我國部分地區(qū)豬流行性腹瀉病毒的分離與進(jìn)化分析
發(fā)布時(shí)間：2018-11-09 13:14
【摘要】：2013年冬及2014年春,吉林省、陜西省豬場疑似發(fā)生流行性腹瀉病毒感染。為確定病原、了解其與國內(nèi)外其他毒株的基因差異,分別采集腹瀉糞便樣品和小腸組織,進(jìn)行病毒分離培養(yǎng)并對(duì)關(guān)鍵基因進(jìn)行測序和分析,獲得如下結(jié)果:1.利用Vero細(xì)胞連續(xù)傳代后,分離到豬流行性腹瀉病毒株SP株(吉林),YL9099、YL9140、YL9234株(陜西)。2.用RT-PCR方法對(duì)分離的到的4株豬流行性腹瀉病毒S基因及SP株的M、ORF3基因進(jìn)行進(jìn)行擴(kuò)增、克隆、測序,用DNAStar、MEGA6.0、Net NGlyc 1.0 Server與Gene Bank中登錄的豬流行性腹瀉病毒株S、M、ORF3序列進(jìn)行序列變化、遺傳進(jìn)化關(guān)系、抗原位點(diǎn),糖基化位點(diǎn)的分析。(1)本實(shí)驗(yàn)的4株病毒與經(jīng)典毒株株CV777、DR13相比,核苷酸同源性在94.0%-99.4%之間。其推導(dǎo)的氨基酸與疫苗株相比有79-84處突變,其中發(fā)生在S1區(qū)的突變較多,占整體突變的70%以上,這也表明其S1區(qū)為該病毒的高變區(qū)。糖基化位點(diǎn)與抗原性分析,該4株病毒與疫苗株對(duì)比在已報(bào)道的抗原表位COE、2C10、S1D6均發(fā)生了幾處突變,在S1D5區(qū)未發(fā)現(xiàn)突變。(2)與國內(nèi)外68株毒株的核苷酸對(duì)比分析后發(fā)現(xiàn),本實(shí)驗(yàn)分離4株病毒與流行毒株AJ1102、CH/JLCC、CH18,同源性較高,在99.5%-100%之間,與經(jīng)典毒株同源性低。(3)SP株ORF3基因核苷酸序列分析表明:SP株與中國毒株JS-HZ2012 China(杭州)、GD-B China(廣東)的核苷酸同源性最高,達(dá)99.7%,與中國株LZC China(蘭州)的同源性最低,為95.6%。SP株M基因核苷酸序列分析表明:SP株M基因與國內(nèi)外13-019349 American、USA Colorado 2013 American、BJ-2011-3 China、GD-B China、HB-2012-1 China、HBMC2012 China、JS-HZ2012 China6株的同源性最高,均達(dá)100%。與中國毒株LZC China的同源性最低,為97.2%。分析結(jié)果提示現(xiàn)階段PEDV呈現(xiàn)快速變異和進(jìn)化的趨勢,其地域性和時(shí)間性愈來愈明顯,目前急需選擇新的疫苗株來控制PEDV的爆發(fā)。由于本研究的SP、YL9099、YL9140、YL9234株具有近年來PEDV變異株流行的普遍分子特征,因而可為新的疫苗毒株篩選提供參考。
[Abstract]:In the winter of 2013 and spring of 2014, swine farms in Jilin Province and Shaanxi Province were suspected to have epidemic diarrhea virus infection. In order to determine the pathogen and understand the gene difference between the pathogen and other strains at home and abroad, the fecal samples and small intestine tissues of diarrhea were collected, the virus was isolated and cultured, and the key genes were sequenced and analyzed. The results were as follows: 1. The porcine epidemic diarrhea virus strains SP (Jilin) and YL9099,YL9140,YL9234 (Shaanxi) were isolated after continuous passage of Vero cells. The S gene of 4 strains of porcine epidemic diarrhea virus and the MNORF3 gene of SP strain were amplified, cloned, sequenced and sequenced by RT-PCR. The sequence changes, genetic evolution, antigenic sites and glycosylation sites of the swine epidemic diarrhoea virus strain Schim MN ORF3, which were registered in Net NGlyc 1.0 Server and Gene Bank, were analyzed. (1) the four strains of the virus and the classical strain of CV777, in this study were selected for the analysis of the sequence changes, genetic evolution, antigenic sites and glycosylation sites. The nucleotide homology of DR13 was 94. 0-99. 4%. Compared with the vaccine strain, the deduced amino acid had 79-84 mutations, among which more mutations occurred in the S1 region, which accounted for more than 70% of the total mutation, which also indicated that the S1 region was the high variable region of the virus. The glycosylation sites and antigenicity of the four strains were compared with those of the vaccine strains. Several mutations were found in the reported epitope COE,2C10,S1D6. No mutation was found in the S1D5 region. (2) the nucleotide analysis of 68 strains of viruses at home and abroad showed that the homology of AJ1102,CH/JLCC,CH18, between the four viruses isolated in this experiment and the prevalent strains was between 99.5- 100%. (3) the nucleotide sequence analysis of ORF3 gene of SP strain showed that SP strain had the highest nucleotide homology with Chinese strains JS-HZ2012 China (Hangzhou) and GD-B China (Guangdong), and the nucleotide sequence of SP strain was 99.7%. The nucleotide sequence analysis of M gene of 95.6%.SP strain showed that M gene of SP strain had the lowest homology with that of Chinese strain LZC China (Lanzhou. It was found that M gene of SP strain had the highest homology with 13-019349 American,USA Colorado 2013 American,BJ-2011-3 China,GD-B China,HB-2012-1 China, at home and abroad. The homology