口蹄疫病毒多基因重组质粒的体外表达及衣壳组装

PCR扩增获得包含口蹄疫病毒P1、2A、3C、3D及部分2B编码区的目的基因片段P12X3C3D，将P12X3C3D经AflⅡ和XbaⅠ双酶切后，定向克隆于真核表达质粒载体pcDNA3.1( )；另将PCR扩增获得的P12X3C3D直接与真核表达质粒载体pTARGET^TM连接，进行筛选、鉴定及DNA序列分析，分别获得重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D。将重组质粒分别转染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，用磷钨酸负染，以电子显微镜观察转染重组质粒的细胞中组装的口蹄疫病毒空衣壳。结果表明，口蹄疫病毒基因组片段正确克隆到真核表达质粒载体上，重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D均可在BHK-21细胞中表达FMDV目的蛋白。其中重组质粒pTARGET／P12X3C3D表达的口蹄疫病毒抗原蛋白，能够在细胞内正确组装成病毒空衣壳。

Expression of Foot-and Mouth Disease Virus Proteins in vitro with Eukaryotic Expression Plasmid

The genomic fragment P12X3C3D that includes full length P1,2A,3C,3D and a part of 2B of foot-and-mouth disease virus (FMDV) was obtained by PCR. After being digested by restriction enzymes respectively, the fragment P12X3C3D was cloned into pcDNA3.1(+) that had been digested by the same enzymes and the recombinant plasmid was named pcDNA3.1/P12X3C3D. Meantime, P12X3C3D was inserted into pTARGET~(TM) expression vector and the resulted recombinant plasmid was named pTARGET/P12X3C3D. Recombinant plasmids were identified by restriction enzyme analysis and nucleic acid sequencing. Further, BHK-21 cells were transfected with pcDNA3.1/P12X3C3D, pTAGRET/P12X3C3D by using lipoid. The proteins of foot-and-mouth disease virus expressed in BKH-21 cells were confirmed by sandwich-ELISA and IFA, and the capsid of FMDV was observed under electron microscope. The results showed P12X3C3D was cloned into eukaryotic expression plasmid correctly. The recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C3D, pTARGET/P12X3C3D could express proteins of foot-and-mouth disease virus in BKH-21 cells, and the expressed proteins by pTARGET/P12X3C3D had immunocompetence and could assembly into empty capsid structures of FMDV.