鲤春病毒血症病毒糖蛋白的高效表达和纯化.pdfVIP

2010, 18( 2): 18- 22 Ch inese Journal ofAn mi al Infectious D iseases 兰文升, 刘 荭, 高隆英, 史秀杰, 郑晓聪, 何俊强, 卢体康 ( , 518010) : 本研究从鲤春病毒血症病毒( Sprin virem ia of carp virus, SVCV) SV CV741毒株克隆鲤春病毒血症病 毒糖蛋白基因( svcg) , 将 svcg 亚克隆到原核表达载体 pET28a, 转化大肠 菌E. coli BL21( DE3) 后, 用 IPTG 诱导培养, 获得菌体总蛋白用 SVCV毒株免疫山羊所得到的抗血清作为一抗进行免疫印迹实验, 结果在硝酸 纤维素滤膜上检测到 50~ 71 kDa之间的特异性免疫条带, 与 SVCV 糖蛋白预测分子量一致, 研究证明了工程 菌表达获得的重组蛋白具有与 SVCV 毒株相同的免疫原性, 也证明了该蛋白的糖基化对其免疫表位是非必需 的本研究进一步通过发酵基因工程菌和蛋白质纯化过程, 获得大量的鲤春病毒血症病毒糖蛋白, 为后期免 疫动物获得抗血清储备了原料, 也为 SVCV 的免疫学检测方法的建立奠定了物质基础 : 糖蛋白; 表达; 鲤春病毒血症病毒 : S852. 659. 5 : A : 2010) 0200 1805 H IGH LEVEL EXPRESSION AND PURIFICATION OF RECOMBINANT GLYCOPROTEIN OF SPRING VIREM IA OF CARP VIRUS ( SVCV) LAN W enshen , LIU Hon , GAO Lon y in , SHI X iujie, ZHENG X iaocon , HE Junq ian , LU T ikan (The Technical Center of A nmi al and P lant Insp ection and Quarantine, Shenzhen Entr Ex it Inspection and Quarantine Bureau, Shenzhen K e L aborator f or RD on D etection Technolog of Exotic Pests, Shenzhen 518010, China) Abstract: G lycoprotein ene ( svc ) of Sprin virem ia of carp virus ( SV CV ) was cloned from isolate SVCV74 1. The svc enew as subcloned into plasm id pET28 and the resu ltan t p lasm id was transformed intoE. coli BL2 1( DE3). The total protein w as obtained from E. coli cultures induced by IPTG. W estern b lot usin oat anti SV CV serum revealed a specific band between 50 kDa and 71 kDa. This proved that tar et protein has the same mi m uno en icity as SVCV strain. It is speculated that lycosylation is not necessary for the epitopes of lycoprotein. A lar e quantity of recom b inant lycoprotein can be produced by ferm entation of recom b inant bacteria. K y w