基于hexon蛋白的检测禽腺病毒抗体的ELISA方法的建立

发布时间：2018-07-01 13:15

  本文选题：禽腺病毒 + hexon　； 参考：《扬州大学》2017年硕士论文

【摘要】：禽腺病毒(Fowl Adenovirus,FAdV)属于腺病毒科禽腺病毒属。基于限制性酶切图谱分析以及血清交叉中和试验,目前可将FAdV划分为5个基因型(A-E),12个血清型(血清型1-7、8a、8b、9-11)。流行病学研究发现,FAdV在世界范围内广泛分布,且不同日龄的家禽均易感。FAdV感染一般引起亚临床症状,而急性感染主要以包涵体肝炎、肝炎-心包积液综合症以及肌胃糜烂等为主。自2013年以来,国内大部分地区鸡群中流行暴发肝炎-心包积液综合症,感染鸡群的死淘率可高达80%,对国内养鸡业造成了严重的经济损失。然目前对FAdV尚无有效的疫苗和药物进行预防和治疗,更缺乏可靠的FAdV血清学检测方法。本研究从禽腺病毒保守基因hexon着手,分别构建了 GST-hexon和pET-hexon、pCold-hexon的分段原核表达载体,进行了融合蛋白的纯化,并利用纯化的融合蛋白建立了检测FAdV抗体的间接ELISA方法。1、hexon基因的分段原核表达及纯化本研究选取禽腺病毒5个基因型毒株,通过比对其hexon蛋白氨基酸序列,选择比较保守的4段表位,设计引物克隆至pGEX-6P-1原核表达载体中,阳性质粒分别命名为GST-hexon1、GST-hexon2、GST-hexon3 和 GST-hexon4。IPTG 诱导表达后,经 SDS-PAGE 分析发现,原核表达的重组蛋白主要在沉淀中以包涵体的形式存在。Western blot鉴定发现,原核表达的重组蛋白只有GST-hexon1与抗FAdV的鸡阳性血清具有良好反应性。进而将hexon1片段克隆至pET-32a和pCold Ⅰ载体中进行蛋白可溶性表达尝试。经SDS-PAGE分析发现,原核表达产物仍然在沉淀中以包涵体的形式存在。随即对包涵体进行了纯化,蛋白浓度可达0.5mg/mL。SDS-PAGE以及Wetern blot分析表明,纯化的重组蛋白具有很好的纯度及良好的抗原反应性。这一纯化的hexon1重组蛋白的获得为进一步建立检测FAdV抗体的间接ELISA方法提供了材料。2、检测FAdV抗体的间接ELISA方法的建立利用纯化的hexon1重组蛋白作为包被抗原,建立了检测FAdV抗体的间接ELISA方法。实验数据表明,抗原的最佳包被浓度为6.2μg/mL;一抗最佳稀释度为1:400,最佳孵育时间为60min;酶标二抗最佳稀释度为1:50000,最佳孵育时间为45min;显色时间为10min。该ELISA的CUT-OFF值为OD(?)0.173。特异性检测发现,建立的ELISA与新城疫病毒、禽白血病病毒、马立克氏病毒、传染性支气管炎、传染性法氏囊病病毒阳性血清以及SPF鸡血清均没有反应,OD值均低于0.1548;仅与检测的抗FAdV的阳性血清反应。临床应用检测表明,该ELISA能用于检测FAdV免疫鸡群抗FAdV抗体水平。这些研究结果表明,本研究建立的基于Hexon蛋白的检测FAdV抗体的间接ELISA方法在FAdV感染及免疫监测中具有一定的应用价值。
[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，

本文编号：2087904