表达绿色荧光蛋白的重组1型禽腺病毒的构建

doi:10.3969/j.issn.1674-5817.2013.04.001

实验动物与比较医学 ›› 2013, Vol. 33 ›› Issue (4): 249-255.DOI: 10.3969/j.issn.1674-5817.2013.04.001

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表达绿色荧光蛋白的重组1型禽腺病毒的构建

周洁¹, 赵莉^1,2,3, 胡建华¹, 高诚¹

1.上海实验动物研究中心, 上海 201203;
2.扬州大学兽医学院, 扬州 225009;
3.中科院上海药物所, 上海 201203

收稿日期:2012-10-15 出版日期:2013-08-25 发布日期:2013-08-25
作者简介:周洁(1978-),女,博士,副研究员;研究方向:动物病毒分子生物学,E-mail:zhoujie0526@163.com
基金资助:
上海市科委基金项目(No. 09140901400)

Construction of Recombinant Fowl Adenovirus Serotype 1 Exprsessing Exotic Green Fluorescence Protein

ZHOU Jie¹, ZHAO Li^1,2,3, HU Jian-hua¹, GAO Cheng¹

1. Shanghai Laboratory Animal Research Center, Shanghai 201203, China;
2. College of Veterinary Medicine,Yangzhou University, Yangzhou 225009, China;
3. Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China

Received:2012-10-15 Online:2013-08-25 Published:2013-08-25

摘要/Abstract

摘要： 目的构建表达绿色荧光蛋白的重组禽腺病毒。方法通过带有禽腺病毒基因组复制非必需区(nt40 065~nt43 684)侧翼序列和增强型绿色荧光蛋白基因的转移质粒与亲本禽腺病毒在鸡肝癌细胞内同源重组,缺失掉了两侧翼序列之间的复制非必需区而替换为增强型绿色荧光蛋白基因的完整表达盒。为提高同源重组率,对质粒转染细胞的条件进行了摸索,确定了最佳转染条件为细胞以3×10⁵/ ml密度接种到6孔板上,36 h后用8 μl lipofectamine 2000试剂和3 μg pUC-LR-eGFP质粒转染,蚀斑方法筛选重组病毒。结果获得了表达EGFP的重组病毒单一克隆株rFAV-I-eGFP。结论研究结果为开展禽类活载体疫苗研制及相关基础研究提供了有利参考。

关键词: 重组I型禽腺病毒, 鸡肝癌细胞系, 增强型绿色荧光蛋白

Abstract: Objective To construct a recombinant fowl adenovirus expressing exotic green fluorescence protein(eGFP). Methods The flanking fragments of non-essential region(nt40 065~nt43 684) in fowl adenovirus genome were cloned into a transfer plasmid as well as eGFP. Through homologous recombination between the transfer plasmid and wild fowl adenovirus in chicken hepatocellular carcinoma cell line(LMH), the non-essential region between the flanking fragments was replaced by the complete eGFP expression cassette. In order to improve the occurrence probability of recombination, the transfection conditions such as logarithmic growth phase of LMH, the ratio of plasmid and lipofectamine, cells seeding density were explored. The optimum transfection condition was determined as follows: Seed 3×10⁵/ml LMH cells into 6 cell culture plate, 36 h later, cells were transfected with 8 μl lipofectamine 2 000 and 3.0 μg plasmid pUC-LR-eGFP. Recombinant virus was screening by plaque purification. Results recombinant adenovirus serotype 1 (rFAdV-1) expressing eGFP was obtained. Conclusion These results provided favorable references for fowl live vaccine development and related fundamental research.

Key words: Recombinant fowl adenovirus serotype 1(rFAdV-1), Chicken hepatocellular carcinoma cell line(LMH), Exotic green fluorescence protein(eGFP)

中图分类号:

Q95-33

周洁, 赵莉, 胡建华, 高诚. 表达绿色荧光蛋白的重组1型禽腺病毒的构建[J]. 实验动物与比较医学, 2013, 33(4): 249-255.

ZHOU Jie, ZHAO Li, HU Jian-hua, GAO Cheng. Construction of Recombinant Fowl Adenovirus Serotype 1 Exprsessing Exotic Green Fluorescence Protein[J]. Laboratory Animal and Comparative Medicine, 2013, 33(4): 249-255.

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表达绿色荧光蛋白的重组1型禽腺病毒的构建

Construction of Recombinant Fowl Adenovirus Serotype 1 Exprsessing Exotic Green Fluorescence Protein

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