發(fā)布時(shí)間：2018-02-24 01:19

  本文關(guān)鍵詞： 鴨甲肝病毒 分離鑒定 序列分析 結(jié)構(gòu)蛋白VP1原核表達(dá) 熒光定量PCR　出處：《廣西大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：鴨病毒性肝炎(DVH)是由鴨肝炎病毒(DHV)感染3周齡以內(nèi)雛鴨引發(fā)的一種急性、高致死性的病毒性傳染病,嚴(yán)重影響?zhàn)B鴨業(yè)的發(fā)展。目前我國(guó)流行的DVH病原主要是鴨甲型肝炎病毒(duck hepatitis A virus, DHAV) 。因此,開展DHAV的病毒分離、遺傳進(jìn)化、主要結(jié)構(gòu)蛋白表達(dá)及病原鑒別診斷方法的研究對(duì)今后DHAV分子流行病學(xué)研究、病原檢測(cè)與疾病防控具有重要意義。本研究采集山東省梁山縣某鴨場(chǎng)發(fā)生的1起疑似DVH病例的臨床病料,通過(guò)鴨胚增殖、動(dòng)物回歸試驗(yàn)、ELDso測(cè)定和RT-PCR方法分離鑒定,得到一株DHAV-3毒株,命名為L(zhǎng)S株。對(duì)分離毒株的全基因組進(jìn)行了RT-PCR擴(kuò)增、基因克隆、序列測(cè)定與分析,結(jié)果表明,LS株整個(gè)基因組全長(zhǎng)7815nt,不含帽子結(jié)構(gòu),其中5’非編碼區(qū)(5'UTR)長(zhǎng)652nt,整個(gè)ORF長(zhǎng)6756nt,3'端非編碼區(qū)(3'UTR)長(zhǎng)366nt,后面帶有至少26個(gè)A的poly(A)尾巴。LS株與DHAV-3參考株核苷酸、氨基酸同源性最高,達(dá)97.4%和99.0%；與C-BLZ株、SD1201株親緣關(guān)系最近,處于同一較小的分枝上。因此證明LS株屬于DHAV-3 。利用相關(guān)分子生物學(xué)技術(shù)對(duì)LS株VP1結(jié)構(gòu)蛋白的氨基酸序列和主要抗原表位進(jìn)行比對(duì)分析,并針對(duì)高變區(qū)使用原核表達(dá)系統(tǒng)進(jìn)行蛋白表達(dá)。結(jié)果顯示,B細(xì)胞抗原表位存在第132-139、178-191、195-203、211-221位氨基酸區(qū)段的可能性最大,同時(shí)這些位點(diǎn)也是最易變異的位點(diǎn)。針對(duì)高變區(qū)選取的第132-240位氨基酸區(qū)段得到成功表達(dá),純化的融合蛋白分子量約為38ku。Western blot分析結(jié)果表明,融合蛋白能被抗GST標(biāo)簽鼠單克隆抗體特異性識(shí)別,證明該蛋白具有良好的反應(yīng)原性。本研究還針對(duì)1、3型鴨甲肝病毒5端保守區(qū)設(shè)計(jì)一對(duì)特異性引物,采用SYBR Green I熒光染料,成功建立了一種基于熒光定量PCR熔解曲線法的DHAV分型檢測(cè)方法。結(jié)果表明,建立的方法特異、快速、敏感,對(duì)1.0×102～1.0×109copies/μL濃度的模板均有較好的檢測(cè)效果,最低檢出量均為100 copies/μL,是普通PCR的100倍；特異性好,排除常見禽源病毒的干擾；對(duì)高、中、低不同濃度質(zhì)粒標(biāo)準(zhǔn)品檢測(cè)表現(xiàn)出良好重復(fù)性。利用建立方法對(duì)DHAV-1和DHAV-3感染雛鴨后在體內(nèi)組織器官的增殖規(guī)律進(jìn)行探究,發(fā)現(xiàn)鴨甲型肝炎病毒廣泛增殖于鴨的大部分組織臟器內(nèi)。兩種病毒在組織器官中的增殖規(guī)律大體一致,其中對(duì)肝臟最具有嗜性,脾臟、腎臟、肺臟中病毒滴度也很高,進(jìn)而從病毒含量角度解釋了DHAV感染雛鴨后在肝臟、脾臟、腎臟、肺臟等主要靶器官引起的特征病變。
[Abstract]:Duck viral hepatitis (DVH) is an acute and highly fatal viral infection caused by duck hepatitis virus (DHV) infection in ducklings within 3 weeks of age. The main DVH pathogen in China is duck hepatitis A virus (DHAV). Therefore, the virus isolation and genetic evolution of DHAV are carried out. Studies on the expression of major structural proteins and the methods for differential diagnosis of the pathogeny in the future study on molecular epidemiology of DHAV. The detection of pathogen and the prevention and control of disease are of great significance. The clinical samples of a suspected case of DVH in a duck farm in Liangshan County Shandong Province were collected and identified by the methods of duck embryo proliferation animal regression test ELDso test and RT-PCR method. A DHAV-3 strain named LS strain was obtained. The whole genome of the isolated strain was amplified by RT-PCR, cloned, sequenced and analyzed. The length of ORF is 652 nt, the length of ORF is 6756 NT / 3 'and the length is 366nt, and there are at least 26 A's tail. LS strain and DHAV-3 reference strain have the highest amino acid homology (97.4% and 99.0), and have the closest phylogenetic relationship with C-BLZ strain SD1201. It is proved that LS strain belongs to DHAV-3. The amino acid sequences and major antigen epitopes of VP1 structural proteins of LS strain were compared with each other by molecular biological techniques. The protein was expressed by prokaryotic expression system in the hypervariable region. The results showed that the amino acid region at the 132-139tl 178-191C 195-203C 211-221 was the most likely to exist in the antigenic epitope of the B cell. The 132-240 amino acid region selected for the hypervariable region was successfully expressed, and the molecular weight of the purified fusion protein was about 38ku.Western blot. The fusion protein can be specifically recognized by monoclonal antibodies against GST labeled mice, which proves that the fusion protein has good reactivity. A pair of specific primers were designed for the conserved region of duck hepatitis A virus type 1 / 3, and SYBR Green I fluorescent dye was used. A DHAV typing method based on fluorescence quantitative PCR melting curve method was successfully established. The results showed that the established method was specific, rapid and sensitive, and had good detection effect on 1.0 脳 10 ~ (2) C _ (10) 脳 10 ~ (9) copi es / 渭 L template. The lowest detectable amount is 100 copias / 渭 L, which is 100 times higher than that of normal PCR. The detection of plasmid standard at different concentrations showed good reproducibility. The rules of tissue and organ proliferation in ducklings infected with DHAV-1 and DHAV-3 were investigated by using the established method. It was found that duck hepatitis A virus proliferates widely in most tissues and organs of duck. The proliferation of the two viruses in tissues and organs is roughly the same, and the virus titer in spleen, kidney and lung is also very high, which is the most eosinophilic to the liver. From the point of view of virus content, the characteristic pathological changes caused by DHAV infection in the liver, spleen, kidney, lung and other main target organs of ducklings were explained.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

