口蹄疫病毒非结构蛋白3abc基因的克隆与表达

口蹄疫病毒(Foot-and-mouth disease virus,FMDV)非结构蛋白(NSP)—3ABC可用于注苗与感染动物的鉴别诊断，合成该基因的PCR引物，并在引物5’端和3'端分别加入含BamHⅠ和HindⅢ限制性酶切位点序列。以FMDV毒株基因组RNA为模板，利用RT-PCR技术扩增3abc基因，得到的基因片段与T载体连接，转化DH5α。提取重组载体pT-3ABC，经BamHⅠ/HindⅢ双酶切后与载体pET32a连接，转化宿主菌BL21(DE3)plysS，IPTG诱导表达目的蛋白。SDS—PAGE及Western Blotting检测和鉴定结果表明，在大肠杆菌中成功表达了NSP—3ABC蛋白，分子量约56kDa，且该表达产物可与FMDV感染的动物血清产生免疫反应。ELISA试验结果显示，表达蛋白可用于FMDV注苗与感染动物的鉴别诊断。

Cloning and Expressing FMDV Non-Structural Protein 3abc Gene in E.coli

The Foot-and-mouth disease virus (FMDV) non-structural protein 3ABC can be used for differentiation of infection from vaccination. The primers of 3ABC gene were designed and synthesized, and the 5?end and 3?end of primers were adding the sequence of restriction endonuclease BamH I and Hind III respectively. The 3ABC gene coding region was obtained from the FMDV genome RNA by RT-PCR. The amplified fragment was cloned into T-vector. The recombinant plasmid pT-3ABC was digested with BamH I and Hind III and then cloned into pET32a. The recombinant plasmid pET3ABC was transformed into BL21 (DE3) plysS and the target protein was induced by IPTG. Expression of NSP-3ABC protein was examined and identificated by SDS-PAGE, Western blotting and ELISA. The results showed that recombinant plasmid pET3ABC was constructed and the NSP-3ABC was expressed in E.coli successfully. A special electrophoretic band in SDS-PAGE (56kDa) was noted, Western blotting showed it can react with FMDV infected animal serum,and ELISA result showed the expressed protein can be used to differentiate the infection from vaccination.