口蹄疫病毒2B基因绿色荧光蛋白融合表达质粒的构建及表达  

Construction of Recombinant Plasmid for Fusion Expression of Foot and Mouth Disease Virus 2B Gene and Green Fluorescent Protein Gene

作　　者：崔丽瑾[1] 王兴龙[2] 梅英武[1] 任林柱[1] 闫广谋[1] 张辉[1] 郎需龙[2] 段小宇[1] 路浩[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]军事医学科学院军事兽医研究所,长春130062

出　　处：《中国生物制品学杂志》2008年第9期756-758,共3页Chinese Journal of Biologicals

基　　金：军事医学科学院科技创新基金资助项目;吉林省科技厅科技发展计划项目(20050549)

摘　　要：目的构建口蹄疫病毒(FMDV)2B基因绿色荧光蛋白(GFP)融合表达质粒,并在BHK-21细胞中表达。方法RT-PCR扩增O型口蹄疫病毒WFL株的2B基因,克隆入表达载体pEGFP-C1,并进行双酶切、PCR及测序鉴定。将阳性重组质粒转染BHK-21细胞,检测绿色荧光蛋白的表达和2B基因转录水平。结果经双酶切及PCR鉴定,目的基因片段大小与预期相符,测序结果与WFL株相应序列一致。荧光显微镜和流式细胞仪均检测到细胞内绿色荧光蛋白的表达,荧光定量PCR检测到细胞内有2B基因的转录。结论已成功构建了FMDV2B基因GFP融合表达质粒,并在BHK-21细胞中获得了表达。Objective To construct a recombinant plasmid for fusion expression of foot and mouth disease virus （FMDV） 2B gene and green fluorescent protein （GFP） gene and express in BHK-21 cells. Methods Amplify 2B gene from WFL strain of FMDV group O by RT-PCR and insert into expression vector pEGFP-C1 containing GFP gene and identify the constructed recombinant plasmid by restriction analysis, PCR and sequencing. Transfect BHK-21 cells with screened positive recombinants and determine the expression of GFP and transcription level of 2B gene. Results Both restriction analysis and PCR proved that the length of target gene fragment was consistent with that expected. The sequence of target gene was identical to that of corresponding gene fragment in WFL strain. The expression of GFP was observed by both fluorescent microscopy and flow cytometry. Fluorescent quantitative PCR proved the transcription of 2B gene in transfected BHK-21 cells. Conclusion The recombinant plasmid for fusion expression of FMDV 2B gene and GFP gene was successfully constructed and expressed in BHK-21 cells.

关 键 词：口蹄疫病毒 2B基因 绿色荧光蛋白 融合表达 

分 类 号：Q786[生物学—分子生物学]