生物素标记重组猪圆环病毒2d基因型毒株感染性克隆的构建与鉴定

Construction and Identification of Biotin-Labeled Recombinant Porcine Circovirus Type 2d Genotype Strain Infectious Clone

本试验旨在获得临床上逐渐增多的猪圆环病毒2d基因型毒株(PCV2d),并对其引入生物素标记。首先从陕西咸阳某临床发病猪场采集了2头发病仔猪和15头未发病仔猪的组织样本,对其进行猪圆环病毒2型(PCV2)检测,并对阳性样本进行测序及序列比对,将获得的PCV2d基因型毒株基因组orf 2中226~246 bp序列替换为生物素受体肽(BAP)编码序列并克隆入质粒载体,同时稳定构建表达原核生物素连接酶(BirA)的PK15细胞系,将替换有BAP的PCV2d基因组酶切并环化后转染稳定表达BirA的PK15细胞,连续盲传5代后鉴定获得的重组病毒。结果显示,检测的组织样本中共有5头仔猪的样本为PCV2阳性,其中1份样本为PCV2d基因型,4份样本为PCV2b基因型;在获得BAP序列替换的PCV2d,构建重组质粒T-PCV2d-BAP和稳定表达BirA的PK15细胞系后,将环化的PCV2d-BAP转染稳定表达BirA的PK15细胞并盲传5代,经PCR和Western blot检测均为PCV2阳性,且能被链酶亲和素识别。本试验成功获得的生物素标记重组PCV2d基因型毒株为后续探究PCV2d基因型毒株免疫逃避机制提供一个非常有效的工具。

This study aimed to generate a biotin-labeled,increasing clinical infective porcine circovirus type 2d(PCV2d)genotype strain.Tissue samples were collected from 2 clinical diseased piglets and 15 clinical healthy piglets from a clinically diseased pig farm in Xianyang of Shannxi for PCV2 test,and PCV2 amplicons were sequenced and aligned.The 226-246 bp of orf 2 in the PCV2d genome was replaced with the sequence of biotin acceptor peptide(BAP),and the recombinant PCV2d sequence was cloned into a plasmid.A PK15 cell line stably expressing the prokaryotic biotin ligase(BirA)was constructed.The PCV2d genome containing BAP was digested and cyclized for transfection into PK15 cells expressing BirA.The recombinant virus was identified after 5 blind passages.The results showed that 5 of piglets tissue samples were PCV2 positive,including one PCV2d genotype and four PCV2b genotype strains.The recombinant plasmid T-PCV2d-BAP in which the sequences were replaced with BAP,and the PK15 cell line stably expressing BirA were successfully constructed.Cyclized PCV2d-BAP was transfected into PK15 cells stably expressing BirA.After 5 blind passages,the resultant PK15 cells were PCR and Western blot positive for PCV2,which could be recognized by streptavidin.The generation of biotin-labeled recombinant PCV2d genotype strain in this study provides a useful tool for further study on its pathogenesis and immune escape mechanism.