1型鸭甲肝病毒和鸭坦布苏病毒液相芯片抗体检测方法的建立

发布时间：2018-04-22 13:30

本文选题：1型鸭甲肝病毒 + 鸭坦布苏病毒　；参考：《山东农业大学》2017年硕士论文

【摘要】：鸭病毒性肝炎(Duck virus hepatitis,DVH)是危害雏鸭的一种急性、高致死性传染病,主要侵害3周龄以内雏鸭,已在国内广泛流行,严重威胁养鸭业的健康发展。鸭坦布苏病毒病(Duck tembusu disease)是一种传播迅速的急性传染病,可使肉鸭表现神经症状、产蛋鸭产蛋量下降等。自2010年在我国爆发以来,影响范围广,已造成巨大经济损失。目前,鸭甲肝病毒(Duck hepatitis A virus,DHAV)和鸭坦布苏病毒(D uck tembusu virus,DTMUV)的抗体检测主要依赖中和试验和酶联免疫吸附试验,批量检测样品时费时费力,因此临床急需一种高效、快速的检测方法对多种病原抗体同时进行检测。VP1基因和E基因分别是鸭甲肝病毒和鸭坦布苏病毒的主要抗原基因。本试验将1型鸭甲肝病毒的VP1基因和鸭坦布苏病毒的E基因分别在BL21(DE3)大肠杆菌中表达,用纯化的重组蛋白为抗原建立1型鸭甲肝病毒与鸭坦布苏病毒的液相芯片抗体检测方法,为DHA-1和DTMU的快速血清学诊断奠定基础。本研究主要包括以下三部分内容:1.鸭甲肝病毒的分离鉴定及VP1基因序列列分析分析收集2016年山东、河南等地区发病鸭场中疑似鸭甲肝病鸭的肝脏组织,共8份,经研磨冻融离心处理后接种鸭胚,中和试验进一步确诊。结果有2株分离毒能被1型鸭甲肝病毒阳性血清中和,有6株分离毒能被3型鸭甲肝病毒阳性血清中和,初步确诊分离毒株中有2株为1型鸭甲肝病毒(DHAV-1),6株为3型鸭甲肝病毒(DHAV-3)。利用设计的特异性引物对1型鸭甲肝病毒和3型鸭甲肝病毒分别进行RT-PCR扩增,将纯化的1型VP1基因和3型VP1基因分别克隆至pMD18-T载体中,构建pMD18-T-1VP1和pMD18-T-3VP1基因克隆重组质粒并测序,利用MegAlign软件对VP1蛋白的氨基酸同源关系进行比对,结果显示:2016年分离的1型鸭甲肝病毒毒株1-JS DH/16株和1-HZYC/16株与经典毒株1-R85952/55的同源性分别是93.8%和93.4%,与其他分离毒株的同源性为最低92%~最高94.5%;3型鸭甲肝病毒毒株3-GD/16株、3-HNSQ/16株、3-SDGT/16株、3-SDXD/16、3-SC1/16株和3-SC2/16株与经典毒株AP04114/03株的同源性分别为92.1%、92.5%、92.4%、93.5、92.4和92.4%,与其他分离株的同源性为最低90.1%~最高99.7%;利用MEGA 4.0生物学软件进行遗传进化分析,绘制进化树,可见:2016年分离毒株1-JSDH/16株和1-HZYC/16株处于同一个分支,与近几年的流行株1-CS/14株的亲缘关系最近,与经典毒株1-R85952/55株亲缘关系相对较远;3-GD/16株与3-SC1/16株、3-SC2/16株三者亲缘关系最近,处于同一个小分支上,3-SDGT/16株与3-HNSQ/16株的亲缘关系近,处于同一分支上,3-SDXD/16株单独处于一个分支,上述分离株与经典毒株3-AP04023/04株的亲缘关系较远。2.pCold-1VP1和pET-28a-E的原核表达将纯化的1VP1基因克隆至pCold-III原核表达载体中,筛选获得含1VP1基因正向插入的、有正确读码框的重组质粒p Cold-1VP1。用Nde I/Hind III进行双酶切鉴定,经琼脂糖凝胶电泳检测出两条条带,分别在4,377bp和714bp左右,与预期大小相符。提取鸭坦布苏病毒(DTMUV)的RNA,用特异性引物进行RT-PCR扩增,得到1,503bp的E基因,将纯化后的E基因克隆至pMD18-T载体中,构建pMD18-T-E重组质粒并保存,然后将其插入pET-28a(+)原核表达载体,筛选阳性重组质粒p ET-28a-E。用EcoR I/Xho I进行双酶切鉴定,经琼脂糖凝胶电泳检测出两条条带,分别在5,285bp和1,503bp左右,与预期大小相符。对获得重组表达载体p Cold-1VP1和pET-28a-E菌液,进行IPTG诱导表达,对表达的重组蛋白进行SDS-PAGE和Western blot分析(一抗分别为1型鸭甲肝病毒阳性血清和鸭坦布苏病毒阳性血清)。结果发现,DHAV-1VP1和DTMUV-E蛋白在大肠杆菌BL21(DE3)中可稳定、高效地表达。将表达的重组蛋白纯化后均得到单一的蛋白条带,分别为26.5kDa和54.8k Da,Western blot检测表明重组蛋白具有良好的免疫原性。3.DHAV-1和DTMUV液相芯片抗体检测方法的建立根据液相蛋白芯片技术原理,以表达的重组蛋白为抗原,分别与不同编号的微球偶联,获得偶联复合体作为捕获载体,建立了分别检测鸭血清中DHAV-1、DTMUV抗体的单一液相芯片检测方法,以及可以同时检测两种抗体的双重液相芯片检测方法,结果表明,建立的液相芯片检测方法特异性、灵敏性和可重复性良好,为DHAV-1和DTMUV抗体监测提供了高通量、重复性好、特异性高的抗体检测体系,有利于鸭甲肝病毒病和鸭坦布苏病毒病的快速检测,给生产生活带来改善,降低经济损失。
[Abstract]:Duck virus hepatitis (DVH) is a kind of acute, high fatal infectious disease which is harmful to ducklings. It mainly infringes ducklings within 3 weeks of age. It is widely prevalent in China. It is a serious threat to the healthy development of duck industry. The duck tanbsu virus (Duck Tembusu disease) is a rapid and acute infectious disease, which can make the duck show nerve. Symptoms, the drop of egg production of laying ducks, and so on. Since the outbreak of China in 2010, it has a wide range of influence and has caused great economic loss. At present, the antibody detection of duck hepatitis A virus (Duck hepatitis A virus, DHAV) and