【作者】 刘震；

【导师】 洪健； 吴建祥；

【作者基本信息】 浙江大学 ， 植物保护， 2016， 硕士

【摘要】 每年柑橘病毒病害都会对世界柑橘生产造成严重的经济损失。由柑橘衰退病毒(Citrus tristeza virus, CTV)引起的柑橘衰退病和由柑橘黄化脉明病毒(Citrus yellow vein clearing virus, CYVCV)引起的柑橘黄化脉明病是目前危害柑橘的两种重要病毒病害。为了掌握这两种病毒病在我国的发生流行规律,进而建立科学防控体系,急需建立对两种病毒的快速、有效的检测方法。而血清学检测方法操作简单、特异性好、灵敏度高,且适合大规模样品检测,因此被广泛应用于植物病毒的诊断、流行规律分析、抗病育种和科学防控等方面。为此,本论文分别制备了CTV和CYVCV的特异、灵敏的单克隆抗体,并以其为核心分别建立了检测这两种病毒的特异、灵敏的血清学方法,从而为这两种病毒病的诊断、流行病学分析和科学防控体系建立提供物质和技术支撑。(1)CTV单抗的制备及其检测应用：CTV重庆分离物的外壳蛋白基因(CP)用原核表达载体pET-28a在大肠杆菌进行表达,表达的重组蛋白通过Ni2+-NTA胶纯化后作为抗原免疫BALB/c小鼠,经细胞融合、筛选和克隆获得4株分泌抗CTV单抗的杂交瘤细胞株系(14B10、14H11、20D5和20G12)。间接ELISA方法测得4株单抗腹水的效价在10-6-10-7之间,Western blot分析显示,4株单抗均能与CTV的CP发生特异性的免疫反应。以单抗为核心建立检测CTV的dot-ELISA、Tissue blot-ELISA、TAS-ELISA三种检测CTV的血清学方法,其中dot-ELISA和TAS-ELISA可以检测感染CTV病叶粗提液的灵敏度分别达到1：2560和1：10240倍稀释(w/v, g/ml)。此外,利用制备的单抗研制出了检测CTV的胶体金免疫试纸条,其检测病叶的灵敏度达到1：1280倍稀释,从而实现CTV检测的田头化、快速化、傻瓜化。TAS-ELISA检测灵敏度最高,可以很好地应用于脱毒树苗的检测。田间调查显示,CTV在我国重庆市、江西省和浙江省柑橘产区发生普遍,且血清学方法的检测结果与RT-PCR检测结果的符合率达到99.5%以上。CTV单抗的制备及其灵敏、特异的血清学检测方法的建立对我国CTV的检测和防控具有重大作用。(2) CYVCV单抗的制备及其检测应用：为了诊断、流行病学分析和控制CYVCV病害,本论文以原核表达的CYVCV外壳蛋白作为抗原免疫小鼠和兔子,分别制备了4株抗CYVCV的特异、灵敏的单抗和兔多抗。4株单抗腹水的间接ELISA效价在10-6-10-7之间,单抗均能特异性地与CYVCV CP蛋白和CYVCV感染的柑橘叶片粗提液反应。以单抗为核心建立检测CYVCV的dot-ELISA、Tissue blot-ELISA、DAS-ELISA、TAS-ELISA四种血清学方法,其中dot-ELISA、DAS-ELISA和TAS-ELISA检测CYVCV病叶的灵敏度分别达到1：2560、1：10240和1：20480倍稀释(w/v, g/ml)。用建立的血清学方法分析了来自我国重庆市、云南省果园125份疑似病毒病样品,检测结果发现,其中46个样品感染CYVCV,发病率达到36.8%,然而在浙江和江西省158个样品中未检测到该病毒,且血清学方法的检测结果与RT-PCR检测结果的符合率达到99.6%以上。这四种血清学方法对我国CYVCV的诊断和检测、流行病学调查、脱毒苗的生产和防控具有重要意义。更多还原