of HBMC2012 China,JS-HZ2012 China6 strain was the highest, which was 100%. The homology with the Chinese strain LZC China was the lowest (97.2%). The results suggest that PEDV presents a trend of rapid variation and evolution, and its regional and temporal characteristics are becoming more and more obvious. It is urgent to select new vaccine strains to control the outbreak of PEDV. Because the SP,YL9099,YL9140,YL9234 strain in this study has the prevalent molecular characteristics of PEDV variant in recent years, it can be used as a reference for the screening of new vaccine strains.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
[Abstract]:In the winter of 2013 and spring of 2014, swine farms in Jilin Province and Shaanxi Province were suspected to have epidemic diarrhea virus infection. In order to determine the pathogen and understand the gene difference between the pathogen and other strains at home and abroad, the fecal samples and small intestine tissues of diarrhea were collected, the virus was isolated and cultured, and the key genes were sequenced and analyzed. The results were as follows: 1. The porcine epidemic diarrhea virus strains SP (Jilin) and YL9099,YL9140,YL9234 (Shaanxi) were isolated after continuous passage of Vero cells. The S gene of 4 strains of porcine epidemic diarrhea virus and the MNORF3 gene of SP strain were amplified, cloned, sequenced and sequenced by RT-PCR. The sequence changes, genetic evolution, antigenic sites and glycosylation sites of the swine epidemic diarrhoea virus strain Schim MN ORF3, which were registered in Net NGlyc 1.0 Server and Gene Bank, were analyzed. (1) the four strains of the virus and the classical strain of CV777, in this study were selected for the analysis of the sequence changes, genetic evolution, antigenic sites and glycosylation sites. The nucleotide homology of DR13 was 94. 0-99. 4%. Compared with the vaccine strain, the deduced amino acid had 79-84 mutations, among which more mutations occurred in the S1 region, which accounted for more than 70% of the total mutation, which also indicated that the S1 region was the high variable region of the virus. The glycosylation sites and antigenicity of the four strains were compared with those of the vaccine strains. Several mutations were found in the reported epitope COE,2C10,S1D6. No mutation was found in the S1D5 region. (2) the nucleotide analysis of 68 strains of viruses at home and abroad showed that the homology of AJ1102,CH/JLCC,CH18, between the four viruses isolated in this experiment and the prevalent strains was between 99.5- 100%. (3) the nucleotide sequence analysis of ORF3 gene of SP strain showed that SP strain had the highest nucleotide homology with Chinese strains JS-HZ2012 China (Hangzhou) and GD-B China (Guangdong), and the nucleotide sequence of SP strain was 99.7%. The nucleotide sequence analysis of M gene of 95.6%.SP strain showed that M gene of SP strain had the lowest homology with that of Chinese strain LZC China (Lanzhou. It was found that M gene of SP strain had the highest homology with 13-019349 American,USA Colorado 2013 American,BJ-2011-3 China,GD-B China,HB-2012-1 China, at home and abroad. The homology of HBMC2012 China,JS-HZ2012 China6 strain was the highest, which was 100%. The homology with the Chinese strain LZC China was the lowest (97.2%). The results suggest that PEDV presents a trend of rapid variation and evolution, and its regional and temporal characteristics are becoming more and more obvious. It is urgent to select new vaccine strains to control the outbreak of PEDV. Because the SP,YL9099,YL9140,YL9234 strain in this study has the prevalent molecular characteristics of PEDV variant in recent years, it can be used as a reference for the screening of new vaccine strains.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
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