期刊论文

中文

中国兽医科学

1673-4696

2012

42

5

495-500

10.16656/j.issn.1673-4696.2012.05.012

(2007FJ1003):湖南省科技重大专项 (04YJ03):湖南农业大学***基金项目

本校为第一机构

为了在大肠杆菌中表达猪札幌病毒VP1基因,并以纯化的重组蛋白为抗原建立猪札幌病毒血清抗体ELISA检测方法。采用RT-PCR技术扩增VP1基因,并将其克隆到pMD18-T载体中,再将所克隆的衣壳蛋白的编码序列亚克隆到表达载体pET-28a(+)中,将成功构建的原核表达质粒pETSAVCAP转化大肠杆菌Rosetta(DE3)进行诱导表达。用纯化的VP1蛋白作为抗原建立了猪札幌病毒血清抗体间接ELISA检测方法并初步用于临床样品的检测。结果显示,猪札幌病毒VP1基因可在大肠杆菌中稳定、高效地表达,Western-blot分析表明该重组蛋白具有良好的反应原性。经对反应条件进行优化,确定间接ELISA的抗原最佳包被浓度为0.5μg/mL,且不与其...

The aim of the present study was to establish an indirect ELISA method for the detection of porcine sapovirus antibody based on the expression of VP1 gene of porcine sapovirus in Escherichia coli.Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank,the VP1 gene of porcine sapovirus was amplified with RT-PCR method from stool samples of piglet suffered from diarrhea.The PCR product was then cloned into a pET-28a（＋） vector to construct a prokaryotic recombinant plasmid pETSAVCAP.The pETSAVCAP was...