牛白血病病毒GP51蛋白在大肠杆菌中表达及胶体金免疫层析试纸条检测方法的建立  

Expression of bovine leukemia virus GP51 protein in E.coli and development of colloidal gold immunochromatographic diagnosis strips for detection of antibody against the virus

作　　者：宋阳威 张鑫宇[2] 张常印[3] 吴亚力 孙怀昌[2] 唐泰山[3] 张敬友[3] 

机构地区：[1]徐州出入境检验检疫局,江苏徐州221006 [2]扬州大学兽医学院,江苏扬州225009 [3]江苏出入境检验检疫局,江苏南京210001

出　　处：《中国预防兽医学报》2010年第4期272-275,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏检验检疫局科研项目(2006KJ04)

摘　　要：为了建立一种快速诊断牛白血病(BLV)的检测方法,本研究从BLV中扩增出其囊膜糖蛋白gp51基因,经测序后克隆于表达载体pET32a(+)中,构建了重组表达质粒pET32a-gp51。将其转化受体菌BL21,用IPTG诱导后表达出约42ku的目的蛋白,经western blot检测表明目的蛋白具良好的反应原性。以胶体金标记的羊抗牛IgG(Fc)抗体作为标记抗体,将纯化的His-gp51重组蛋白和羊抗牛IgG分别标记于硝酸纤维素膜上作为检测线和质控线,各部件按序装配形成快速诊断试纸条。对22份样本分别用试纸条和ELISA试验进行检测,2种方法的阳性符合率为88.9%(8/9),表明本研究建立的胶体金免疫层析方法简便、快捷,具有较好的特异性和一定的敏感性,适宜基层初筛诊断和现场应用。The gp51 gene of BLV was amplified from the bovine leukaemia virus（BLV） ,cloned into the expression vector pET32a（＋） ,and expressed in E.coli BL21 cells. The recombinant protein was detected by western blot and showed similar immuno-reactiongenicity to native GP51. A rapid gold immumochromatographic（GICA） strip was assembled by spraying the purified recombinant protein on the nitrocellulose membrane as the test line（T line） ,and the Gold-conjugated goat anti-bovine IgG as the control line（C line） . Test on a total of 22 field samples showed that the GICA assay and ELISA had a positive coincidence rate of 88.9 %（8/9） ,and a negative coincidence rate of 84.62 %（11/13） . The GICA established in this study is simple,rapid,sensitive and specific,which could be easily applieded in the field.

关 键 词：牛白血病病毒 gp51蛋白 表达 胶体金 免疫层析法 

分 类 号：S852.65[农业科学—基础兽医学]