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针对非洲猪瘟病毒p30基因CRISPR/Cas9基因编辑系统的构建和初步鉴定

文献类型: 中文期刊

作者: 于海男 1 ; 崔帅 1 ; 王洋 1 ; 刘雪婷 1 ; 高新桃 2 ; 房立春 3 ; 郭晓宇 1 ; 朱鸿飞 1 ;

作者机构: 1.中国农业科学院北京畜牧兽医研究所

2.中国农业科学院生物技术研究所

3.山东省农业科学院家禽研究所

关键词: 非洲猪瘟病毒;p30基因;CRISPR/Cas9;单导向RNA;真核表达

期刊名称: 中国动物传染病学报

ISSN: 1674-6422

年卷期: 2023 年 31 卷 002 期

页码: 59-66

摘要: 构建针对非洲猪瘟病毒(ASFV)p30(CP204L)基因的CRISPR/Cas9基因编辑系统,探究该系统对p30蛋白真核表达的影响.以含p30基因序列的质粒p30-IRES为模板,定向扩增目的基因片段并克隆至pmCherry-N1载体,构建含有p30、红色荧光报告基因的融合表达质粒;通过网站http://crispr.mit.edu/设计靶向CP204L的单导向RNA(sgRNA),并克隆至Cas9表达质粒eSpCas9-2A-GFP(PX458)中.将融合表达质粒与Cas9表达质粒共转染HEK 293T细胞,同时设置对照组,转染48 h后通过Western blot和荧光定量PCR(qPCR)方法分别检测转染细胞中p30蛋白和mRNA表达水平.由于CRISPR/Cas9基因编辑系统靶向切割p30基因,Western blot和qPCR结果显示转染靶向p30基因的Cas9质粒后,p30蛋白的真核表达受到抑制,具有显著性差异(P<0.05).结果表明,CRISPR/Cas9基因编辑系统可以高效切割非洲猪瘟p30基因,抑制该蛋白的真核表达水平,该体系为抑制病毒生长和疫情防控提供了新的研究思路.

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