山东地区猪流行性腹泻病毒遗传变异分析及其单克隆抗体的制备

发布时间：2018-04-04 07:56

本文选题：猪流行腹泻病毒　切入点：S基因　出处：《中国兽医学报》2017年05期

【摘要】：为了分析猪流行腹泻病毒(PEDV)在山东地区的流行和遗传变异情况,对2013—2015年收集的临床腹泻样品,参照GenBank中已经发表的PEDV毒株的N基因序列,设计1对特异性引物,利用RT-PCR技术检测PEDV抗原,并对其中的16份阳性样品进行病毒分离与S1基因测序分析。将含有PEDV流行株S1基因其中1个抗原表位(83~276aa)的重组蛋白免疫BALB/c小鼠,通过杂交瘤细胞融合技术,筛选获得2株针对PEDV S蛋白抗原表位的杂交瘤细胞,其中1株(1E5)可以区分PEDV经典毒株和流行毒株。临床检测结果表明:PEDV临床检出率由2013年的57.8%提高到2015年的75.68%,PEDV在山东地区的发病季节除春冬季节,近年夏季也呈高发趋势,且各地区均有不同程度的感染。PEDV S1基因序列分析结果表明:分离到的16株PEDV毒株间的S1基因的核苷酸序列同源性为91%~99.8%,氨基酸序列同源性为87.8%~99.7%;山东分离毒株与15个国内外参考毒株的核苷酸和氨基酸序列同源性分别为90.5%~100%和88.4%~99.9%。对S基因分子流行病学分析发现,7个国外参考毒株、8个国内参考毒株与本次分离到的16个PEDV毒株被分为2个不同的进化分支G1和G2。在2013-2015年分离到的16株毒株中,有3株位于G1中,且距离CV777经典毒株较近;剩余的13株位于G2中,G2则以近年流行毒株为主,而山东分离株主要在G2分支中,这也解释了现有CV777疫苗毒株在山东地区的免疫保护力不佳的原因。本研究结果不仅从分子流行病学角度分析了山东地区PEDV的流行和变异情况,为指导该地区PEDV疫病防制提供了新的参考,并且制备了能够区分PEDV经典毒株和流行毒株的S蛋白单克隆抗体,为本病的实验室鉴别诊断提供了有效的检测工具和重要的理论基础。
[Abstract]:In order to analyze the prevalence and genetic variation of porcine epidemic diarrhea virus (PEV) in Shandong province, a pair of specific primers were designed for clinical diarrhea samples collected from 2013 to 2015. According to the N gene sequence of PEDV strain published in GenBank, a pair of specific primers were designed.RT-PCR technique was used to detect PEDV antigen, and 16 positive samples were isolated and S1 gene sequenced.The recombinant protein containing one antigen epitope of S1 gene of PEDV strain was immunized with BALB/c mice. Two hybridoma cells targeting the antigen epitopes of PEDV S protein were obtained by hybridoma fusion technique.One strain of E5 can distinguish the classical PEDV strain from the epidemic strain.The clinical test results showed that the clinical detection rate of PEDV increased from 57.8% in 2013 to 75.68% in 2015. The incidence rate of PEDV in Shandong was also high in recent years, except spring and winter.The homology of nucleotide and amino acid sequences of reference strains were 90.5% and 88.4%, respectively.According to the molecular epidemiology analysis of S gene, 7 foreign reference strains, 8 domestic reference strains and 16 PEDV strains were divided into two different evolutionary branches: G1 and G2.Of the 16 strains isolated from 2013 to 2015, 3 were located in G1 and close to the classical CV777 strains, while the remaining 13 in G2 were mainly prevalent in recent years, while those in Shandong were mainly in G2 branches.This also explains why the existing CV777 vaccine strains have poor immune protection in Shandong.The results of this study not only analyzed the prevalence and variation of PEDV in Shandong Province from the point of view of molecular epidemiology, but also provided a new reference for the prevention and control of PEDV epidemic in this area.The monoclonal antibody of S protein which can distinguish the classical PEDV strain from the prevalent strain was prepared, which provides an effective detection tool and an important theoretical basis for the laboratory differential diagnosis of the disease.
【作者单位】：山东农业大学动物科技学院;山东省农业科学院畜牧兽医研究所;山东省畜禽疫病防治与繁育重点实验室;
【基金】：山东省现代农业产业技术体系资助项目(SDAIT-08-06) 泰山学者特聘专家工程经费资助项目(ts201511069)
【分类号】：S852.651

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