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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。
In order to analyze the prevalence and genetic variation of swine porcine epidemic diarrhea virus (PEDV) in Shandong province, we designed a pair of specific primers for clinical diarrhea samples collected in 2013-2015 according to the N gene sequence of PEDV strains already published in GenBank , PEDV antigen was detected by RT-PCR and virus isolation and S1 gene sequencing analysis of 16 positive samples. BALB / c mice were immunized with a recombinant protein containing one antigenic epitope (83-276aa) of the S1 gene of PEDV strain, and two hybridoma cells against the antigenic epitope of PEDV S protein were screened by hybridoma cell fusion technique , One of which (1E5) can distinguish PEDV classic strains and epidemic strains. The clinical test results showed that the clinical detection rate of PEDV increased from 57.8% in 2013 to 75.68% in 2015. The onset season of PEDV in Shandong Province except for the spring and winter seasons also showed a trend of high incidence in recent years, with different degrees in all regions Infection. The results of PEDV S1 gene sequence analysis showed that the nucleotide sequence homology of S1 gene among the 16 PEDV isolates was 91% -99.8% and the amino acid sequence identity was 87.8% -99.7% The nucleotide and amino acid sequence homologies between the strain and 15 reference strains at home and abroad were 90.5% -100% and 88.4% -99.9%, respectively. The epidemiological analysis of S gene revealed that seven foreign reference strains, eight domestic reference strains and the 16 isolated PEDV strains were divided into two different evolutionary branches G1 and G2. Of the 16 isolates isolated in 2013-2015, 3 were located in G1 and closer to the classical strain of CV777; the remaining 13 were located in G2, with G2 being predominantly endemic in recent years and Shandong The isolate is mainly in the G2 branch, which explains why the existing CV777 vaccine strain is poorly immunoprotective in Shandong. The results of this study not only analyzed the prevalence and variation of PEDV in Shandong from the perspective of molecular epidemiology, provided a new reference for the prevention and control of PEDV in Shandong, and also prepared S Protein monoclonal antibody provides an effective detection tool and an important theoretical basis for the laboratory differential diagnosis of this disease.