本文選題：豬流行腹瀉病毒　切入點(diǎn)：S基因　出處：《中國獸醫(yī)學(xué)報(bào)》2017年05期

【摘要】：為了分析豬流行腹瀉病毒(PEDV)在山東地區(qū)的流行和遺傳變異情況,對(duì)2013—2015年收集的臨床腹瀉樣品,參照GenBank中已經(jīng)發(fā)表的PEDV毒株的N基因序列,設(shè)計(jì)1對(duì)特異性引物,利用RT-PCR技術(shù)檢測(cè)PEDV抗原,并對(duì)其中的16份陽性樣品進(jìn)行病毒分離與S1基因測(cè)序分析。將含有PEDV流行株S1基因其中1個(gè)抗原表位(83~276aa)的重組蛋白免疫BALB/c小鼠,通過雜交瘤細(xì)胞融合技術(shù),篩選獲得2株針對(duì)PEDV S蛋白抗原表位的雜交瘤細(xì)胞,其中1株(1E5)可以區(qū)分PEDV經(jīng)典毒株和流行毒株。臨床檢測(cè)結(jié)果表明:PEDV臨床檢出率由2013年的57.8%提高到2015年的75.68%,PEDV在山東地區(qū)的發(fā)病季節(jié)除春冬季節(jié),近年夏季也呈高發(fā)趨勢(shì),且各地區(qū)均有不同程度的感染。PEDV S1基因序列分析結(jié)果表明:分離到的16株P(guān)EDV毒株間的S1基因的核苷酸序列同源性為91%~99.8%,氨基酸序列同源性為87.8%~99.7%;山東分離毒株與15個(gè)國內(nèi)外參考毒株的核苷酸和氨基酸序列同源性分別為90.5%~100%和88.4%~99.9%。對(duì)S基因分子流行病學(xué)分析發(fā)現(xiàn),7個(gè)國外參考毒株、8個(gè)國內(nèi)參考毒株與本次分離到的16個(gè)PEDV毒株被分為2個(gè)不同的進(jìn)化分支G1和G2。在2013-2015年分離到的16株毒株中,有3株位于G1中,且距離CV777經(jīng)典毒株較近;剩余的13株位于G2中,G2則以近年流行毒株為主,而山東分離株主要在G2分支中,這也解釋了現(xiàn)有CV777疫苗毒株在山東地區(qū)的免疫保護(hù)力不佳的原因。本研究結(jié)果不僅從分子流行病學(xué)角度分析了山東地區(qū)PEDV的流行和變異情況,為指導(dǎo)該地區(qū)PEDV疫病防制提供了新的參考,并且制備了能夠區(qū)分PEDV經(jīng)典毒株和流行毒株的S蛋白單克隆抗體,為本病的實(shí)驗(yàn)室鑒別診斷提供了有效的檢測(cè)工具和重要的理論基礎(chǔ)。
[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.
【作者單位】： 山東農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院;山東省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所;山東省畜禽疫病防治與繁育重點(diǎn)實(shí)驗(yàn)室;
【基金】：山東省現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系資助項(xiàng)目(SDAIT-08-06) 泰山學(xué)者特聘專家工程經(jīng)費(fèi)資助項(xiàng)目(ts201511069)
【分類號(hào)】：S852.651

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