基于单克隆抗体的南方菜豆花叶病毒血清学检测技术

为建立南方菜豆花叶病毒(southern bean mosaic virus,SBMV)的快速、简便、高通量检测技术,加强该病毒的口岸检验检疫,以提纯的SBMV粒子为免疫原免疫BALB/C小鼠,利用杂交瘤技术获得3株杂交瘤细胞株19C3、19H9和20G4,其分泌的SBMV腹水单抗效价均达到10-7,且3个单抗与感染SBMV大豆叶片组织粗提液有强烈的特异性免疫反应,而不与感染南方豇豆花叶病毒(southern cowpea mosaic virus,SCPMV)的豇豆、健康的大豆、毛豆、豌豆、蚕豆和菜豆叶片组织粗提液发生免疫反应。以制备的单抗为核心,建立了检测植物中SBMV的ACP-ELISA和dotELISA两种血清学方法。3个单抗中19H9单抗的检测灵敏度最高,以其建立的ACP-ELISA和dot-ELISA方法检测大豆病叶粗提液的灵敏度分别达到1∶163 840和1∶10 240稀释浓度。利用建立的dot-ELISA方法可从上海口岸截获的大豆种子中检测出SBMV,且该检测结果得到RT-PCR方法验证。表明制备的SBMV单抗及建立的SBMV血清学检测技术可有效用于我国SBMV的口岸检验检疫。     

Detection and characterization of an isolate of Tomato mottle mosaic virus infecting tomato in China

Tomato (Solanum lycopersicum) plants exhibiting severe leaf distortion, mottle and systemic crinkling symptoms were identified in Hainan province in China in 2016. To survey and control the disease, it is necessary to identify and characterize the pathogen causing the disease. Dot enzyme-linked immunosorbent assay showed that the crude saps of the infected tomato samples reacted positively with the monoclonal antibody against Tobacco mosaic virus which indicated that one or more tobamoviruses are likely associated with the disease. RT-pCR and DNA sequence analysis results further elucidated that Tomato mottle mosaic virus (ToMMV) in Tobamovirus was the pathogen causing the mottle disease in tomato. We amplified and sequenced the full-length sequence of the genome which showed the highest nucleotide identity with ToMMV YYMLJ and ToMMV TiLhaLJ isolates. The putative virus isolate was named ToMMV Hainan. Biological indexing studies showed that ToMMV Hainan can infect Nicotiana benthamiana, Capsicum annuum and Solanum lycopersicum showing serious symptoms. This was the first identification and characterization of ToMMV infecting tomato in Hainan of China.     

甜瓜黄斑病毒外壳蛋白的原核表达、多克隆抗体制备及其应用

本研究将甜瓜黄斑病毒(Melon yellow spot virus,MYSV)外壳蛋白基因克隆到原核表达载体pET32a(+)中,利用大肠杆菌系统表达该蛋白,免疫家兔制备其多克隆抗体。本文通过多克隆抗体,建立了间接ELISA(ID-ELISA)、免疫捕获PCR(IC-RT-PCR)、Western blot及dot-ELISA的MYSV检测方法,以上各种方法均能特异的检测MYSV。检测结果表明:ID-ELISA方法检测效价大于1∶6 400;Western blot检测灵敏度达到1∶320(W/V,g·mL-1);Dot-ELISA检测灵敏度达到1∶80(W/V,g·mL-1),且用牙签等非实验室工具便能检测该病毒,能高效地应用于田间样品的检测。     

犬恶丝虫抗体dot-ELISA检测方法的建立

为快速诊断犬恶丝虫病，建立了犬恶丝虫抗体dot—ELISA检测方法，并与阻断试验、交叉反应试验、重复性试验、琼脂扩散试验和平行对照试验进行了比较。结果显示，制备的犬恶丝虫虫体粗制抗原与犬钩虫、犬弓首蛔虫和犬蠕形螨感染犬的阳性血清无交叉反应，检出犬恶丝虫阳性血清的最高抗体效价为1：1024，重复性好；建立的犬恶丝虫病dot—ELISA诊断方法的灵敏度比琼脂扩散试验和美国IDEXX实验室制备的犬恶丝虫病诊断试剂盒分别高2048倍和48倍。     

