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

【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。

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

【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.