猪肠病毒RT-PCR方法的建立和一株野猪肠病毒全基因组分析以及抗PEDV变异株卵黄抗体的制备

发布时间：2018-05-16 19:26

本文选题：猪肠病毒 + RT-PCR　；参考：《江西农业大学》2017年硕士论文

【摘要】：猪肠病毒(Porcine enteroviruses,PEVs)现被分类为肠病毒G型(Porcine enteroviruses G,EV-G),属于小RNA病毒科(Picornaviridae)。猪是EV-G唯一的自然宿主,且各个年龄段猪只对EV-G敏感,猪感染EV-G后大部分不会表现出明显的临床症状。目前为止,在北美、欧洲和亚洲等地区均有猪群检测到EV-G型的报道,但是我国江西省尚未见相关研究报道。本试验针对EV-G参考序列的保守区域设计一对特异性检测引物,建立了一种EV-G的RT-PCR快速检测方法,并运用此方法检测了2015年来自江西省九江(25份)和鹰潭(18份)两个地区共43份腹泻仔猪的粪便样品。检测结果显示EV-G总阳性率为11.6%(5/43),其中九江某猪场检测阳性率为12.0%(3/25),鹰潭某猪场检测阳性率为11.1%(2/18)。为了研究江西省EV-G的分子流行病学和基因组遗传变异特征,根据EV-G的参考序列保守区域设计了3对扩增EV-G全长的引物,利用设计引物成功扩增得到一株来自九江市的野猪EV-G毒株全基因组序列。测序结果发现该毒株的基因组全长为7,404 nt,包含一个6501nt的大开放阅读框,编码2167个氨基酸。通过DNAStar Lasergene 7.10软件对测序株与20条参考毒株进行全基因组序列和推定氨基酸序列比对,结果表明测序株与8株EV-G型参考株的核苷酸序列同源性为69.8%~78.4%,氨基酸序列同源性为71.8%~77.9%,其中与EV-G2型(登录号为AF363455.1)的序列同源性最高;基于全基因组水平及其编码蛋白的遗传进化树分析也显示测序株与EV-G2型的遗传距离最近。猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)是引起仔猪腹泻的重要肠道病毒之一,发病猪只的临床症状与猪传染性胃肠炎十分类似,主要通过粪口途径传播。2010年末后变异株PEDV引起的腹泻在我国大面积爆发,呈现高发病率及高死亡率。该病至今依然流行且呈愈演愈烈的趋势,给我国养猪业造成了严重的损失。本试验使用PEDV变异毒株免疫海兰褐母鸡,通过酸化水提法、PEG-6000法以及酸化水提法结合硫酸铵的二次沉淀法等三种方法提取卵黄抗体,基于PEDV江西变异株全病毒之上,建立了一套快速准确的间接ELISA方法,用以测定卵黄抗体(Egg Yolk Antibody,IgY)效价,并评估IgY的耐热性、耐酸碱性以及模拟胃酸条件下三种不同保护剂的保护效果等。结果表明酸化水提法纯化的蛋白浓度较高,但提取的特异性IgY较少;PEG-6000法提取的IgY纯度较高,但蛋白浓度较酸化水提法要低;酸化水提法结合硫酸铵的二次沉淀法提取的IgY纯度最高,但是获得的蛋白浓度较低。试验结果还表明抗PEDV变异毒株IgY在高温下(70℃)和pH4和pH11的条件下易发生变性,在37~70℃和pH 4~11的范围内可保持稳定。此外,本试验还测试了IgY在模拟胃酸环境下(pH 2.0和3.0),添加20%蔗糖、20%葡萄糖和20%硫糖铝等三种糖类保护剂后,对IgY活性情况的影响。结果显示在三种糖类中,20%的蔗糖与葡萄糖在pH2.0和3.0的条件下不能提高抗体的稳定性,但是20%的硫糖铝对IgY有一定的保护作用。
[Abstract]:Porcine enteroviruses (PEVs) is now classified as enterovirus G (Porcine enteroviruses G, EV-G), belonging to the small RNA virus family (Picornaviridae). Pigs are the only natural host of EV-G, and pigs are sensitive to EV-G in all ages. Most of the pigs are infected without obvious clinical symptoms. So far, in North America, Europe The reports of EV-G type were reported in the region of Asia and other regions, but there were no related reports in Jiangxi Province in China. This experiment designed a pair of specific detection primers for the conservative region of the EV-G reference sequence and established a rapid detection method of RT-PCR for EV-G. The method was used to detect the 2015 from Jiujiang (25 copies) from Jiangxi province. The fecal samples of 43 diarrhoea piglets in two regions of Yingtan (18) showed that the total positive rate of EV-G was 11.6% (5/43), of which the positive rate of a pig farm in Jiujiang was 12% (3/25), and the positive rate of a pig farm in Yingtan was 11.1% (2/18). In order to study the molecular epidemiology and genomic genetic variation characteristics of EV-G in Jiangxi Province, according to the reference of EV-G 3 pairs of primers for the full length of EV-G were designed in the conservative region. The whole genome sequence of a wild boar EV-G strain from Jiujiang was amplified by the design primers. The whole genome length of the strain was 7404 NT, including a large open reading frame of 6501nt, 2167 amino acids encoded by DNAStar Lasergene. 7.10 the whole genome sequence and the deduced amino acid sequence were compared to the sequencing and 20 reference strains. The results showed that the nucleotide sequence of the sequence and 8 EV-G reference strains was 69.8%~78.4%, the homology of the amino acid sequence was 71.8%~77.9%, and the sequence homology with the EV-G2 type (the login number AF363455.1) was the highest. The genetic evolution tree analysis of the genome level and its encoded protein also showed that the genetic distance between the sequence and the EV-G2 type was the closest. The Porcine epidemic diarrhea virus (PEDV) is one of the important enterovirus causing piglet diarrhea. The clinical symptoms of the pigs are very similar to the swine infectious gastroenteritis, mainly through the feces. The oral route spread the diarrhea caused by the mutant strain PEDV of the late.2010 year in China, which has a high incidence and high mortality. This disease is still prevalent and increasingly intense, causing serious loss to the pig industry in China. This experiment uses PEDV variant strains to immunization the brown hens by the acidified water method and the PEG-6000 method. The egg yolk antibody was extracted with three methods such as acidification water extraction combined with two precipitation method of ammonium sulfate. Based on the PEDV Jiangxi mutant strain, a set of rapid and accurate indirect ELISA method was established to determine the titer of Egg Yolk Antibody (IgY), and to evaluate the heat resistance of IgY, acid alkali resistance and three of simulated gastric acid conditions. The results showed that the concentration of protein purified by acidified water extraction was higher, but the specificity of IgY extracted was less; the purity of IgY extracted by PEG-6000 was higher, but the concentration of protein was lower than that of acidified water; the purity of IgY extracted by acidified water extraction combined with the two precipitation method of ammonium sulfate was the highest, but the protein concentration was obtained. The results also showed that the anti PEDV mutant strain IgY was easily denatured under the conditions of high temperature (70 C) and pH4 and pH11, and remained stable at 37~70 and pH 4~11. In addition, the test also tested three sugar protectant of IgY in simulated gastric acid environment (pH 2 and 3), 20% sugarcane sugar, 20% glucose and 20% glucosamine, and so on. The effects on the activity of IgY showed that in the three sugars, 20% of sucrose and glucose could not improve the stability of the antibody under the conditions of pH2.0 and 3, but 20% of the glucosalfate had a protective effect on IgY.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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