口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达  

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

作　　者：郭慧琛[1] 刘在新[1] 孙世琪[1] 卢增军[1] 周广清[1] 祁淑云[1] 靳野[1] 刘湘涛[1] 谢庆阁[1] 

机构地区：[1]中国农业科学院兰州兽医研究所农业部畜禽重点实验室,兰州730046

出　　处：《生物工程学报》2003年第3期376-379,共4页Chinese Journal of Biotechnology

基　　金：国家重大基础研究发展规划( 973 )基金资助 (No .G19990 1190 3 )~~

摘　　要：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pcDNA3.1 P12X3C转染BHK 2 1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 2In 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.

关 键 词：口蹄疫病毒 真核表达质粒 细胞 表达 

分 类 号：Q786[生物学—分子生物学]