【相似文獻(xiàn)】

相關(guān)期刊論文 前5條

1 吳朝軍;劉金鳳;馮濤;馬秀麗;;鴨甲型肝炎病毒1型3D基因克隆與表達(dá)[J];家禽科學(xué);2013年09期

2 黃秋雪;湯承;岳華;;基因C型鴨甲型肝炎病毒分子生物學(xué)研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2013年02期

3 張冰;張煥容;楊發(fā)龍;湯承;岳華;;基因C型鴨甲型肝炎病毒在鴨胚原代肝細(xì)胞中的增殖規(guī)律[J];中國(guó)獸醫(yī)科學(xué);2013年08期

4 任麗倩;李晶;畢玉海;陳璨;張大丙;劉文軍;;鴨甲型病毒性肝炎的研究進(jìn)展[J];生物工程學(xué)報(bào);2012年07期

5 ;[J];;年期

相關(guān)碩士學(xué)位論文 前4條

1 胡文;鴨甲型肝炎病毒2A蛋白的生物學(xué)功能的初步研究[D];甘肅農(nóng)業(yè)大學(xué);2014年

2 張映;1型和3型鴨甲型肝炎病毒逆轉(zhuǎn)錄環(huán)介導(dǎo)等溫?cái)U(kuò)增檢測(cè)研究[D];中國(guó)獸醫(yī)藥品監(jiān)察所;2016年

3 黃云秀;DHAV-3 LS株遺傳變異分析、VP1蛋白表達(dá)及熒光定量PCR分型方法的建立[D];廣西大學(xué);2015年

4 朱寅彪;鴨甲型肝炎病毒RT-PCR檢測(cè)方法的建立及華東地區(qū)2009-2011年鴨甲型肝炎病毒的流行病學(xué)調(diào)查[D];揚(yáng)州大學(xué);2011年

，

本文編號(hào)：1528338