發(fā)布時間：2019-06-18 08:17

【摘要】：豬博卡病毒(Porcine Bocavirus,PBoV),自2009年被首次發(fā)現(xiàn)以后,在世界范圍內(nèi)陸續(xù)被報道。為有效檢測PBoV,制定相關(guān)檢測方法十分必要。本試驗建立了PBoV間接ELISA檢測方法。試驗以選取PBoV VP2基因中一段抗原性較佳且保守的基因區(qū)域進行原核表達。從PBoV陽性病料中提取目的基因,并通過測序驗證目的基因準確性。構(gòu)建pET-32a-PBoV-VP2(pET-VP2)重組表達質(zhì)粒,將重組質(zhì)粒導(dǎo)入BL21(DE3)表達菌,在37℃下,加入IPTG誘導(dǎo)表達出含有6個重組蛋白,大小約為41 kD。經(jīng)鑒定重組蛋白主要以包涵體形式存在,用透析復(fù)性法對其進行純化復(fù)性。復(fù)性后進行Western blot實驗,結(jié)果表明其與PBoV陽性血清結(jié)合特異性良好,為下一步間接ELISA方法建立的提供材料。用純化復(fù)性后的重組蛋白作為間接ELISA的包被抗原,選取抗原包被濃度8.25?g/m L、一抗稀釋比例1:100、二抗稀釋比例1:4000、2%明膠溶液封閉、封閉時間2 h、顯色時間15 min等為最佳條件建立PBoV的間接ELISA方法。該方法進行特異性試驗、陰陽血清臨界值試驗、批內(nèi)批間重復(fù)性試驗,結(jié)果發(fā)現(xiàn)該方法特異性良好,不跟其他豬病毒交叉反應(yīng);陰陽性血清臨界值為0.203;批內(nèi)及批間重復(fù)性良好。使用本試驗中建立的PBoV間接ELISA方法檢測江西各地區(qū)采集的90份豬血清樣品,陽性檢出率為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，

本文編號：2501310