猪伪狂犬病野毒感染与疫苗免疫鉴别诊断方法的建立及初步临床应用

发布时间：2018-02-26 06:25

本文关键词： PRV新流行毒株血清流行病学调查和分子进化分析鉴别诊断ELISA 免疫抗体水平评价ELISA 病毒抗原检测的双抗夹心 ELISA和胶体金试纸　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：伪狂犬病（PR）是多种动物共患的传染病；其病原体伪狂犬病病毒（PRV）是一种猪的疱疹病毒，分类学命名为猪疱疹病毒I型（Suid herpesvirus1）。猪是PRV的传染源和自然贮存宿主，感染后表现为发热、仔猪神经症状和高死亡率、大中猪出现呼吸道症状等临床特征。消化道、呼吸道、精液、胎盘及空气等是PRV的主要传播途径。PRV对于口鼻部呼吸道的上皮细胞具有非常强的嗜性；其基因组是大小约为150kb的线性双链DNA，编码70-100余种蛋白。 2011年底，在河北、河南、山东、山西等多个省份许多使用Bartha-K61疫苗免疫猪场爆发了疑似PR的疫病，发病猪出现高温(40-42°C）、食欲不振、咳嗽、腹泻并伴随有系统性神经症状，新生仔猪死亡率高；病毒分离等实验证实引起这场疫病爆发的源头是变异的PRV病毒，这对PR的防控提出了严峻挑战。因此本研究对河南省及周边部分省份PRV的流行进行了血清学调查和分子进化分析；表达了PRV新流行毒株的gE蛋白和gB蛋白并制备了单抗，以期建立的PRV抗原抗体ELISA检测方法和制备的胶体金快速检测试纸能在PRV鉴别诊断和免疫抗体水平评价等方面发挥作用。血清流行病学分析结果表明河南省PRV gE抗体阳性率在2012年升高至38.0%，2013年仍居高不下（31.3%）。种猪群阳性率为36.3%，育肥后期（19-25周龄）阳性率高达59.3%，后备种猪阳性率为23.8%。在2013年检测的347个猪场中，猪场阳性率为83.6%，其中豫北猪场阳性率最高为93.8%。这些数据表明PR在河南省呈严重流行态势。基于分离的12株PRV新流行株的gE、gB和gC基因开展的分子进化分析显示新流行株处于独立的进化分支，，与国内早期PRV分离株和其它新流行株进化关系较近，与Bartha疫苗株进化关系较远。为建立针对PRV新流行毒株抗体检测的ELISA方法，我们表达了PRV新流行株（汤阴株）的gE蛋白和gB蛋白。其中gE蛋白为可溶表达，gB蛋白为包涵体表达；利用Ni-NTA亲和层析和离子交换纯化的gE蛋白和gB蛋白分别建立了gE抗体检测的间接ELISA（gE-ELISA）和gB抗体检测的间接ELISA（gB-ELISA）方法。gE-ELISA与商品化试剂盒gpI-ELISA（IDEXX）的符合率为88.76%；特异性（DSP）为79.15%；敏感性（DSN）为94.03%。gB-ELISA与商品化试剂盒gB-ELISA（IDEXX）的符合率为93.14%；DSP为77.78%；DSN为93.64%。在检测的66个阳性场中，阳性活跃场占95.5%，阳性稳定场占4.5%。在PRV gE抗体阴性样品中，gB抗体阳性率占到了94.9%。制备了针对gE蛋白和gB蛋白上具有免疫优势的表位的单抗并建立了单抗阻断ELISA检测方法。gE单抗阻断ELISA（blocking gE-ELISA）与商品化试剂盒gpI-ELISA的符合率为94.10%；DSP为89.90%；DSN为96.18%。gB单抗阻断ELISA（blocking gB-ELISA）与商品化试剂盒gB-ELISA的符合率为94.07%；DSP为85.37%；DSN为95.90%；两种单抗阻断ELISA较间接ELISA在符合率、特异性和敏感性三方面性能均有所提高。在检测的72个阳性场中，阳性活跃场占95.8%，阳性稳定场占4.2%。在PRV gE抗体阴性样品中，gB抗体阳性率占到了98.0%，与间接ELISA结果相符合；说明各个猪场采用的常规疫苗免疫诱导产生了gB抗体，并能够在一定程度上预防PRV的感染或流行。同时我们利用gE蛋白或gB蛋白高免猪血清IgG及特异性单抗酶标物，以NP-40处理的PRV病毒抗原为检测靶标建立了双抗体夹心ELISA并制备了胶体金快速检测试纸。以gE高免猪血清IgG建立的夹心ELISA和试纸均只与PRV野毒抗原反应，不与gE缺失疫苗株和正常BHK-21细胞对照反应；可检测到的病毒样品的最低检测限约为105.0TCID50/0.1mL左右。以gB高免IgG建立的夹心ELISA和试纸可同时与PRV野毒抗原和gE缺失的疫苗株反应，不和正常BHK-21细胞对照反应。本研究中的血清和分子流行病学调查将为河南省PRV的流行和进化关系提供参考；建立的间接和单抗阻断ELISA可用于区分疫苗免疫抗体与野毒感染抗体，并为PRV疫苗免疫抗体水平评价提供手段；建立的检测PRV病毒抗原的双抗体夹心ELISA和制备的胶体金快速检测试纸能够直观简便地进行PRV野毒感染的检测和疫苗质量评估。因此，本研究为我国PR的净化提供了抗原、抗体快速检测的方法。
[Abstract]:Pseudorabies virus (PR) is a variety of animal zoonotic infectious disease; the pathogen of pseudorabies virus (PRV) is a swine herpesvirus taxonomy named pig herpes simplex virus type I (Suid herpesvirus1). The pig is PRV source of infection and the natural reservoir, after infection characterized by fever, neurological symptoms and high mortality in piglets the clinical features of respiratory symptoms, and other large and medium-sized pig. The digestive tract, respiratory tract, semen, placenta and air is.PRV PRV main transmission way has a very strong tropism for the nose and mouth of respiratory epithelial cells; its genome is about the size of 150kb linear double stranded DNA, encoding 70-100 kinds of protein.
By the end of 2011, in Hebei, Henan, Shandong, Shanxi and other provinces to use many Bartha-K61 vaccine of swine outbreak of suspected PR disease, swine high temperature (40-42 degrees C), loss of appetite, cough, diarrhea and accompanied by systemic neurological symptoms, high rate of death of newborn piglets; virus isolation experiments confirmed by the source the outbreak is a variant of the PRV virus, which has posed a severe challenge to prevent and control PR. This study analyzed the serological investigation and molecular evolution of Henan province and surrounding provinces part of the popularity of PRV; expression of PRV monoclonal antibody preparation of new strains of gE protein and gB protein and preparation, in order to establish the PRV ELISA antigen antibody detection method and preparation of colloidal gold strip for rapid detection can play a role in the differential diagnosis of PRV and antibody level of evaluation.
