| 伪狂犬病病毒IE180基因启动子缺失克隆的构建与鉴定 |
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| 引用本文： | 韩冬梅,阮可悦,曹云雷,张丽荣,童武,李国新,郑浩,童光志.伪狂犬病病毒IE180基因启动子缺失克隆的构建与鉴定[J].中国动物传染病学报,2021(2):1-8. |
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| 作者姓名： | 韩冬梅 阮可悦 曹云雷 张丽荣 童武 李国新 郑浩 童光志 |
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| 作者单位： | 中国农业科学院上海兽医研究所;扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心 |
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| 基金项目： | 国家重点研发计划项目(2016YFD0500100);中国农业科学院科技创新工程专项(0201007001002);上海市科技兴农重点攻关项目(沪农科攻字(2016)第4-2号)。 |
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| 摘 要： | 在本实验室已有的PRV JS-2012感染性克隆pBAC-JS2012及突变筛选菌株SW102-PRVBAC基础上,利用galk正筛选和卡那霉素的筛选标记基因,构建IE180两个拷贝启动子均删除的缺失克隆。利用同源重组将galk表达盒替换掉IE180TATA-box及左边297个碱基和右边172个碱基,经PCR鉴定和筛选获得阳性克隆SW102-galk。通过Western blot分析,IE180仍然存在表达。在此基础上再利用相同的方法将Kna筛选标记基因替换掉另一个拷贝IE180TATA-box及其左边134个碱基和右边309个碱基,经过PCR鉴定、Western blot分析以及转染后绿色荧光表达的分析,证实IE180不再表达。本研究成功筛选到IE180启动子缺失的阳性克隆pPRV-DIE180-BAC,为后期IE180缺失互补系统的构建及更深入的探究IE180的生物学功能奠定基础。
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| 关 键 词： | 伪狂犬病病毒 IE180 启动子 缺失克隆 |
Construction and Characterization of a Promoter Deletion Clone of IE180 Gene from Pseudorabies Virus |
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| HAN Dongmei,RUAN Keyue,CAO Yunlei,ZHANG Lirong,TONG Wu,LI Guoxin,ZHENG Hao,TONG Guangzhi.Construction and Characterization of a Promoter Deletion Clone of IE180 Gene from Pseudorabies Virus[J].Chinese Journal of Animal Infectious Diseases,2021(2):1-8. |
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| Authors: | HAN Dongmei RUAN Keyue CAO Yunlei ZHANG Lirong TONG Wu LI Guoxin ZHENG Hao TONG Guangzhi |
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| Institution: | (Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou University,Yangzhou 225009,China) |
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| Abstract: | The deletion clone of IE 180 both promoter was constructed using the infectious clone pBAC-JS2012 of PRV JS-2012 and positive clone SW102-PRV BAC in our laboratory by balk positive screening and screening marker gene of Kandinsky.The galk expression gene was used to replace the 297 bp base on the left and 172 bp base on the right of IE180TATA-box.The positive clone SW102-galk was identified by PCR.The expression of IE 180 was detected by Western blot.Furthermore,the kanamycin screening gene was used to replace other copy of IE180TATA-box,134 bp on the left side and 309 bp on the right side using the same method.After PCR amplification,Western blot analysis and green fluorescent expression post transfection,the IE 180 promotor was confirmed no longer expressed.The availability of the IE180 promoter deletion positive clone PPRV-DIE180-BAC laid a foundation to further explore its biological function and construct a complementary system. |
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| Keywords: | Pseudorabies virus IE 180 promoter deletion clone |
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