科学基金 |
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| 摘 要： | 棉铃虫核型多角体病毒（HaSNPV）ORF27编码区全长768bp，编码255个氨基酸残基组成的多肽，预计分子量29.5kD。PCR扩增获得该基因，克隆到融合表达载体pGEX-4t-2中，表达蛋白分子量55.5kD，表达量约占细菌总蛋白的9.2%。割胶透析法纯化融合蛋白，免疫家兔制备多克隆抗体。再把该基因插入到转移载体pFastBacHTb，转化大肠杆菌DH10Bac感受态细胞，构建重组病毒；提取病毒基因组DNA，在Lipofectin介导下转染昆虫细胞Tn-5B1-4。表达蛋白分子量为32kD，表达量约占细胞总蛋白的2.9%。将ORF27在昆虫细胞中表达产物固定，免疫电镜法确定表达蛋白主要存在于细胞的细胞质中。ORF27基因表达及在细胞内定位为其进一步研究提供了基础。
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| 关 键 词： | 棉铃虫核型多角体病毒 ORF27基因 表达 定位 |
| 文章编号： | 1006-1304（2007）03-0503-05 |
| 收稿时间： | 2006-10-23 |
| 修稿时间： | 2006-10-232006-12-20 |
Expression of the Open Reading Frame 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedro virus in Escherichia coli and Its Cell Localization |
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| LIU Yan-he,CHEN Yan-xia,ZHANG Yi-qing,XU Hai-jun,ZHANG Chuan-xi.Expression of the Open Reading Frame 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedro virus in Escherichia coli and Its Cell Localization[J].Journal of Agricultural Biotechnology,2007,15(3):503-507. |
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| Authors: | LIU Yan-he CHEN Yan-xia ZHANG Yi-qing XU Hai-jun ZHANG Chuan-xi |
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| Institution: | 1. Institute of Insect Sciences, Zhejiang University, Hangzhou 310029, China; 2. Agronomy College, Guangdong Ocean University, Zhanjiang 524088, China |
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| Abstract: | The open reading frame (orf) 27 of Helicoverpa armigera single nucleocapsid nucleopolyhedro virus is 768 bp in length and potential to encode a polypeptide of 255 amino acids, with a molecular weight of about 29.5 kD. The coding region of orf27 was amplified from HaSNPV genome DNA by PCR, and cloned into the expression vector pGEX-4t-2 for bacterial expression. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. The molecular weight of expressed GST-ORF27 fusion protein was 55.5 kD. Western blotting analysis showed that the recombinant protein could react with GST antibody. The fusion protein was retrieved from the SDS-PAGE gel and used to immunize the rabbits to raise the antibody against HaSNPV ORF27. orf27 gene was also recombined into the baculovirus vector Bacmid form the recombinant Bacmid-orf27 and then expressed in Tn-5B1-4 cells of cabbage looper (Trichopolusia ni). SDS-PAGE analysis showed the expressed His-ORF27 was 32 kD and identical to the predicted molecular weight. Western blotting analysis with anti-ORF27 further confirmed the correct expression. Immunoelectron microscopy demonstrated His-ORF27 fusion protein was mainly localized in the cytoplasm. |
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| Keywords: | Helicoverpa armigera single nucleopolyhedro virus orf27 expression localization |
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