本文選題：山東省 + 偽狂犬病病毒��； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：偽狂犬病(pseudorabies,PR)是由偽狂犬病毒(pseudorabies virus,PRV)引起的多種家畜以及野生動物的一種急性致死性傳染病,只有豬可以在急性感染中存活,是該病的自然儲存宿主。豬偽狂犬病是當(dāng)前我國豬場一種非常重要的病毒性疾病,給感染豬場造成嚴(yán)重的經(jīng)濟(jì)損失。2011年前由于基因缺失苗的應(yīng)用以及鑒別診斷方法gE-ELISA的配合使用,PR在我國得到了有效的控制。然而,山東省從2011年開始各地陸續(xù)在疫苗免疫豬場發(fā)生PR,造成母豬流產(chǎn)死胎,哺乳仔豬呈現(xiàn)嘔吐腹瀉及神經(jīng)癥狀,并導(dǎo)致高死亡率。本研究主要分為兩部分:第一部分山東省免疫豬場12株P(guān)RV分離和鑒定2013年12月份至2014年5月,采集山東省濟(jì)南、德州、臨沂、濰坊、東營、青島等地12個經(jīng)PRV疫苗(Bartha-K61)免疫豬場的PR疑似病料,病料研磨處理,過濾除菌后接種到BHK-21細(xì)胞上。連續(xù)在BHK-21細(xì)胞上增殖6代,第五代和第六代細(xì)胞培養(yǎng)液的DNA使用偽狂犬病毒熒光PCR檢測試劑盒檢測,并將第六代的病毒液接種成兔。結(jié)果12份病料的組織勻漿液接種后的BHK細(xì)胞以及之后增殖病毒的每一代細(xì)胞均出現(xiàn)了典型的PRV感染所致的細(xì)胞病變。提取第五代和第六代細(xì)胞培養(yǎng)液的DNA,經(jīng)偽狂犬病毒熒光PCR檢測試劑盒檢測,12株P(guān)RV的第五代和第六代細(xì)胞培養(yǎng)液全部為陽性。12株P(guān)RV第六代病毒液接種成兔后均出現(xiàn)奇癢并最終死亡。TCID50測定結(jié)果顯示,12株P(guān)RV的TCID50介于10-7.1/0.1ml與10-9.5/0.1ml之間。以上結(jié)果表明,本實驗自山東省免疫豬場分離到12株P(guān)RV。第二部分12株P(guān)RV gE、gC、gD和TK基因的序列分析本研究對2013和2014年從山東省免疫豬場分離到的12株P(guān)RV的gE、gC、gD和TK基因進(jìn)行PCR擴(kuò)增、序列測定與分析。結(jié)果顯示,12株P(guān)RV的gE基因核苷酸和氨基酸同源性分別為99.9%~100%和99.7%~100%;與亞洲毒株的核苷酸和氨基酸同源性均高于歐美毒株。gE基因的氨基酸進(jìn)化樹分析表明,包括本研究分離的12株P(guān)RV在內(nèi)的所有42株亞洲毒株屬于GⅠ型,所有歐美毒株屬于GⅡ型。這兩個基因型之間分別在第58、105、148、178、180、214、215、470、500、505、518、522和569位存在明顯的氨基酸差異,可以作為鑒別PRV歐美毒株與亞洲毒株的遺傳標(biāo)志。12株P(guān)RVgC基因的核苷酸和氨基酸同源性分別為99.8%~100%和99.6%~100%。gC基因氨基酸進(jìn)化樹顯示,包括本研究分離的12株P(guān)RV在內(nèi)41株國內(nèi)外分離株分為3個基因型:GⅠ型、GⅡ型和GIII型。而GⅠ型又分為S1和S2兩個亞型,除WY、LJN01-2014和SC1301的國內(nèi)2011年后分離株位于S1亞型。12株P(guān)RV的gD基因核苷酸和氨基酸同源性均為99.8%~100%,國內(nèi)分離株的核苷酸同源性均高于三株歐美毒株。國內(nèi)毒株與歐美毒株在340位和347位的氨基酸存在穩(wěn)定的突變。gD基因的氨基酸進(jìn)化樹顯示所有的國內(nèi)分離株與三株歐美毒株處于不同的分支上,三株歐美毒株中的美國毒株又位于一個單獨的分支上,其它兩株匈牙利毒株位于同一個分支上,這表明gD基因有明顯的地域特征。對12株P(guān)RV的TK基因與國內(nèi)以及歐美毒株的序列分析并沒有發(fā)現(xiàn)明顯的不同。
[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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 王利軍;;豬偽狂犬病綜合防治措施[J];當(dāng)代畜牧;2012年04期

2 王勤;豬偽狂犬病病毒gE基因的研究進(jìn)展[J];畜牧與獸醫(yī);2002年10期

3 關(guān)偉杰,徐家宰;關(guān)于豬“奧氏病”(偽狂犬病)的報導(dǎo)[J];畜牧與獸醫(yī);1959年05期

，

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