duck tanburu virus (D uck Tembusu virus, DTMUV) mainly depends on the test of Lai neutralization and enzyme linked immunosorbent assay to detect samples in batch. It is time-consuming and laborious, so it is urgent to detect the.VP1 gene and E gene, the main antigen gene of duck hepatitis A virus and duck tanb virus, respectively. The VP1 gene of duck hepatitis A virus type 1 and the E gene of duck Tanbu Su virus are in BL21 (DE3) Escherichia coli, respectively. Expression, using the purified recombinant protein as antigen to establish a liquid chip antibody detection method for duck hepatitis A virus type 1 and duck Tanbu Su virus, which lays the foundation for rapid serological diagnosis of DHA-1 and DTMU. This study mainly includes the following three parts: isolation and identification of 1. duck hepatitis A virus and sequence analysis and analysis of VP1 gene in Shandong in 2016 The liver tissues of ducks suspected to be duck hepatitis a disease in Henan and other regions of Henan, were inoculated with duck embryos by lapping and thawing centrifugation, and the neutralization test was further confirmed. The results showed that 2 isolates could be neutralized by the positive sera of type 1 duck hepatitis A virus and 6 isolates could be neutralized by the positive sera of type 3 duck hepatitis A virus (HCV), and the isolated strains were preliminarily confirmed. 2 of them were type 1 duck hepatitis A virus (DHAV-1) and 3 type of duck hepatitis A virus (DHAV-3). The specific primers were designed to amplify RT-PCR of duck hepatitis A virus type 1 and type 3 duck hepatitis A virus, respectively. The purified VP1 gene and the 3 VP1 gene were cloned into the pMD18-T carrier, and the cloning and recombination of pMD18-T-1VP1 and pMD18-T-3VP1 genes were constructed. Plasmid and sequencing, using MegAlign software to compare the amino acid homology of VP1 protein, the results showed that the homology of 1-JS DH/16 strain and 1-HZYC/16 strain of the 1 type duck hepatitis A virus strain of 2016 was 93.8% and 93.4% respectively, and the homology of the other isolates was the lowest 94.5% of the lowest 92%~, 3 type of duck liver disease. The homology of virus strain 3-GD/16 strain, 3-HNSQ/16 strain, 3-SDGT/16 strain, 3-SDXD/16,3-SC1/16 strain and 3-SC2/16 strain and the classical strain AP04114/03 strain were 92.1%, 92.5%, 92.4%, 93.5,92.4 and 92.4% respectively. The homology of the other isolates was the lowest 99.7% of the lowest 90.1%~, and the genetic evolution analysis was carried out using the MEGA 4 biological software to draw the evolutionary tree. See: the isolated strain 1-JSDH/16 and 1-HZYC/16 strains were in the same branch in 2016, closely related to the 1-CS/14 strain of the epidemic strain of recent years, and relative to the classic strain 1-R85952/55 strain. The relationship between 3-GD/16 strain and 3-SC1/16 strain and 3-SC2/16 strain three was recently on the