| 鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定 |
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| 引用本文： | 杨森,滕蔓,刘金玲,周子誉,郑鹿平,楚钰淑,丁轲,余祖华,罗俊.鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定[J].畜牧兽医学报,2020,51(8):1970-1976. |
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| 作者姓名： | 杨森 滕蔓 刘金玲 周子誉 郑鹿平 楚钰淑 丁轲 余祖华 罗俊 |
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| 作者单位： | 1. 河南科技大学动物科技学院, 动物疫病与公共卫生重点实验室, 洛阳 471003;2. 河南省农业科学院动物免疫学重点实验室, 农业部动物免疫学重点实验室, 河南省动物免疫学重点实验室, 郑州 450002;3. 河南省农业科学院, 中英禽病国际研究中心, 郑州 450002;4. 河南农业大学牧医工程学院, 郑州 450002 |
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| 基金项目： | 国家自然科学基金（U1604232）；中原千人计划-中原基础研究领军人才；河南省重点研发与推广专项（192102110072）；河南省农业科学院杰出青年科技基金（2019JQ01）；河南省农业科学院科研发展专项资金（2019CY012） |
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| 摘 要： | meq是鸡马立克病病毒（MDV）最重要的致瘤基因，在马立克病（MD）肿瘤发生中发挥关键作用。同时，它在疫苗株和强毒株之间具有明显的序列差异性。本文利用CRISPR/Cas9基因编辑技术，以MDV疫苗株CVI988/Rispens meq基因为靶点，设计合成gRNA，克隆构建pX459-gRNA质粒，转染CEF并感染CVI988/Rispens，然后对meq基因编辑的病毒噬斑进行克隆纯化，经过PCR扩增、测序分析及IFA鉴定，成功构建1株meq基因编辑的缺失毒株CVI988Δmeq-C7，为后续筛选和鉴定抗MD疫苗株MEQ单抗及鉴别诊断研究奠定了基础。
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| 关 键 词： | 马立克病 CVI988/Rispens meq基因 CRISPR/Cas9 基因编辑 |
| 收稿时间： | 2020-02-08 |
Construction of meq Deleted strain by Gene Editing of Marek's Disease Vaccine Strain CVI988/Rispens via the CRISPR/Cas9 System and Identification |
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| YANG Sen,TENG Man,LIU Jinling,ZHOU Ziyu,ZHENG Luping,CHU Yushu,DING Ke,YU Zuhua,LUO Jun.Construction of meq Deleted strain by Gene Editing of Marek's Disease Vaccine Strain CVI988/Rispens via the CRISPR/Cas9 System and Identification[J].Acta Veterinaria et Zootechnica Sinica,2020,51(8):1970-1976. |
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| Authors: | YANG Sen TENG Man LIU Jinling ZHOU Ziyu ZHENG Luping CHU Yushu DING Ke YU Zuhua LUO Jun |
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| Abstract: | Meq gene is commonly regarded as the main oncogene that plays critical role in the tumorigenesis of Marek's disease (MD). Evolutionarily, there is an obvious sequence difference between the meq genes of vaccine and pathogenic strains with differ virulence. In this study, we have designed the specific guide RNAs (gRNAs) targeting to the meq gene of the vaccine strain CVI988/Rispens, to make the plasmid pX459-gRNA. The CEF cells were transfected with pX459-gRNA plasmids and were infected with CVI988/Rispens virus to produce meq-gene mutated viral particles. Utilizing the CRISPR/Cas9-based gene editing technique, together with the viral plaque purification, PCR analysis, DNA sequencing and indirect immunofluorescence assay (IFA), we successfully generated a meq-deleted mutant named as CVI988Δmeq-C7. Our data provides an important basis for further screening of the monoclonal antibodies against MD vaccine MEQ protein and the development of differential diagnostic reagents. |
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| Keywords: | Marek's disease CVI988/Rispens meq gene CRISPR/Cas9 gene editing |
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