摘要
从持续感染牛白血病病毒(BLV)的羊胎肾细胞(FLK-BLV)中提取前病毒DNA,用PCR方法扩增编码牛白血病病毒gp51蛋白的env(gp51)基因,序列测定结果表明扩增片段全长828 bp。将扩增基因插入原核表达载体pET-32a构建pET-32a-gp51重组质粒,转化BL21(DE3)进行诱导表达,SDS-PAGE电泳结果表明重组融合蛋白大小约为43 000,Western-blot结果表明重组表达蛋白具有免疫反应性。以纯化的重组蛋白gp51作为抗原,建立检测BLV抗体的间接ELISA诊断方法。结果表明,抗原gp51的最佳包被浓度为2.19μg.mL-1,血清的最佳稀释倍数为1∶80,阳性判定标准确定为样品OD450 nm大于0.451。用该方法对12份参考血清进行检测,准确率为91.67%。
The DNA of bovine leukemia virus（BLV） provirus was extracted from fetal lamb kidney（FLK） cell line persistently infected by BLV.The env（gp51）gene which encode gp51 protein was amplified by PCR.Sequencing results showed that the amplified env gene was a 828 bp fragment,the gene was inserted into the expression vector pET-32a,the recombinant plasmid was named pET-32a-gp51.A fusion protein was expressed in BL21（DE3） that transfected by pET-32a-gp51 and induced by IPTG.The molecular weight of the recombinant protein was about 43 000 by SDS-PAGE,and the immunoreaction activity of the recombinant protein was confirmed by Western-blot,in which positive serum against BLV was used.An indirect enzyme-linked immunosorbent assay for detecting antibody against BLV was developed by using expressed protein gp51 as coating antigen.Results showed that the optimal coating concentration for gp51 is 2.19 μg·mL-1 and the serum dilution fold is 1∶80.The positive criterion for this ELISA assay is OD450 nm〉 0.451.Accuracy rate for detecting 12 standard BLV sera is 91.67%.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2009年第4期538-543,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
江苏出入境检验检疫局科研项目(2005KJ01)
湖北省武汉市科技攻关项目(20022002062)
