山东省免疫猪场猪伪狂犬病毒分离鉴定及gE、gC、gD和TK基因的序列分析

发布时间：2018-04-15 04:29

本文选题：山东省 + 伪狂犬病病毒　；参考：《山东农业大学》2015年硕士论文

【摘要】：伪狂犬病(pseudorabies,PR)是由伪狂犬病毒(pseudorabies virus,PRV)引起的多种家畜以及野生动物的一种急性致死性传染病,只有猪可以在急性感染中存活,是该病的自然储存宿主。猪伪狂犬病是当前我国猪场一种非常重要的病毒性疾病,给感染猪场造成严重的经济损失。2011年前由于基因缺失苗的应用以及鉴别诊断方法gE-ELISA的配合使用,PR在我国得到了有效的控制。然而,山东省从2011年开始各地陆续在疫苗免疫猪场发生PR,造成母猪流产死胎,哺乳仔猪呈现呕吐腹泻及神经症状,并导致高死亡率。本研究主要分为两部分:第一部分山东省免疫猪场12株PRV分离和鉴定2013年12月份至2014年5月,采集山东省济南、德州、临沂、潍坊、东营、青岛等地12个经PRV疫苗(Bartha-K61)免疫猪场的PR疑似病料,病料研磨处理,过滤除菌后接种到BHK-21细胞上。连续在BHK-21细胞上增殖6代,第五代和第六代细胞培养液的DNA使用伪狂犬病毒荧光PCR检测试剂盒检测,并将第六代的病毒液接种成兔。结果12份病料的组织匀浆液接种后的BHK细胞以及之后增殖病毒的每一代细胞均出现了典型的PRV感染所致的细胞病变。提取第五代和第六代细胞培养液的DNA,经伪狂犬病毒荧光PCR检测试剂盒检测,12株PRV的第五代和第六代细胞培养液全部为阳性。12株PRV第六代病毒液接种成兔后均出现奇痒并最终死亡。TCID50测定结果显示,12株PRV的TCID50介于10-7.1/0.1ml与10-9.5/0.1ml之间。以上结果表明,本实验自山东省免疫猪场分离到12株PRV。第二部分12株PRV gE、gC、gD和TK基因的序列分析本研究对2013和2014年从山东省免疫猪场分离到的12株PRV的gE、gC、gD和TK基因进行PCR扩增、序列测定与分析。结果显示,12株PRV的gE基因核苷酸和氨基酸同源性分别为99.9%~100%和99.7%~100%;与亚洲毒株的核苷酸和氨基酸同源性均高于欧美毒株。gE基因的氨基酸进化树分析表明,包括本研究分离的12株PRV在内的所有42株亚洲毒株属于GⅠ型,所有欧美毒株属于GⅡ型。这两个基因型之间分别在第58、105、148、178、180、214、215、470、500、505、518、522和569位存在明显的氨基酸差异,可以作为鉴别PRV欧美毒株与亚洲毒株的遗传标志。12株PRVgC基因的核苷酸和氨基酸同源性分别为99.8%~100%和99.6%~100%。gC基因氨基酸进化树显示,包括本研究分离的12株PRV在内41株国内外分离株分为3个基因型:GⅠ型、GⅡ型和GIII型。而GⅠ型又分为S1和S2两个亚型,除WY、LJN01-2014和SC1301的国内2011年后分离株位于S1亚型。12株PRV的gD基因核苷酸和氨基酸同源性均为99.8%~100%,国内分离株的核苷酸同源性均高于三株欧美毒株。国内毒株与欧美毒株在340位和347位的氨基酸存在稳定的突变。gD基因的氨基酸进化树显示所有的国内分离株与三株欧美毒株处于不同的分支上,三株欧美毒株中的美国毒株又位于一个单独的分支上,其它两株匈牙利毒株位于同一个分支上,这表明gD基因有明显的地域特征。对12株PRV的TK基因与国内以及欧美毒株的序列分析并没有发现明显的不同。
[Abstract]:Pseudorabies (pseudorabies, PR) is composed of pseudorabies virus (pseudorabies virus, PRV) a variety of domestic and wild animal caused an acute and fatal infectious disease, only pigs can survive in the acute infection, the disease is the natural reservoir. Pseudorabies is when our farm is a very important a viral disease, causing serious economic losses due to the use of.2011 years ago with the application of gene deletion vaccine and the diagnostic method of gE-ELISA infected farms, PR has been effectively controlled in China. However, Shandong province began around 2011 in succession in the vaccine from swine PR, causing sow abortion stillbirth, lactation piglets showed diarrhea and vomiting symptoms, and leads to high mortality. This research is mainly divided into two parts: the first part of Shandong province pig immune isolation and identification of 12 strains of PRV from 2013 December to May 2014, acquisition Shandong Province, Ji'nan, Dezhou, Linyi, Weifang, Dongying, Qingdao and other places 12 by PRV vaccine (Bartha-K61) immune swine suspected PR disease, disease material grinding, filtration and inoculated into BHK-21 cells. The proliferation of BHK-21 cells continuously in the 6 generation, fifth generation and sixth generation of cell culture fluid DNA using Pseudorabies virus fluorescence PCR detection kit, and the sixth generation of the virus were inoculated into rabbits. Results 12 tissue homogenate inoculation disease material after BHK cell and cell proliferation after each generation virus showed typical PRV infected cells caused by lesions. Extraction of the fifth and sixth generation cell culture medium DNA, the pseudorabies virus fluorescence PCR detection kit, 12 strains of PRV fifth generation and sixth generation cells were all positive.12 strains of PRV virus were inoculated into the sixth generation of rabbit occurred after itching and eventual death.TCID50 results. In TCID50 between 10-7.1/0.1ml and 10-9.5/0.1ml 12 strains of PRV. These results indicate that the Shandong province pig immune isolate 12 PRV. second part of 12 strains of PRV gE, gC, gD and sequence analysis of TK gene in this study, 12 strains of PRV in 2013 and 2014 from Shandong province pig immune gE gC, gD, and TK genes were amplified by PCR, sequenced and analyzed. The results showed that the gE gene nucleotide and amino acid homology of 12 PRV strains were 99.9%~100% and 99.7%~100%; and the nucleotide and amino acid homology of Asian strains were higher than that of European and American strains.GE gene phylogenetic tree analysis showed that this study includes the separation of the 12 line PRV, all 42 strains of Asian strain belongs to G type, G type II strains belonging to all of Europe and the United States. Between the two genotypes respectively in the 58105148178180214215470500505518522 and 569 obvious amino acids Differences can be used to identify PRV strains and strains of the Asia Europe genetic marker.12 strain PRVgC gene nucleotide and amino acid homology were 99.8%~100% and 99.6%~100%.gC gene phylogenetic tree showed that this study included 12 PRV strains, 41 strains of isolates were divided into 3 genotypes: G type, G type and GIII. And G type can be divided into S1 and S2 two subtypes, except WY, LJN01-2014 and SC1301 in China after 2011 isolates gD gene nucleotide and amino acid in S1 subtype.12 strain PRV homology were 99.8%~100%, the nucleotide homology was higher than that of strains isolated from three strains and strains. The domestic and European strains in 340 strains and 347 amino acid mutations exist stable phylogenetic tree.GD gene showed that all isolates and three strains and strains in different branches, three strains and strains in the virus The strain is also located on a separate branch. The other two Hungarian strains are located on the same branch. This indicates that the gD gene has obvious regional characteristics. There is no obvious difference in the sequence analysis of the TK gene between the 12 PRV and domestic and European and American strains.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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