口蹄疫病毒VP1基因重组慢病毒载体的构建及VP1基因稳定表达细胞系的建立  

Construction of VP1 Recombinant Lentivirus Vector and Establishment of BHK-21 Cell Lines Stably Expressing VP1 Gene of FMDV

作　　者：代文君[1,2] 王洪梅[1] 刘晓[1] 高运东[1] 于力[3] 王立群[2] 仲跻峰[1] 何洪彬[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,济南250100 [2]东北农业大学生命科学学院,哈尔滨150030 [3]中国农业科学院哈尔滨兽医研究所,哈尔滨150001

出　　处：《中国农业科学》2010年第16期3455-3460,共6页Scientia Agricultura Sinica

基　　金：山东省自然科学基金(Y2008D20);国家转基因重大专项(2009ZX08007-006B);山东省科技攻关项目(2009GG20002032);山东省农业重大应用技术创新课题(何洪彬);兽医生物技术国家重点开放实验室开放基金(SKLVBF200806)

摘　　要：【目的】构建口蹄疫病毒VP1基因重组慢病毒载体FG9-VP1,并建立稳定表达VP1基因的BHK-21细胞系。【方法】采用RT-PCR技术从口蹄疫病毒材料中扩增出VP1基因,并将其连入慢病毒载体FG9中,经PCR、酶切和测序鉴定正确后,转染BHK-21细胞,96h后经流式细胞分选筛选GFP阳性细胞,细胞增殖后经WB检测VP1基因的表达。【结果】VP1基因重组慢病毒载体FG9-VP1测序正确,转染BHK-21细胞经流式细胞仪筛选的GFP阳性细胞后可稳定表达VP1基因。【结论】VP1基因重组慢病毒载体构建成功并获得了稳定表达VP1基因的BHK-21细胞系。【Objective】 To construct the lentivirus vector containing VP1 gene and to establish the cell line with stable expression of VP1 gene of foot-and-mouth disease virus （FMDV）. 【Method】 The full-length VP1 gene was amplified by RT-PCR,and VP1 gene was cloned into FG9 vector,then recombinant vector was confirmed by restricting enzyme digestion and DNA sequence. The recombinant plasmid was transfected into BHK-21 cells through LipofectamineTM 2000,and the GFP positive cells were screened via FACS after 96h. The expressions of VP1 gene were confirmed by Western-blot. 【Result】 FG9-VP1 was constructed successfully and the VP1 gene could be stably expressed in BHK-21 cel1s. 【Conclusion】 The recombinant lentivirus vector containing VP1 gene was cloned successfully and the stable cell line expressing the VP1 gene was established.

关 键 词：口蹄疫病毒 VP1 转染 稳定细胞系 

分 类 号：S852.65[农业科学—基础兽医学]