牛嵴病毒VP3蛋白的原核表达及初步应用

Prokaryotic expression and primary application of VP3 of bovine kobuvirus

牛嵴病毒(BKV)是近几年在牛消化道中新发现的病毒,其致病性尚不清楚。我们的相关研究已表明,该病毒在我国的牛群中普遍存在。为重组表达BKV VP3蛋白,本研究采用RT-PCR方法从牛腹泻粪便样品中扩增BKV VP3蛋白的编码基因,将其克隆至pET-28a(+)载体中构建原核重组表达质粒pET-VP3。将pET-VP3转化至大肠杆菌BL21(DE3)中。经IPTG诱导表达及SDS-PAGE分析表明重组蛋白分子量约为27.8 ku,以包涵体形式存在。采用电洗脱方法纯化该重组蛋白并免疫BALB/c小鼠,制备抗BKV VP3的多克隆抗体。以纯化的重组蛋白作为包被抗原,初步建立了间接ELISA抗体检测方法。小范围调查结果显示,在检测的牛群中BKV感染率较高。

Bovine kobuvirus （BKV） was newly isolated and identified virus in the digestive tract of cattle, but the pathogenicity of BKV has remained unknown. Our previous studies showed that this virus was ubiquitous among eagles in China. In this study, the gene encoding VP3 protein of BKV was amplified by RT-PCR from the fecal samples of cattle affected with diarrhea, which was cloned into the pET-28a（＋） and expressed in BL21 （DE3）. SDS-PAGE analysis showed that the expression product had MW of 27.8 ku. The BALB/C mice were inoculated with the purified protein, and the polyclonal antibodies against BKV VP3 protein were prepared. An indirect ELISA based on the recombinant 3/P3 protein was established for detection of antibodies against BKV. The serologically surveys in a cattle farm showed that the infection of BKV in cattle was rather high.