马立克氏病病毒囊膜糖蛋白B基因原核细胞表达产物单克隆抗体的制备

用大肠杆菌 BL2 1表达了马立克氏病病毒 (MDV)强毒 GA株的囊膜糖蛋白 B(g B)基因 ,通过 SDS- PAGE电泳分离表达蛋白条带 ,切下并碾碎后作为免疫原制备单克隆抗体。通过 EL ISA和间接免疫荧光试验 (IFA) ,得到 1株阳性单克隆抗体杂交瘤细胞株 7C8。该单抗腹水的 EL ISA效价为 1∶ 2 1 2 ,IFA效价为 1∶ 80 0 ,与 MDV不同致病型毒株CVI988、GA、RB1B、MD11、6 48A株感染的鸡胚成纤维细胞 (CEF)均呈 IFA阳性。1∶ 10 0稀释时与 GA株感染的 CEF在斑点酶联免疫吸附试验 (dot- EL ISA)中呈阳性。     

玉米花粉传播SCMV的遗传学鉴定

利用类似杂交的方法,在试验田中,分健康植株自交、发病植株自交、父本感染病毒而母本是健康的、父本健康而母本是感染病毒的4个处理,开展了玉米花粉能否传播SCMV的试验。结果表明,健康植株自交得到的种子和父本感染病毒而母本健康的处理得到的种子利用dot-ELISA没有检测到病毒,而发病植株自交和父本健康而母本感染病毒的处理所获得的种子则检测到SCMV。由此可见花粉不能传播SCMV。     

Monoclonal antibody-based serological detection of potato virus M in potato plants and tubers

Potato virus M(PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide. To investigate and control this viral disease, efficient and specific detection techniques are needed. In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies(MAbs). Four highly specific and sensitive murine MAbs, i.e., 1 E1, 2 A5, 8 A1 and 17 G8 were prepared through a conventional hybridoma technology. Using these four MAbs, we have developed an antigen-coated plate(ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers. PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240(w/v, g mL~(–1)) by the dot-ELISA or at 1:163 840(w/v, g mL~(–1)) by the ACP-ELISA. The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection. Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China. The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing. We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM. These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.     

SRBSDV结构蛋白P10和非结构蛋白P9-1抗体的制备及应用

为丰富南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的预测预报体系,本研究利用Gateway原核表达系统,将SRBSDV结构蛋白P10和非结构蛋白P9-1进行了大量表达,纯化的表达蛋白分别免疫新西兰大白兔(Oryctolagus cuniculus)获得抗血清,抗血清的间接酶联免疫吸附分析(indirect enzyme-linked immunosorbent assay,in-ELISA)效价分别为1∶6 400和1∶3 200,Western blot检测结果表明,制备的2种抗体均具特异性.利用免疫捕获RT-PCR(immunocapture RT-PCR,IC-RT-PCR)和斑点酶联免疫吸附分析(dot enzyme-linked immunosorbent assay,dot-ELISA)方法比较两种抗体检测水稻(Oryza sativa)和白背飞虱(Sogatella furcifera,WBPH)带毒率的应用效果,结果表现一致,说明SRBSDV结构蛋白P10和非结构蛋白P9-1均可用于病毒的田间检测,为病害的预测预报奠定了基础.     