【Abstract】 Every year, citrus virus diseases cause serious economic loss in citrus industry worldwide. Now, Citrus tristeza disease caused by Citrus tristeza virus (CTV) and Citrus yellow vein clearing disease caused by Citrus yellow vein clearing virus (CYVCV) are two important virus diseases of citrus trees. In order to understand the epidemic laws and establish the scientific prevention and control system of the two citrus virus diseases, rapid and efficient detection techniques should be urgently developed. Serological assays for plant virus detection are simple, rapid, sensitive, specific and suitable to detect large-scale field samples. So it has been widely used in plant virus disease diagnosis, epidemic rule analyses, resistive breeding, scientific prevention and control and so on. Therefore, specific and sensitive monoclonal antibodies (MAbs) against CTV and CYVCV were respectively produced, and specific and sensitive serological assays based on the prepared MAbs were developed for rapid and reliable virus detection in this thesis. The result of this study has provided technology and materiel for diagnoses, epidemiological analyses and establishments of scientific prevention and control systems of the two virus diseases.(1) Preparation and application of MAbs against CTV:The major coat protein gene(CP) of CTV Chongqing isolate was expressed in Escherichia coli BL21 (DE3) using the expression vector pET-28a. The recombinant protein was purified through Ni2+-NTA affinity column and used to immunize BALB/c mice. Four hybridoma cell lines (14B10,14H11,20D5 and 20G12) secreting monoclonal antibodies (MAbs) against CTV were obtained though cell fusion、cell screening and cell cloning. The titers of ascitic fluids of MAbs secreted by four hybridomas ranged from 10-6 to 10-7 by indirect-ELISA. Western blot analysis indicated that all four MAbs could specifically react with CTV CP. Using MAbs, dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot enzyme-linked immunosorbent assay (Tissue blot-ELISA) and triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) were developed to detect CTV in citrus trees. The developed dot-ELISA and TAS-ELISA methods could detect virus in CTV-infected citrus leaf crude extracts diluted 1:2560 and 1:10240 (w/v, g/mL), respectively. Furthermore, the immune colloidal gold strip was developed to detect CTV in citrus trees using the prepared two MAbs, and could detect virus in CTV-infected leaf crude extracts diluted 1:1280 (w/v, g/ml). The establishment of the immune colloidal gold strip has realized the on-site, fast, fool detection of CTV. The sensitivity of TAS-ELISA is so high that can easy determine virus-free seedling. The field survey revealed that CTV was prevalent in citrus trees in Chongqing municipality, Jiangxi and Zhejiang provinces of China, and the coincidence rate of serological and RT-PCR test results reached more than 99.5%. The prepared MAbs against CTV and established sensitive and specific serological assays have a significant role in the detection and prevention and control of CTV in our country.(2) Preparation and application of MAbs against CYVCV:To detect, analyze epidemiology and control CYVCV, capsid protein (CP) of CYVCV were prepared using a prokaryotic expression system and used as the immunogen to immunitze mice or rabbits, and four highly specific and sensitive murine MAbs and one polyclonal antibody (PAb) were produced in this thesis. Titers of the four MAbs in ascites fluids ranged 10-6 to 10-7 by an indirect-ELISA. All four prepared MAbs could strongly and specifically react with CYVCV CP and CYVCV-infected citrus leaf tissue crude extracts. Based on prepared antibodies, four serological assays, dot-ELISA, tissue blot-ELISA, DAS-ELISA and TAS-ELISA were developed. Sensitivities of the developed dot-ELISA, DAS-ELISA and TAS-ELISA for CYVCV-infected citrus leaf tissues had reached 1:2560,1:10240 and 1:20480 (w/v, g/ml), respectively. Using the developed serological assays,125 suspected virus disease citrus samples from groves in Yunnan Province and Chongqing Municipality were detected. The detection results demonstrated that 46 of 125 samples were infected by CYVCV with a 36.8% incidence rate, while none of 158 samples from groves in Zhejiang or Jiangxi provinces had contained CYVCV. The coincidence rate of serological and RT-PCR test results reached more than 99.6%. These four developed detection assays have great signaficance in CYVCV diagnosis and detection, epidemiological studies and production of virus-free citrus seedlings.更多还原