本文選題：Orfv　切入點(diǎn)：B2L基因　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究從內(nèi)蒙古赤峰、武川和山東青島地區(qū)疑似羊口瘡的發(fā)病羊群采集病料,通過(guò)剪碎、研磨、凍融三次離心取上清液加雙抗進(jìn)行無(wú)菌處理后在犢牛睪丸細(xì)胞、MDBK細(xì)胞上接種傳代,同時(shí)根據(jù)GenBank中發(fā)表的的羊口瘡病毒B2L蛋白基因設(shè)計(jì)1對(duì)引物,對(duì)細(xì)胞培養(yǎng)物、病料中疑似羊口瘡病毒B2L基因的PCR擴(kuò)增,病料處理差速離心后經(jīng)負(fù)染透射電子顯微鏡觀察。在此基礎(chǔ)上根據(jù)GeneBank中羊口瘡病毒的B2L、F1L、VIR基因設(shè)計(jì)3對(duì)引物,對(duì)三個(gè)地方的羊口瘡病毒進(jìn)行DNA提取、PCR擴(kuò)增、連接到pMD19-T載體、轉(zhuǎn)化到DH5a感受態(tài)細(xì)胞、通過(guò)藍(lán)白斑篩選陽(yáng)性質(zhì)粒和重組質(zhì)粒的鑒定后測(cè)序。用DNAstar軟件進(jìn)行三個(gè)不同地區(qū)分離株之間核苷酸同源性比較分析,再將測(cè)序的結(jié)果利用NCBI上的BLAST的方法與GenBank中公布的Orfv的B2L、F1L、VIR基因核苷酸序列進(jìn)行同源性比較分析。最后應(yīng)用DNA Star程序中的Meg Align軟件包進(jìn)行分析,通過(guò)Clustal-V Method構(gòu)建系統(tǒng)進(jìn)化樹(shù)。結(jié)果表明：從三個(gè)不同地區(qū)采集的病料在犢牛睪丸細(xì)胞和MDBK細(xì)胞上接種傳代時(shí)第一、二代出現(xiàn)明顯的細(xì)胞病變。從第三代開(kāi)始細(xì)胞病變不明顯,但細(xì)胞培養(yǎng)物仍能用PCR擴(kuò)增出明顯的條帶；透射電鏡觀察到橢圓形、8字形的毛線團(tuán)樣典型的羊口瘡病毒粒子；這些結(jié)果表明本次成功分離到Orfv,分別命名為Orfv-W、rfv-C和Orfv-Q。三個(gè)分離株之間B2L、F1L和VIR基因核苷酸同源性分別為94.8%-97.4%、93.2%～98%和95.1%-100%。三個(gè)分離株與國(guó)內(nèi)外不同來(lái)源的Orfv病毒株B2L、F1L和VIR基因核苷酸同源性分別為82.1%～98.0%、93.4%～98.0%和93.8%～99.1%。對(duì)B2L、F1L和VIR基因進(jìn)行系統(tǒng)進(jìn)化樹(shù)分析后顯示,Orfv-W毒株與山羊分離株親緣關(guān)系近；Orfv-Q毒株與綿羊分離株親緣關(guān)系近；Orfv-C毒株既與綿羊分離株親緣關(guān)系近,也與山羊分離株親緣關(guān)系近,且三個(gè)基因分支均與國(guó)內(nèi)西北部地區(qū)分離株的親緣關(guān)系接近。
[Abstract]:In this study, sheep diseases were collected from suspected sheep and mouth ulcers in Chifeng, Wuchuan and Qingdao, Shandong Province, and were shredded and ground. The supernatant of frozen thawed three times centrifugation and double antibody were inoculated on the MDBK cells of calf testicular cells after sterile treatment. At the same time, a pair of primers were designed according to the B2L protein gene of sheep aphthoea virus published in GenBank. The B2L gene of suspected sheep mouth sore virus was amplified by PCR, and observed by negative staining transmission electron microscope after differential centrifugation. Based on this, three pairs of primers were designed according to the B _ 2LN _ (F1L) VIR gene of sheep mouth sore virus in GeneBank. Sheep aphtha virus was amplified by DNA and ligated to pMD19-T vector and transformed into DH5a receptive cells. The positive plasmids and recombinant plasmids were screened by blue and white spot and sequenced. The nucleotide homology was analyzed by DNAstar software. The results of sequencing were compared and analyzed by using the method of BLAST on NCBI and the nucleotide sequence of Orfv gene B2LF1L1 / VIR published in GenBank. Finally, the Meg Align software package of DNA Star program was used to analyze the nucleotide sequence of VIR gene. Phylogenetic tree was constructed by Clustal-V Method. The results showed that obvious cytopathic changes appeared in the first and second passages of calf testicular cells and MDBK cells inoculated from three different regions, but the cytopathic changes were not obvious from the third generation. But the cell culture could still amplify the obvious bands by PCR, and the typical sheep aphtha virus particles were observed by transmission electron microscope. These results indicate that Orfv-C and Orfv-Q.The nucleotide homology of B2LF1L and VIR genes between the three isolates were 94.8-97.4% and 95.1-100%, respectively. The nucleotide homology of the three isolates was 94.8% -97.4% and 95.1-100%, respectively. The nucleotide homology of the three isolates was 94.8% -97.4% and 95.1-100% respectively with the Orfv virus strains B2LLF1L and VIR genes from different sources at home and abroad. The homology of glycoside was 98.0% and 99.1.The phylogenetic tree analysis of B2LF1L and VIR genes showed that Orfv-W strain was closely related to goat isolate and Orfv-Q strain was closely related to sheep isolate, and Orfv-C strain was closely related to sheep strain. It was also closely related to goat isolates, and the three gene branches were close to those of the isolates in northwestern China.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.654

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 田婷婷;李杰;姚運(yùn)亮;李前瑞;許君艷;趙燕青;陳德坤;;羊口瘡病毒滅活疫苗的制備及免疫效果觀察[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2013年06期

2 姜艷;賈福英;;羊口瘡的防制[J];農(nóng)技服務(wù);2008年12期

3 孫德云;;淺析羊口瘡病的綜合防治[J];草業(yè)與畜牧;2010年03期

4 ;Identification and Phylogenetic Analysis of an Orf Virus Isolated from an Outbreak in Boer Goat in Shanxi Province[J];Agricultural Sciences in China;2011年06期

5 向智龍;卓建華;鮮思美;歐德淵;李啟發(fā);徐雄真;;羊口瘡病毒環(huán)介導(dǎo)等溫?cái)U(kuò)增快速檢測(cè)方法的建立及應(yīng)用[J];中國(guó)獸醫(yī)科學(xué);2011年06期

6 黃引賢,李乃津;羊傳染性膿皰病毒某些特性的初步研究[J];中國(guó)獸醫(yī)雜志;1986年04期

7 李慧霞;朱學(xué)亮;駱學(xué)農(nóng);;羊口瘡病的研究進(jìn)展[J];中國(guó)預(yù)防獸醫(yī)學(xué)報(bào);2013年05期

相關(guān)碩士學(xué)位論文 前1條

1 張海瑞;羊口瘡病毒的分離鑒定及其ELISA檢測(cè)方法的建立[D];中國(guó)農(nóng)業(yè)科學(xué)院;2011年

，

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