發(fā)布時(shí)間：2018-07-01 13:15

  本文選題：禽腺病毒 + hexon�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：禽腺病毒(Fowl Adenovirus,FAdV)屬于腺病毒科禽腺病毒屬�；谙拗菩悦盖袌D譜分析以及血清交叉中和試驗(yàn),目前可將FAdV劃分為5個(gè)基因型(A-E),12個(gè)血清型(血清型1-7、8a、8b、9-11)。流行病學(xué)研究發(fā)現(xiàn),FAdV在世界范圍內(nèi)廣泛分布,且不同日齡的家禽均易感。FAdV感染一般引起亞臨床癥狀,而急性感染主要以包涵體肝炎、肝炎-心包積液綜合癥以及肌胃糜爛等為主。自2013年以來(lái),國(guó)內(nèi)大部分地區(qū)雞群中流行暴發(fā)肝炎-心包積液綜合癥,感染雞群的死淘率可高達(dá)80%,對(duì)國(guó)內(nèi)養(yǎng)雞業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失。然目前對(duì)FAdV尚無(wú)有效的疫苗和藥物進(jìn)行預(yù)防和治療,更缺乏可靠的FAdV血清學(xué)檢測(cè)方法。本研究從禽腺病毒保守基因hexon著手,分別構(gòu)建了 GST-hexon和pET-hexon、pCold-hexon的分段原核表達(dá)載體,進(jìn)行了融合蛋白的純化,并利用純化的融合蛋白建立了檢測(cè)FAdV抗體的間接ELISA方法。1、hexon基因的分段原核表達(dá)及純化本研究選取禽腺病毒5個(gè)基因型毒株,通過(guò)比對(duì)其hexon蛋白氨基酸序列,選擇比較保守的4段表位,設(shè)計(jì)引物克隆至pGEX-6P-1原核表達(dá)載體中,陽(yáng)性質(zhì)粒分別命名為GST-hexon1、GST-hexon2、GST-hexon3 和 GST-hexon4。IPTG 誘導(dǎo)表達(dá)后,經(jīng) SDS-PAGE 分析發(fā)現(xiàn),原核表達(dá)的重組蛋白主要在沉淀中以包涵體的形式存在。Western blot鑒定發(fā)現(xiàn),原核表達(dá)的重組蛋白只有GST-hexon1與抗FAdV的雞陽(yáng)性血清具有良好反應(yīng)性。進(jìn)而將hexon1片段克隆至pET-32a和pCold Ⅰ載體中進(jìn)行蛋白可溶性表達(dá)嘗試。經(jīng)SDS-PAGE分析發(fā)現(xiàn),原核表達(dá)產(chǎn)物仍然在沉淀中以包涵體的形式存在。隨即對(duì)包涵體進(jìn)行了純化,蛋白濃度可達(dá)0.5mg/mL。SDS-PAGE以及Wetern blot分析表明,純化的重組蛋白具有很好的純度及良好的抗原反應(yīng)性。這一純化的hexon1重組蛋白的獲得為進(jìn)一步建立檢測(cè)FAdV抗體的間接ELISA方法提供了材料。2、檢測(cè)FAdV抗體的間接ELISA方法的建立利用純化的hexon1重組蛋白作為包被抗原,建立了檢測(cè)FAdV抗體的間接ELISA方法。實(shí)驗(yàn)數(shù)據(jù)表明,抗原的最佳包被濃度為6.2μg/mL;一抗最佳稀釋度為1:400,最佳孵育時(shí)間為60min;酶標(biāo)二抗最佳稀釋度為1:50000,最佳孵育時(shí)間為45min;顯色時(shí)間為10min。該ELISA的CUT-OFF值為OD(?)0.173。特異性檢測(cè)發(fā)現(xiàn),建立的ELISA與新城疫病毒、禽白血病病毒、馬立克氏病毒、傳染性支氣管炎、傳染性法氏囊病病毒陽(yáng)性血清以及SPF雞血清均沒(méi)有反應(yīng),OD值均低于0.1548;僅與檢測(cè)的抗FAdV的陽(yáng)性血清反應(yīng)。臨床應(yīng)用檢測(cè)表明,該ELISA能用于檢測(cè)FAdV免疫雞群抗FAdV抗體水平。這些研究結(jié)果表明,本研究建立的基于Hexon蛋白的檢測(cè)FAdV抗體的間接ELISA方法在FAdV感染及免疫監(jiān)測(cè)中具有一定的應(yīng)用價(jià)值。
[Abstract]:Avian adenovirus (Fowl AdenovirusFAdV) belongs to the genus Avian adenovirus. FAdV can be divided into 5 genotypes (A-E) and 12 serotypes (serotypes 1-7 ~ (8) A ~ (8) b ~ (9) based on restriction enzyme restriction map analysis and serum cross-neutralization test. Epidemiological studies showed that FAdV was widely distributed in the world, and all fowls of different ages were susceptible. FAdV infection generally caused subclinical symptoms, while acute infection was mainly in inclusion body hepatitis. Hepatitis-pericardial effusion syndrome and gastric erosion. Since 2013, the prevalence of hepatitis-pericardial effusion syndrome in chickens in most areas of China has caused serious economic losses to domestic chicken industry. The death rate of infected chickens can be as high as 80%. However, there are no effective vaccines and drugs for prevention and treatment of FAdV, and there is no reliable method for FAdV serological detection. In this study, the fragment prokaryotic expression vectors of GST-hexon and pET-hexonpCold-hexon were constructed from the conserved gene hexon of avian adenovirus, and the fusion protein was purified. Using the purified fusion protein, an indirect Elisa method for detection of FAdV antibody was established. The prokaryotic expression of the FAdV gene was carried out. In this study, five genotypes of avian adenovirus were selected, and the amino acid sequences of the hexon protein were compared. Four conservative epitopes were selected and cloned into prokaryotic expression vector pGEX-6P-1. The positive plasmids were named GST-hexon1, GST-hexon2, GST-hexon3 and GST-hexon4.IPTG, respectively. Western blot analysis showed that only GST-hexon1 could react well with anti-FAdV positive serum. Then the hexon1 fragment was cloned into pET-32a and pCold 鈪，

本文編號(hào)：2087904