狂犬病病毒糖蛋白膜外区基因在大肠埃希氏菌中的高效表达

为获得狂犬病病毒(RV)糖蛋白抗原,采用RT-PCR方法从狂犬病病毒ERA株中扩增了编码RV糖蛋白的全长基因,将其克隆于pMD18-T载体,再经PCR扩增出糖蛋白全长基因和膜外区基因,将其分别亚克隆至原核表达载体pET-28a( )、pET-32a( )和pGEX-4T-1中,经PCR 和双酶切鉴定以及序列分析,表明已成功构建了重组质粒。将重组质粒转化到大肠埃希氏菌BL21 (DE3)中进行表达,结果显示,克隆到pET-32a( )的糖蛋白膜外区基因表达量最高,目的蛋白表达量占菌体总蛋白的45．4％。经Western-blotting检测,不同载体表达的糖蛋白膜外区产物均可与兔抗RV多抗发生特异性反应,表明,重组蛋白具有良好的反应原性。

High level expression of rabies virus glycoprotein extracellular domain gene in Escherichia coli

To obtain glycoprotein of rabies virus(RV) for diagnostic purpose, the glycoprotein gene was amplified from rabies virus which was cultivated in Vero cells by RT-PCR. The PCR product was inserted initially into pMD18-T vector. The full-length glycoprotein gene and its extracellular domain gene were amplified and then sub-cloned into the prokaryotic expression vector pET-28a( ),pET-32a( ) and pGEX-4T-1,respectively.PCR identification,digestion with restriction enzyme and sequencing analysis showed that the recombinant plasmids were obtained. Then they were transformed into E.coli BL21 (DE3) competent cells for expression. The results showed that E. coli BL21(DE3) harboring the recombinant plasmid pET32a-GP expressed the glycoprotein at high level(45. 4%). In Western-blotting analysis, the expressed proteins were recognized specifically by rabbit anti-RV polyclonal antibody.