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

摘要

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 RTPCR, and its complete cDNA was cloned into pIRES2-EGFP vector. The recombinant pIRES2EGFP-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 VP 2 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 VP 2 gene of FMDV were established, and it provides a good platform to study the role of VP2 gene during FMDV induces apoptosis.%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LipofeetamineTM 2000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Western Blot技术检测其VP2的表达,最终筛选到稳定表达FMDV VP2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60 d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.