Comparative Evaluation of a Field-based Dot-ELISA Kit with Three Other Serological Tests for the Detection of Brucella Antibodies in Goats

A dot-ELISA (d-ELISA) test was evaluated and compared with the serum agglutination test (SAT), micro-complement fixation test (CFT) and a plate-ELISA (p-ELISA) for field use in screening herds of goats against brucellosis. During the standardization of the dot-ELISA kit on 1732 caprine serum samples, 1571 samples out of 1666 were found to be negative in d-ELISA, SAT and micro-CFT, while 59 were positive in different combinations. Of a further 66 serum samples, 34 were negative and 31 were positive in different combinations in d-ELISA, SAT, micro-CFT and p-ELISA. A total of 1584 goats belonging to different herds were then screened for brucellosis. Of the 694 serum samples screened in the first batch using d-ELISA, a positive reaction was observed in 26 cases. Further screening of these cases revealed 13 and 21 goats as positive reactors in SAT and CFT, respectively. In a second batch of 890 goats there were 109 positive reactors in d-ELISA. Among these 109 goats, 34, 40 and 80 goats were positive reactors in SAT, CFT and p-ELISA, respectively. The results of d-ELISA correlated well with those of p-ELISA. Dot-ELISA was found to be a more suitable and rapid test for screening large numbers of goats in the field.     

西瓜花叶病毒（WMV）单克隆抗体的制备及其应用

【目的】制备抗西瓜花叶病毒（Watermelon mosaic virus, WMV）的特异性单克隆抗体,并以其为核心建立能快速有效地检测WMV的血清学方法,从而为中国田间西瓜花叶病毒病的诊断和检测、预测预警及科学防控体系的建立提供物质和技术支撑。【方法】用提纯的WMV病毒粒子免疫BALB/c小鼠,经细胞融合和细胞培养、抗体筛选和细胞克隆等杂交瘤细胞技术,获得能稳定分泌抗WMV单克隆抗体的杂交瘤细胞株,将杂交瘤细胞注射入BALB/c小鼠腹腔制备其单抗腹水,并以制备的单抗为核心建立能准确、特异、灵敏地检测田间植物中WMV的ACP-ELISA、DAS-ELISA、dot-ELISA、Tissue blot-ELISA和IC-RT-PCR方法,以及能检测单头传毒介体蚜虫体内WMV的dot-ELISA方法。【结果】3株能稳定分泌WMV单克隆抗体的杂交瘤细胞株（2C8、15A8和16C12）及其单抗腹水被制备,3株杂交瘤细胞分泌的单抗腹水的间接ELISA效价均达到了10-6以上,抗体类型及亚类均为IgG1、kappa轻链。Western blot分析发现,这3个单抗均与WMV的外壳蛋白亚基有特异性反应。灵敏度分析结果表明,ACP-ELISA、DAS-ELISA、dot-ELISA和IC-RT-PCR方法检测WMV病叶的灵敏度分别达到1﹕163 840、1﹕327 680、1﹕5 120和1﹕1 310 720倍稀释（w/v,g/mL）。特异性分析结果表明,以这3个单抗为核心建立的ACP-ELISA、DAS-ELISA、dot-ELISA、Tissue blot-ELISA和IC-RT-PCR 5种血清学检测方法均能与感染WMV的病叶发生特异性免疫反应,而与感染小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)、马铃薯Y病毒(Potato virus Y,PVY)的植物样品、健康白南瓜、西瓜、葫芦和烟草的植物组织均呈阴性反应,且dot-ELISA方法还能特异性地检测单头蚜虫体内的WMV,而在检测无毒蚜虫时呈阴性反应。利用建立的血清学检测方法对采自浙江省、江苏省、山东省和海南省的275株葫芦科疑似病株进行检测,结果发现187株植物感染WMV,发病率达68%,说明WMV在中国田间葫芦科植物中广泛流行发生,且血清学方法的检测结果与RT-PCR方法的检测结果完全一致,将部分PCR产物进行核酸测序和序列比对分析,结果表明这些PCR产物是WMV CP基因片段,证明血清学方法检测阳性的样品确实感染WMV。【结论】获得了3株特异、灵敏的WMV单克隆抗体,以其为核心建立的5种血清学方法能准确、灵敏、可靠地应用于田间样品中WMV的检测,从而为中国WMV田间样品的大规模快速检测和诊断、该病害预报预警和科学防控提供物质和技术支撑。