靶向PRRSV的FnCas9-rgRNA敲除载体构建研究  

Construction of FnCas9-rgRNA knockout vector system targeting to PRRSV

作　　者：赵姝灿 刘彦均 郑桂纯 肖文 傅建华 王丙云[1] 陈志胜[1] ZHAO Shucan;LIU Yanjun;ZHENG Guichun;XIAO Wen;FU Jianhua;WANG Bingyun;CHEN Zhisheng(Foshan University,Foshan 528200,China)

机构地区：[1]佛山科学技术学院,广东佛山528200

出　　处：《黑龙江畜牧兽医》2019年第3期20-23,176,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：广东省教育厅科研项目(2014KTSCX154);广东省教育厅预防兽医学重点实验室项目(2014KTSPT037)

摘　　要：为了检测靶向猪繁殖与呼吸综合征病毒(PRRSV)的FnCas9-rgRNA敲除载体是否构建成功,试验先构建FnCas9-rgRNA敲除载体骨架并对其进行测序,然后将黄色荧光蛋白(EYFP)、PRRSV分别与各自的FnCas9-rgRNA敲除载体转染至HEK293、Marc145细胞内,通过流式细胞仪分析HEK293细胞内的荧光强弱及Marc145细胞的病变程度,并对FnCas9-rgRNA敲除载体的初步构建、载体活性进行研究。结果表明:FnCas9-rgRNA敲除载体骨架测序正确,流式细胞仪分析显示FnCas9-rgRNA敲除载体对HEK293细胞内EYFP基因的表达产生明显抑制作用,表达荧光的细胞数量明显下降,3种针对PRRSV设计的rgRNA分别与FnCas9表达载体共转染细胞,接种病毒后Marc145细胞病变程度明显降低。说明靶向PRRSV的FnCas9-rgRNA敲除载体构建方式正确且FnCas9-rgRNA敲除载体具有活性。To detect the construction of FnCas9-rgRNA knockout vector targeting PRRSV,the FnCas9-rgRNA vector skeleton system was set up and sequenced in this experimant. Then,the enhanced yellow fluorescent protein ( EYFP) and PRRSV were transfected into HEK293 and Marc145 cells respectively with FnCas9 - rgRNA knockout vector,the fluorescence intensity in HEK293 cells and the degree of lesion of Marc145 cells were analyzed by flow cytometry. The preliminary construction of FnCas9-rgRNA knockout vector and its activity was sudied. The results showed that the FnCas9-rgRNA vector skeleton system was constructed correctly.The flow cytometry analysis showed that Fncas9-rgRNA system inhibited the expression of EYFP gene in HEK293 cells significantly,and the number of cells expressing fluorescence decreased. Three rgRNAs designed for PRRSV were co-infectecl with FnCas9 expression vector,and the degree of Marc145 cell lesion decresed after inoculation. It indicats that the FnCas9-rgRNA knockout vector targeting PRRSD is constructed correctly and it is active.

关 键 词：FnCas9-rgRNA敲除载体 黄色荧光蛋白 猪繁殖与呼吸综合征病毒 Marc145细胞 HEK293细胞 

分 类 号：Q782[生物学—分子生物学]