same small branch, 3-SDGT/16 strain and 3-HNSQ/16 strain. The relationship is close to the same branch, and the 3-SDXD/16 strain is in a single branch. The relationship between the above isolated strain and the classic strain 3-AP04023/04 strain is far from.2.pCold-1VP1 and pET-28a-E. The purified 1VP1 gene is cloned into the pCold-III prokaryotic expression vector, and the positive insertion of the 1VP1 gene is screened for the correct reading of the 1VP1 gene. The recombinant plasmid P Cold-1VP1. of the code frame was identified by double enzyme cutting with Nde I/Hind III, and two bands were detected by agarose gel electrophoresis, which were in 4377bp and 714bp, respectively, in accordance with the expected size. The RNA of duck tanbsu virus (DTMUV) was extracted. The 1503bp E gene was obtained by specific primers and 1503bp E genes were obtained. The purified gene was cloned. To pMD18-T vector, pMD18-T-E recombinant plasmid was constructed and preserved, and then inserted into pET-28a (+) prokaryotic expression vector, the positive recombinant plasmid P ET-28a-E. was screened by EcoR I/Xho I for double enzyme digestion, and two bands were detected by agarose gel electrophoresis, which were in 5285bp and 1503bp, respectively. The recombinant expression was obtained. The vector p Cold-1VP1 and pET-28a-E bacteria were induced to induce the expression of IPTG, and the expression of recombinant protein was analyzed by SDS-PAGE and Western blot (one anti 1 duck hepatitis A virus positive serum and duck tanbsu virus positive serum respectively). The results showed that DHAV-1VP1 and DTMUV-E protein could be expressed steadily and efficiently in the BL21 (DE3) of Enterobacteriaceae. The recombinant protein was purified with a single protein band, 26.5kDa and 54.8k Da, respectively, and Western blot detection showed that the recombinant protein has a good immunogenic.3.DHAV-1 and DTMUV liquid chip antibody detection method based on the principle of liquid protein chip technology, with the recombinant protein as the antigen, respectively, and different numbered. By coupling the microspheres and obtaining the coupling complex as the capture carrier, a single liquid phase chip detection method for detecting DHAV-1 and DTMUV antibodies in duck serum, and a dual liquid phase chip detection method for detecting two kinds of antibodies at the same time were established. The results showed that the detection method of liquid phase core was specific, sensitive and reproducible. It provides a high throughput, reproducible and specific antibody detection system for DHAV-1 and DTMUV antibody monitoring, which is beneficial to the rapid detection of duck hepatitis A virus disease and duck Tanbu Su virus disease, to the improvement of production and life, and to reduce economic loss.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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