Serum epidemiological analysis results show that the PRV of Henan province gE antibody positive rate in 2012 increased to 38%, 2013 is still high (31.3%). Among the positive rate was 36.3%, the latter fattening period (19-25 weeks) positive rate was as high as 59.3%, the positive rate for 23.8%. reserve pigs on 347 farms in 2013 detection, positive rate pig is 83.6%, the positive rate of 93.8%. was the highest in swine these data indicate that PR is a serious epidemic situation in Henan province. 12 strains of gE isolated PRV strains based on the molecular evolution of gB gene and gC gene in the analysis show the new strains in the evolution of branch independence, and domestic PRV isolates and other early the new epidemic strain evolution relationship is near, far from the relationship between Bartha vaccine strains evolution.
In order to establish the ELISA method for detection of PRV new strains of antibodies, we expressed PRV new strains (Tang Yinzhu) of the gE protein and gB protein. The expression of gE protein was soluble, gB protein expressed as inclusion bodies; indirect ELISA gE antibody detection was established using Ni-NTA affinity chromatography and ion exchange of gE protein and gB protein white purified respectively (gE-ELISA) and gB antibody detection by indirect ELISA (gB-ELISA).GE-ELISA method and commercial gpI-ELISA Kit (IDEXX) and the coincidence rate was 88.76%; the specificity was 79.15% (DSP); sensitivity (DSN) for 94.03%.gB-ELISA with a commercial gB-ELISA Kit (IDEXX) with the rate of 93.14%; DSP 77.78%; DSN is 93.64%. in the detection of 66 positive field, positive active field accounted for 95.5%, accounted for 4.5%. of PRV in the stability field of positive gE antibody negative samples, the positive rate of gB antibody accounted for 94.9%.
The preparation of the gE protein and gB protein with immunodominant epitope McAbs and established the monoclonal antibody blocking ELISA detection method of.GE monoclonal antibody blocking ELISA (blocking gE-ELISA) with a commercial gpI-ELISA kit with the rate of 94.10%; DSP 89.90%; DSN 96.18%.gB ELISA (blocking gB-ELISA) monoclonal antibody blocking and commercial reagent box of gB-ELISA with the rate of 94.07%; DSP 85.37%; DSN 95.90%; two kinds of monoclonal antibody blocking ELISA than indirect ELISA in coincidence rate, sensitivity and specificity of the three aspects of performance are improved. In the detection of 72 positive field, positive active field accounted for 95.8%, accounted for 4.2%. of PRV in the stability field of positive gE antibody negative in the sample, gB antibody positive rate accounted for 98%, consistent with the results of indirect ELISA; the conventional vaccine induced by pig each gB antibody, and to prevent PRV in the extent of infection or Popular.
At the same time, we use the gE protein or gB protein from porcine serum IgG and specific monoclonal antibody ELISA, the double antibody sandwich ELISA and preparation of colloidal gold strip for rapid detection of PRV virus antigen based on NP-40 processing for target detection. With gE high immune serum IgG to establish sandwich ELISA and test paper are only with PRV wild virus antigen, and the deletion of gE vaccine strain and normal BHK-21 cells in the control reaction; the minimum detection limit of the virus can be detected in the sample is about 105.0TCID50/0.1mL. The sandwich ELISA and test gB high free IgG can be established at the same time with PRV wild virus antigen and gE deletion vaccine strain response, with normal BHK-21 cells the control reaction.
Serum and molecular epidemiological survey in this study will provide a reference for the epidemic and evolutionary relationship of PRV in Henan province; the establishment of indirect and blocking ELISA can be used to distinguish between antibody vaccine and wild virus infection and PRV antibody, vaccine antibody levels provide a means of evaluation; quality inspection and evaluation of PRV vaccine virus antigen set the double antibody sandwich ELISA and preparation of colloidal gold strip for rapid detection of PRV can directly and simply wild virus infection. Therefore, this study provides for our PR antigen purification method, rapid detection of antibodies.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

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