本文選題：豬腸病毒 + RT-PCR��； 參考：《江西農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬腸病毒(Porcine enteroviruses,PEVs)現(xiàn)被分類為腸病毒G型(Porcine enteroviruses G,EV-G),屬于小RNA病毒科(Picornaviridae)。豬是EV-G唯一的自然宿主,且各個(gè)年齡段豬只對(duì)EV-G敏感,豬感染EV-G后大部分不會(huì)表現(xiàn)出明顯的臨床癥狀。目前為止,在北美、歐洲和亞洲等地區(qū)均有豬群檢測(cè)到EV-G型的報(bào)道,但是我國(guó)江西省尚未見相關(guān)研究報(bào)道。本試驗(yàn)針對(duì)EV-G參考序列的保守區(qū)域設(shè)計(jì)一對(duì)特異性檢測(cè)引物,建立了一種EV-G的RT-PCR快速檢測(cè)方法,并運(yùn)用此方法檢測(cè)了2015年來自江西省九江(25份)和鷹潭(18份)兩個(gè)地區(qū)共43份腹瀉仔豬的糞便樣品。檢測(cè)結(jié)果顯示EV-G總陽(yáng)性率為11.6%(5/43),其中九江某豬場(chǎng)檢測(cè)陽(yáng)性率為12.0%(3/25),鷹潭某豬場(chǎng)檢測(cè)陽(yáng)性率為11.1%(2/18)。為了研究江西省EV-G的分子流行病學(xué)和基因組遺傳變異特征,根據(jù)EV-G的參考序列保守區(qū)域設(shè)計(jì)了3對(duì)擴(kuò)增EV-G全長(zhǎng)的引物,利用設(shè)計(jì)引物成功擴(kuò)增得到一株來自九江市的野豬EV-G毒株全基因組序列。測(cè)序結(jié)果發(fā)現(xiàn)該毒株的基因組全長(zhǎng)為7,404 nt,包含一個(gè)6501nt的大開放閱讀框,編碼2167個(gè)氨基酸。通過DNAStar Lasergene 7.10軟件對(duì)測(cè)序株與20條參考毒株進(jìn)行全基因組序列和推定氨基酸序列比對(duì),結(jié)果表明測(cè)序株與8株EV-G型參考株的核苷酸序列同源性為69.8%~78.4%,氨基酸序列同源性為71.8%~77.9%,其中與EV-G2型(登錄號(hào)為AF363455.1)的序列同源性最高;基于全基因組水平及其編碼蛋白的遺傳進(jìn)化樹分析也顯示測(cè)序株與EV-G2型的遺傳距離最近。豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)是引起仔豬腹瀉的重要腸道病毒之一,發(fā)病豬只的臨床癥狀與豬傳染性胃腸炎十分類似,主要通過糞口途徑傳播。2010年末后變異株P(guān)EDV引起的腹瀉在我國(guó)大面積爆發(fā),呈現(xiàn)高發(fā)病率及高死亡率。該病至今依然流行且呈愈演愈烈的趨勢(shì),給我國(guó)養(yǎng)豬業(yè)造成了嚴(yán)重的損失。本試驗(yàn)使用PEDV變異毒株免疫海蘭褐母雞,通過酸化水提法、PEG-6000法以及酸化水提法結(jié)合硫酸銨的二次沉淀法等三種方法提取卵黃抗體,基于PEDV江西變異株全病毒之上,建立了一套快速準(zhǔn)確的間接ELISA方法,用以測(cè)定卵黃抗體(Egg Yolk Antibody,IgY)效價(jià),并評(píng)估IgY的耐熱性、耐酸堿性以及模擬胃酸條件下三種不同保護(hù)劑的保護(hù)效果等。結(jié)果表明酸化水提法純化的蛋白濃度較高,但提取的特異性IgY較少;PEG-6000法提取的IgY純度較高,但蛋白濃度較酸化水提法要低;酸化水提法結(jié)合硫酸銨的二次沉淀法提取的IgY純度最高,但是獲得的蛋白濃度較低。試驗(yàn)結(jié)果還表明抗PEDV變異毒株IgY在高溫下(70℃)和pH4和pH11的條件下易發(fā)生變性,在37~70℃和pH 4~11的范圍內(nèi)可保持穩(wěn)定。此外,本試驗(yàn)還測(cè)試了IgY在模擬胃酸環(huán)境下(pH 2.0和3.0),添加20%蔗糖、20%葡萄糖和20%硫糖鋁等三種糖類保護(hù)劑后,對(duì)IgY活性情況的影響。結(jié)果顯示在三種糖類中,20%的蔗糖與葡萄糖在pH2.0和3.0的條件下不能提高抗體的穩(wěn)定性,但是20%的硫糖鋁對(duì)IgY有一定的保護(hù)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.
【學(xué)位授予單位】：江西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.651

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