非洲猪瘟病毒NP419L基因shRNA表达质粒的构建与筛选  

Construction and screening of shRNA plasmid targeting on NP419L gene of African swine fever virus

作　　者：宋红芹[1] 姜翠翠[1] 张鑫宇[1] 夏晓莉[1] 孙怀昌[1] 

机构地区：[1]扬州大学兽医学院禽类预防医学教育部重点实验室,江苏扬州225009

出　　处：《中国兽医科学》2013年第1期9-14,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31001052);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0978);江苏省优势学科建设工程资助项目(2010)

摘　　要：为构建非洲猪瘟病毒(ASFV)shRNA表达质粒,以PCR扩增出非洲猪瘟病毒NP419L基因,并插入真核表达载体pEGFP-N1中,构建重组真核表达质粒pNP419L-GFP,将其转染NIH-3T3细胞,NP419L-GFP融合基因在细胞中获得表达。针对NP419L基因设计并合成5对干扰序列,将其连接到RNAi表达载体pSuper中,构建干扰质粒pSuper-shNP419L-A,B,C,D,E,将质粒pNP419L-GFP和pSu-per-shNP419L-A,B,C,D,E分别共转染NIH-3T3细胞,通过荧光显微镜观察和流式细胞仪分析干扰效率。结果显示,5个干扰质粒均能抑制NIH-3T3细胞中NP419L-GFP融合基因的表达,其中pSuper-shNP419L-A,B的抑制效果较好,在87%以上,与pSuper-shNP419L-C,D,E之间均存在显著性差异(P<0.05)。表明,本试验成功构建出针对ASFV NP419L基因的RNA干扰质粒,并筛选出了有效沉默NP419L基因的干扰序列,为进一步体内试验研究奠定了基础。To construct RNA interfering plasmid containing NP419L gene of African swine fever virus（ASFV）,NP419L gene was amplified by PCR,then cloned into the expression vector pEGFP-N1,resulting in construction of a report vector pNP419L-GFP.The pNP419L-GFP was transfected into NIH-3T3 cells and the fusion gene NP419L-GFP was expressed.After that,five pairs of shRNAs targeting on NP419L gene were designed and synthesized,then cloned into the RNAi vector pSuper.Consequentially,the RNA interfering plasmids pSuper-shNP419L-A,B,C,D and E were constructed,respectively.The plasmids pSuper-shNP419L-A,B,C,D,E and pNP419L-GFP were co-transfected into NIH-3T3 cells,and the interfe-rence efficiencies of RNA interfering plasmids were tested by fluorescent microscopy and flowcytometry,respectively.In result,all the five interfering plasmids could effectively silence the expression of NP419L-GFP fusion gene in NIH-3T3 cells,and the pSuper-shNP419L-A,B had the better inhibition efficiency than the pSuper-shNP419L-C,D and E（P 0.05）.The result showed that the construction of RNA interfering plasmids successfully interfered in the NP419L gene expression in vitro,which provide a basis for a further investigation in vivo.

关 键 词：非洲猪瘟病毒 NP419L基因 RNA干扰 筛选 

分 类 号：S852.659.1[农业科学—基础兽医学]