口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达

利用定点突变的原理，获得包含有口蹄疫病毒P1，2A，3C及部分2B编码区的目的基因片段，KpnⅠ和XbaⅠ双酶切后，定向克隆于真核表达质粒载体pcDNA3.1（+），经筛选、鉴定及DNA序列分析后，将重组质粒pcDNA3.1/P12X3C转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法，检测细胞中表达的口蹄疫病毒抗原。结果表明，口蹄疫病毒基因片段正确克隆到真核表达质粒载体上，重组质粒pcDNA3.1/P12X3C可在BHK-21细胞中表达FMDV目的蛋白。

Expression of Recombinant Plasmid pcDNA3.1/P12X3C with Multi-genes of Foot-and-mouth Disease Virus in BHK-21 Cells

In order to obtain the gene P12X3C of Foot-and-Mouth Disease Virus (FMDV) that includes full length P1, 2A, 3C and a part of 2B, the site mutation strategy was used. After being digested by Kpn I and Xba I respectively, the gene P12X3C was cloned into the pcDNA3.1 (+) expression vector. The recombinant plasmid was checked by restriction enzyme analysis and nucleic acid sequencing, and then named pcDNA3.1/P12X3C. Further, BHK-21 cells was transfected with pcDNA3.1/P12X3C by using lipoid. The proteins of Foot-and-Mouth Disease Virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy. The result shows the gene P12X3C is cloned into eukaryotic expression plasmid, and the recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C could express proteins of Foot-and-Mouth Disease Virus in BHK-21 cells, which have immunocompetence. This study demonstrates that delivery of a recombinant eukaryotic expression plasmid containing P12X3C coding regions results in the assembly of FMDV capsid structures, which will offer experimental base to DNA vaccine of FMDV.