| 牛乳状瘤病毒13型pEGFP-E5-N1真核表达载体的构建及其在HEK293细胞中的表达 |
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| 引用本文： | 庞峰,史巧芸,朱华培,徐开莲,赵天靖,李亚颖,彭冬梅,李国华,周海龙,王凤阳. 牛乳状瘤病毒13型pEGFP-E5-N1真核表达载体的构建及其在HEK293细胞中的表达[J]. 中国畜牧兽医, 2015, 42(5): 1088-1092. DOI: 10.16431/j.cnki.1671-7236.2015.05.008 |
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| 作者姓名： | 庞峰 史巧芸 朱华培 徐开莲 赵天靖 李亚颖 彭冬梅 李国华 周海龙 王凤阳 |
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| 作者单位： | 海南大学农学院, 海南省热带动物繁育与疫病研究重点实验室, 海口市动物基因工程重点实验室, 海口 570228 |
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| 基金项目： | 海南大学"中西部"重点学科项目(ZXBJH-XK002) |
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| 摘 要： | 基于海口市动物基因工程重点实验室获得的牛乳状瘤病毒13型(BPV-13)基因组序列信息(GenBank登录号:KM258443.2),以基因组为模板,扩增E5基因片段,将其连入pEGFP-N1质粒,构建重组质粒pEGFP-E5-N1,测序正确后瞬时转染HEK293细胞,应用荧光显微镜、流式细胞仪、Western blotting 鉴定融合蛋白的表达。结果显示,E5片段大小为135 bp;重组质粒pEGFP-E5-N1构建正确;荧光显微镜下观察转染重组质粒pEGFP-E5-N1的HEK293细胞,可见明亮的绿色荧光;流式细胞术分析结果显示,转染重组质粒pEGFP-E5-N1 48 h后,约55.59% 的细胞表达绿色荧光蛋白;Western blotting结果表明,表达的融合蛋白大小约为32 ku。本试验结果为下一步研究E5基因的作用机理奠定了基础。
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| 关 键 词： | 牛乳状瘤病毒13型(BPV-13) E5 EGFP 融合蛋白 真核表达 |
| 收稿时间： | 2014-11-03 |
Construction of Eukaryotic Expression Vector pEGFP-E5-N1 of Bovine Papillomavirus Type 13 and its Expression in HEK293 Cells |
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| PANG Feng,SHI Qiao-yun,ZHU Hua-pei,XU Kai-lian,ZHAO Tian-jing,LI Ya-ying,PENG Dong-mei,LI Guo-hua,ZHOU Hai-long,WANG Feng-yang. Construction of Eukaryotic Expression Vector pEGFP-E5-N1 of Bovine Papillomavirus Type 13 and its Expression in HEK293 Cells[J]. China Animal Husbandry & Veterinary Medicine, 2015, 42(5): 1088-1092. DOI: 10.16431/j.cnki.1671-7236.2015.05.008 |
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| Authors: | PANG Feng SHI Qiao-yun ZHU Hua-pei XU Kai-lian ZHAO Tian-jing LI Ya-ying PENG Dong-mei LI Guo-hua ZHOU Hai-long WANG Feng-yang |
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| Affiliation: | Animal Genetic Engineering Key Laboratory of Haikou City, Hainan Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research, College of Agriculture, Hainan University, Haikou 570228, China |
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| Abstract: | The PCR technology was used to amplify the E5 gene according to the genome sequence of bovine papillomavirus type 13 (BPV-13) Hainan strain with the GenBank accession number KM258443.2.Then it was ligated into pEGFP-N1 vector.The recombinant plasmid pEGFP-E5-N1 was transfected into HEK293 cells with liposomes by transient transfection.The expression of fusion protein was identified by fluorescence microscope, flow cytometry and Western blotting.The results showed that the E5 gene was 135 bp with the 100% homology with the sequence deposited in GenBank.Bright green fluorescence could be seen in HEK293 cells transfected by pEGFP-E5-N1.The expressed fusion protein was about 32 ku.This experiment laid the foundation for further study of the functions of E5 gene in eukaryotic cells. |
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| Keywords: | bovine papillomavirus type 13 (BPV-13) E5 EGFP fusion protein eukaryotic expression |
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