水稻条纹病毒（RSV）单克隆抗体的制备及检测应用
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摘要
近年来，由水稻条纹病毒(Rice stripe virus，RSV)引起的水稻条纹病在江苏省的一些地区广泛流行，已成为江苏省水稻生产前期的一个重要病害。目前对于水稻条纹病的防治主要还是以种植抗病品种和适时治虫为主。灰飞虱的带毒虫量是条纹叶枯病发生流行的主要影响因子，因此建立一种快速、灵敏检测灰飞虱带毒率的方法是迫切需要的。为此，我们制备了RSV单克隆抗体，并建立了RSV的检测方法。
用水稻条纹病毒(RSV)免疫的BALB／c小鼠脾细胞与SP2／0鼠骨髓瘤细胞融合，经筛选克隆，获得4株能稳定传代且分泌抗RSV单克隆抗体的杂交瘤细胞。各株单抗腹水的ELISA效价均在1：80000-1：5120000之间。建立了间接ELISA测定RSV的方法，4株单抗检测病汁液的稀释度能达到2560倍以上，与其它病毒无交叉反应。Western blot分析表明4株单抗均与RSV的35 kD的外壳蛋白亚基有特异反应。用建立的间接ELISA对江苏省部分县市大田灰飞虱带毒率进行检测，结果显示灰飞虱的带毒率为12.5％-41.5％。
按常规饱和硫酸铵沉淀法纯化了3株单抗3B9、2H2和2E5，测定其IgG含量分别为：6.52 mg／ml、10.25 mg／ml和9.64 mg／ml。利用过碘酸钠法用辣根过氧化物酶标记了3株单抗的酶标抗体HRP-3B9、HRP-2H2和HRP-2E5，测定其效价分别达到1：25600、1：1600和1：3200，直接ELISA方阵试验确定了酶标抗体HRP-3B9、HRP-2H2和HRP-2E5的最佳工作浓度分别为1：5000、1：1000和1：1500。利用HRP-3B9在硝酸纤维素膜上采用直接和间接斑点免疫结合试验(DIBA)对灰飞虱体内RSV进行了检测。结果表明，DIBA法可有效的用于检测灰飞虱的带毒率，直接DIBA法比间接DIBA法灵敏度高。利用直接DIBA法对田间灰飞虱带毒率进行了检测。
Rice stripe disease is caused by Rice stripe virus (RSV), the disease epidemic is common in some parts of Jiangsu province, and has become a major disease to rice production in the province. The most effective ways for controlling the disease are cultivation of resistant varieties and killing the insect vector. The proportion of the viruliferous Laodelphax striatellus is the most important factor for the epidemic of the disease, so a rapid and sensitive method is necessary for detection of RSV in Laodelphax striatellus. Monoclonal antibodies (MAbs) of RSV were produced and detection methods are established using the MAbs.
Four hybridoma cell lines secreting MAbs against RSV were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized by the RSV particles. The titres of ascitic fluids of four MAbs ranged from 1:80000 to 1:5120000 when tested by indirect ELISA. Indirect ELISA was established with the produced MAbs for RSV detection, and the four MAbs could successfully detect RSV in plant sap with 1:2560 dilution. The MAbs didn't cross-react with other tested plant viruses. The result of Western-blot showed that the four MAbs could react with the 35 kD RSV coat protein specifically. Indirect ELISA was then used for detection of RSV in Laodelphax striatellus Fallen. The proportion of the viruliferous Laodelphax striatellus in Jiangsu province ranged from 12.5% to 41.5%.
Monoclonal antibodies 3B9, 2H2 and 2E5 were purified by saturated (NH4)2SO4 The content of IgG was 6. 52 mg/ml, 10.25 mg/ml and 9.64 mg/ml, respectively. The horseradish peroxidase-linked antibodies HRP-3B9, HRP-2H2 and HRP-2E5 were produced by NaIO4 method. The titres of the three enzyme-linked antibodies were 1:25600, 1:1600 and 1:3200, respectively. In direct ELISA, the suitable diluted times of the three enzyme-linked antibodies were 1:5000, 1:1000 and 1:1500, respectively. Enzyme-linked antibody HRP-3B9 was selected to detect RSV in Laodelphax striatellus Fallen using direct dot immunobinding assay (DIBA) and indirect DIBA. Detection results showed that DIBA could detect RSV in the insect and direct DIBA was more sensitive than indirect DIBA. The direct DIBA was then used to detect the virus in field collected Laodelphax striatellus Fallen.
