口蹄疫病毒VP2基因重组表达载体的构建及其稳定表达细胞系的建立  

Construction of Recombinant Vector of VP2 Gene of Foot-and-mouth Disease Virus and Establishment of Stable Cell Line for Its Stable Expression

作　　者：谢金鹿[1,2,3] 王洪梅[2] 刘国艺[1] 武建明[2] 刘晓[2] 高运东[2] 于力[4] 仲跻峰[2] 何洪彬[2] 

机构地区：[1]哈尔滨医科大学基础医学院,哈尔滨150086 [2]山东省农业科学院奶牛研究中心,济南250100 [3]秦皇岛市卫生学校,秦皇岛066000 [4]中国农业科学院哈尔滨兽医研究所,哈尔滨150001

出　　处：《畜牧兽医学报》2011年第4期521-526,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家转基因重大专项(2009ZX08007-006B);山东省农业重大应用技术创新(何洪彬);山东省科技攻关项目(2009GG20002032);兽医生物技术国家重点开放实验室开放基金(SKLVBF200806);济南市高校院所自主创新计划(201004027);泰山学者海外特聘专家(何洪彬);国家现代农业(奶牛)产业技术体系岗位科学家(何洪彬);国家自然科学基金(31072160);山东省自然基金(ZR2010CQ029)

摘　　要：本试验旨在构建Asia 1型口蹄疫病毒(FMDV)的VP2基因重组表达载体,并建立稳定表达VP2基因的BHK-21细胞系。利用RT-PCR方法扩增Asia 1型FMDV的cDNA,获得VP2基因完整的编码区,构建可表达VP2的带有绿色荧光蛋白标记基因的pIRES2-EGFP-VP2重组表达载体。经鉴定正确后,利用LipofectamineTM2000将重组质粒转染293T细胞,通过Western Blot技术检测VP2蛋白的瞬时表达;然后再转染BHK-21细胞,通过G418筛选,形成单克隆细胞系,经荧光筛选到表达EGFP的抗性单克隆细胞,扩繁培养克隆细胞,再通过West-ern Blot技术检测其VP2的表达,最终筛选到稳定表达FMDVVP2基因的BHK-21细胞系。结果表明,VP2基因重组表达载体pIRES2-EGFP-VP2构建正确,能够在293T细胞内瞬时表达;转染BHK-21细胞,经G418筛选到表达VP2基因的BHK-21细胞克隆,经长达60d的传代,获得了稳定表达VP2基因和EGFP的BHK-21细胞系。上述结果表明,我们建立了稳定表达VP2基因的BHK-21细胞系,为进一步探讨VP2基因在FMDV诱导细胞凋亡中的作用奠定基础。The aim of the study was to construct a recombinant vector,which including VP2 gene of Asia I strain of foot-and-mouth disease virus（FMDV）,and to establish the BHK-21 cell line,which stably expressing VP2 gene.VP2 gene of FMDV was amplified from Asia I strain by RT-PCR,and its complete cDNA was cloned into pIRES2-EGFP vector.The recombinant pIRES2-EGFP-VP2 vector,which could express VP2 and EGFP proteins,was confirmed by sequencing analysis,the transient expression of VP2 in 293T cells transfected with the recombinant plasmid via LipofectamineTM 2000 was then determined by Western blot.Furthermore,BHK-21 cells were transfected with pIRES2-EGFP-VP2,and anti-G418 cell clones were screened using G418,both G418 and GFP positive cell clones were continually cultivated after GFP detection,their expressions of VP2 gene were confirmed by Western blot.The results showed that the recombinant plasmid pIRES2-EGFP-VP2 correctly encoded VP2 gene and transiently expressed in 293T cells.BHK-21 cells were transfected with recombinant plasmid,then BHK-21 cell clones expressing VP2 were obtained by G418 pressure selection,after continuous passage for 60 days,BHK-21 cell lines stably expressing VP2 gene of FMDV were established.The above results indicated that BHK-21 cell lines stably expressing VP2 gene of FMDV were established,and it provides a good platform to study the role of VP2 gene during FMDV induces apoptosis.

关 键 词：口蹄疫病毒 VP2 转染 稳定细胞系 

分 类 号：S852.659.6[农业科学—基础兽医学]