CAJ | 학술논문

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42 ku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.
위료건립일충쾌속진단우백혈병(BLV)적검측방법,본연구종BLV중확증출기낭막당단백gp51기인,경측서후극륭우표체재체pET32a(+)중,구건료중조표체질립pET32a-gp51.장기전화수체균BL21,용IPTG유도후표체출약42 ku적목적단백,경western blot검측표명목적단백구량호적반응원성.이효체금표기적양항우IgG(Fc)항체작위표기항체,장순화적His-gp51중조단백화양항우IgG분별표기우초산섬유소막상작위검측선화질공선,각부건안서장배형성쾌속진단시지조.대22빈양본분별용시지조화ELISA시험진행검측,2충방법적양성부합솔위88.9%(8/9),표명본연구건립적효체금면역층석방법간편、쾌첩,구유교호적특이성화일정적민감성,괄의기층초사진단화현장응용.
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.