用水稻条纹病毒(RSV)免疫的BALB／c小鼠脾细胞与SP2／0鼠骨髓瘤细胞融合，经筛选克隆，获得4株能稳定传代且分泌抗RSV单克隆抗体的杂交瘤细胞。各株单抗腹水的ELISA效价均在1：80000-1：5120000之间。建立了间接ELISA测定RSV的方法，4株单抗检测病汁液的稀释度能达到2560倍以上，与其它病毒无交叉反应。Western blot分析表明4株单抗均与RSV的35 kD的外壳蛋白亚基有特异反应。用建立的间接ELISA对江苏省部分县市大田灰飞虱带毒率进行检测，结果显示灰飞虱的带毒率为12.5％-41.5％。
按常规饱和硫酸铵沉淀法纯化了3株单抗3B9、2H2和2E5，测定其IgG含量分别为：6.52 mg／ml、10.25 mg／ml和9.64 mg／ml。利用过碘酸钠法用辣根过氧化物酶标记了3株单抗的酶标抗体HRP-3B9、HRP-2H2和HRP-2E5，测定其效价分别达到1：25600、1：1600和1：3200，直接ELISA方阵试验确定了酶标抗体HRP-3B9、HRP-2H2和HRP-2E5的最佳工作浓度分别为1：5000、1：1000和1：1500。利用HRP-3B9在硝酸纤维素膜上采用直接和间接斑点免疫结合试验(DIBA)对灰飞虱体内RSV进行了检测。结果表明，DIBA法可有效的用于检测灰飞虱的带毒率，直接DIBA法比间接DIBA法灵敏度高。利用直接DIBA法对田间灰飞虱带毒率进行了检测。
Rice stripe disease is caused by Rice stripe virus (RSV), the disease epidemic is common in some parts of Jiangsu province, and has become a major disease to rice production in the province. The most effective ways for controlling the disease are cultivation of resistant varieties and killing the insect vector. The proportion of the viruliferous Laodelphax striatellus is the most important factor for the epidemic of the disease, so a rapid and sensitive method is necessary for detection of RSV in Laodelphax striatellus. Monoclonal antibodies (MAbs) of RSV were produced and detection methods are established using the MAbs.
Four hybridoma cell lines secreting MAbs against RSV were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized by the RSV particles. The titres of ascitic fluids of four MAbs ranged from 1:80000 to 1:5120000 when tested by indirect ELISA. Indirect ELISA was established with the produced MAbs for RSV detection, and the four MAbs could successfully detect RSV in plant sap with 1:2560 dilution. The MAbs didn't cross-react with other tested plant viruses. The result of Western-blot showed that the four MAbs could react with the 35 kD RSV coat protein specifically. Indirect ELISA was then used for detection of RSV in Laodelphax striatellus Fallen. The proportion of the viruliferous Laodelphax striatellus in Jiangsu province ranged from 12.5% to 41.5%.
Monoclonal antibodies 3B9, 2H2 and 2E5 were purified by saturated (NH4)2SO4 The content of IgG was 6. 52 mg/ml, 10.25 mg/ml and 9.64 mg/ml, respectively. The horseradish peroxidase-linked antibodies HRP-3B9, HRP-2H2 and HRP-2E5 were produced by NaIO4 method. The titres of the three enzyme-linked antibodies were 1:25600, 1:1600 and 1:3200, respectively. In direct ELISA, the suitable diluted times of the three enzyme-linked antibodies were 1:5000, 1:1000 and 1:1500, respectively. Enzyme-linked antibody HRP-3B9 was selected to detect RSV in Laodelphax striatellus Fallen using direct dot immunobinding assay (DIBA) and indirect DIBA. Detection results showed that DIBA could detect RSV in the insect and direct DIBA was more sensitive than indirect DIBA. The direct DIBA was then used to detect the virus in field collected Laodelphax striatellus Fallen.
引文
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