DHAV-3 LS株遗传变异分析、VP1蛋白表达及荧光定量PCR分型方法的建立

发布时间：2018-02-24 01:19

本文关键词： 鸭甲肝病毒分离鉴定序列分析结构蛋白VP1原核表达荧光定量PCR　出处：《广西大学》2015年硕士论文　论文类型：学位论文

【摘要】：鸭病毒性肝炎(DVH)是由鸭肝炎病毒(DHV)感染3周龄以内雏鸭引发的一种急性、高致死性的病毒性传染病,严重影响养鸭业的发展。目前我国流行的DVH病原主要是鸭甲型肝炎病毒(duck hepatitis A virus, DHAV) 。因此,开展DHAV的病毒分离、遗传进化、主要结构蛋白表达及病原鉴别诊断方法的研究对今后DHAV分子流行病学研究、病原检测与疾病防控具有重要意义。本研究采集山东省梁山县某鸭场发生的1起疑似DVH病例的临床病料,通过鸭胚增殖、动物回归试验、ELDso测定和RT-PCR方法分离鉴定,得到一株DHAV-3毒株,命名为LS株。对分离毒株的全基因组进行了RT-PCR扩增、基因克隆、序列测定与分析,结果表明,LS株整个基因组全长7815nt,不含帽子结构,其中5’非编码区(5'UTR)长652nt,整个ORF长6756nt,3'端非编码区(3'UTR)长366nt,后面带有至少26个A的poly(A)尾巴。LS株与DHAV-3参考株核苷酸、氨基酸同源性最高,达97.4%和99.0%；与C-BLZ株、SD1201株亲缘关系最近,处于同一较小的分枝上。因此证明LS株属于DHAV-3 。利用相关分子生物学技术对LS株VP1结构蛋白的氨基酸序列和主要抗原表位进行比对分析,并针对高变区使用原核表达系统进行蛋白表达。结果显示,B细胞抗原表位存在第132-139、178-191、195-203、211-221位氨基酸区段的可能性最大,同时这些位点也是最易变异的位点。针对高变区选取的第132-240位氨基酸区段得到成功表达,纯化的融合蛋白分子量约为38ku。Western blot分析结果表明,融合蛋白能被抗GST标签鼠单克隆抗体特异性识别,证明该蛋白具有良好的反应原性。本研究还针对1、3型鸭甲肝病毒5端保守区设计一对特异性引物,采用SYBR Green I荧光染料,成功建立了一种基于荧光定量PCR熔解曲线法的DHAV分型检测方法。结果表明,建立的方法特异、快速、敏感,对1.0×102～1.0×109copies/μL浓度的模板均有较好的检测效果,最低检出量均为100 copies/μL,是普通PCR的100倍；特异性好,排除常见禽源病毒的干扰；对高、中、低不同浓度质粒标准品检测表现出良好重复性。利用建立方法对DHAV-1和DHAV-3感染雏鸭后在体内组织器官的增殖规律进行探究,发现鸭甲型肝炎病毒广泛增殖于鸭的大部分组织脏器内。两种病毒在组织器官中的增殖规律大体一致,其中对肝脏最具有嗜性,脾脏、肾脏、肺脏中病毒滴度也很高,进而从病毒含量角度解释了DHAV感染雏鸭后在肝脏、脾脏、肾脏、肺脏等主要靶器官引起的特征病变。
[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.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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