牛传染性鼻气管炎病毒糖蛋白gC基因抗原活性区的克隆与表达

通过聚合酶链式反应从牛传染性鼻气管炎病毒Bartha Nu/67株中扩增得到病毒gC基因并克隆至T载体pMD20T,再以后者为模板扩增gC蛋白第15~177位氨基酸对应的抗原活性区片段gCd。将gCd插入原核表达载体pET32a构建重组表达质粒pET32a-bhv1gCd。限制性内切酶以及序列分析鉴定表明重组表达质粒克隆片段序列与阅读框正确。对重组质粒转化大肠杆菌BL21(DE3)的培养物进行蛋白电泳,可检测到分子量约45ku的目的产物,IPTG诱导后5h表达量最高。免疫印迹试验结果证实,gCd重组蛋白与牛传染性鼻气管炎标准阳性血清发生特异性反应,表明gC抗原活性区片段在原核获得表达并具有良好的抗原性。该重组蛋白可用于建立ELISA等免疫诊断方法。

Cloning and expression of antigenic region of glycoprotein gC gene from infectious bovine rhinotrachetis virus

The glycoprotein gene gC of infectious bovine rhinotrachetis virus(IBRV)was amplified from the Bartha Nu/67 srain viral DNA by polymerase chain reaction and cloned into pMD20-T vector to construct pMD20-TgC.The antigenic functional fragment gCd encoding amino acid residues 15~177 of the gC gene was then amplified from pMD20-TgC and inserted into prokaryotic expression vector pET32a.Restriction enzyme analysis and sequencing of the recombinant plasmid confirmed the correct insertion of gCd gene.The recombinant plasimid pET32a-bhv1gCd was transformed into E.coli BL21(DE3)cells.The targeted protein of 45 ku was detected by SDS-PAGE and the highest expression was found at 5h after IPTG induction.The recombinant protein reacted with IBRV positive serum in Western-blotting,indicating that gCd fragment with antigenic function was expressed in prokaryotic system.The gCd recombinant protein can be used as the specific diagnosis antigen in immunoassay such as ELISA.