猪博卡病毒VP2蛋白的原核表达与间接ELISA检测方法的建立及应用
发布时间：2019-06-18 08:17
【摘要】：猪博卡病毒(Porcine Bocavirus,PBoV),自2009年被首次发现以后,在世界范围内陆续被报道。为有效检测PBoV,制定相关检测方法十分必要。本试验建立了PBoV间接ELISA检测方法。试验以选取PBoV VP2基因中一段抗原性较佳且保守的基因区域进行原核表达。从PBoV阳性病料中提取目的基因,并通过测序验证目的基因准确性。构建pET-32a-PBoV-VP2(pET-VP2)重组表达质粒,将重组质粒导入BL21(DE3)表达菌,在37℃下,加入IPTG诱导表达出含有6个重组蛋白,大小约为41 kD。经鉴定重组蛋白主要以包涵体形式存在,用透析复性法对其进行纯化复性。复性后进行Western blot实验,结果表明其与PBoV阳性血清结合特异性良好,为下一步间接ELISA方法建立的提供材料。用纯化复性后的重组蛋白作为间接ELISA的包被抗原,选取抗原包被浓度8.25?g/m L、一抗稀释比例1:100、二抗稀释比例1:4000、2%明胶溶液封闭、封闭时间2 h、显色时间15 min等为最佳条件建立PBoV的间接ELISA方法。该方法进行特异性试验、阴阳血清临界值试验、批内批间重复性试验,结果发现该方法特异性良好,不跟其他猪病毒交叉反应;阴阳性血清临界值为0.203;批内及批间重复性良好。使用本试验中建立的PBoV间接ELISA方法检测江西各地区采集的90份猪血清样品,阳性检出率为68%(61/90)。
[Abstract]:Pig Boca virus (Porcine Bocavirus,PBoV) has been reported worldwide since it was first discovered in 2009. It is necessary to develop related detection methods for effective detection of PBoV,. In this experiment, an indirect ELISA detection method for PBoV was established. A segment of PBoV VP2 gene with better antigenicity and conserved gene was selected for prokaryotic expression. The target gene was extracted from PBoV positive disease material and the accuracy of the target gene was verified by sequencing. The recombinant expression plasmid pET-32a-PBoV-VP2 (pET-VP2) was constructed and introduced into BL21 (DE3) expression strain. At 37 鈩，
本文编号：2501310
[Abstract]:Pig Boca virus (Porcine Bocavirus,PBoV) has been reported worldwide since it was first discovered in 2009. It is necessary to develop related detection methods for effective detection of PBoV,. In this experiment, an indirect ELISA detection method for PBoV was established. A segment of PBoV VP2 gene with better antigenicity and conserved gene was selected for prokaryotic expression. The target gene was extracted from PBoV positive disease material and the accuracy of the target gene was verified by sequencing. The recombinant expression plasmid pET-32a-PBoV-VP2 (pET-VP2) was constructed and introduced into BL21 (DE3) expression strain. At 37 鈩，
本文编号：2501310
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