H5N1亚型禽流感病毒NS1基因的原核表达及其ELISA检测方法的建立

采用RT-PCR技术扩增了禽流感病毒（AIV）A／Goose／HLJ／p46／2003（H5N1）的NSl基因，将其克隆于融合蛋白表达载体pMAL-c2X上，转化DH5α大肠埃希氏菌感受态细胞，经BamHⅠ和HindⅢ双酶切鉴定及序列分析，表明筛选到了重组质粒pc2X-NS1。SDS-PAGE电泳结果显示，重组质粒转化TB1大肠埃希氏菌后，经0．3mmol／L的IPTG诱导，融合蛋白MBP-NS1得到大量表达，融合蛋白以可溶形式存在，分子质量约为67ku。Western-blotting检测结果表明，融合蛋白MBP-NS1能够与H5N1亚型AIV活病毒感染康复鸭血清发生特异性反应，而不能够与H5N1亚型AIV灭活疫苗免疫鸭血清发生反应。试验初步建立了以纯化的融合蛋白MBP-NS1为包被抗原的间接ELISA检测方法，为AIV灭活疫苗免疫家禽与AIV感染家禽的鉴别诊断奠定了基础。     

陕西线辣椒病毒病病原检测简报

在陕西省线辣椒主产区多点随机取样,采用酶联免疫吸附法对陕西线辣椒病毒病毒原进 行了鉴定。结果表明,陕西线辣椒病毒病毒原有BBWV、PMMV、CMV、ToMV、TMV、PVY和 CVMV。优势毒原是BBWV、PMMV和CMV。     

Systemic spread of Plum pox virus (PPV) in Mariana plum GF 8-1 in relation to shoot growth

Infection of Prunus spp. by Plum pox virus (PPV) is characterized by an uneven distribution of the virus within the tree and branches. In order to gain a better understanding of this distribution, a method for modelling tree growth was used. PPV spread was followed within susceptible Mariana plum clone GF 8-1 shoots for 4 months after inoculation. Shoot growth was unaffected by the presence of the virus. Symptoms appeared on leaves produced in the most actively growing parts of the shoots, i.e. at the beginning of the season. PPV was detected in leaves other than those showing symptoms. The proportion of leaves with detectable virus decreased from the zone showing symptoms, with 100% ELISA-positive responses, to the shoot tip with no detectable virus in leaves produced between 111 and 127 days after inoculation. Furthermore, a higher proportion of positive ELISA results was obtained below the zone showing symptoms (77%) compared with 50% above. PPV was detected in 95% of the most vigorous shoots 71 days after inoculation compared with 37% of slower-growing, later-produced shoots.     

晋南冬麦区大麦黄矮病毒流行株系监测及防治策略探讨

连续5年(1996～2000年)采集晋南冬麦区小麦黄矮病标样,采用生物学和血清学(酶联免疫吸附法)相结合的诊断方法对该地区的大麦黄矮病毒流行株系进行了鉴别。结果表明,该小麦黄矮病流行区近五年以GAV株系为主流株系,兼有少量GPV、PAV和混合株系存在。同时对小麦抗黄矮病新品种“临抗1号”进行了GPV和GAV两种株系的抗性测定,明确了该品种兼抗GPV和GAV两种株系。根据小麦黄矮病发生现状,提出了一套以选育推广抗耐病品种为主,以药剂防治为辅的综合防治措施。以期为当地小麦生产服务。     

胶体金免疫层析法快速检测烟草环斑病毒

采用柠檬酸三钠还原法制备胶体金颗粒 ,标记烟草环斑病毒的抗体 ,制成免疫层析检测试纸条。检测粗提纯病毒的灵敏度为 1 0 0 0ng/ml,病汁液稀释 1 0 0 0倍后仍可快速检出。对大豆病种子、烟草冻干病叶等不同材料进行检测也有良好的效果 ,1～ 2min即可出现结果。用 9种不同的病毒进行测试 ,未出现非特异性反应。     

甲基对硫磷人工抗原的合成与鉴定

根据甲基对硫磷的结构特征,分别从甲基对硫磷的硝基一端或磷酸酯基甲氧基一端引入一定长度的间隔臂和活性基团,设计并合成了4种结构的半抗原;针对各种半抗原所带活性基团的性质,通过多种方法制备了与不同的载体蛋白偶联的人工抗原。进行动物免疫后,得到了针对甲基对硫磷的高效价抗体。     

梨火疫细菌实时荧光PCR和诱捕PCR-ELISA检测方法的建立

根据梨火疫细菌中独特而保守存在的质粒pEA29，设计了1对引物和3条探针，建立了实时荧光PCR检测方法和诱捕PCR-ELISA检测方法。实时荧光PCR采用带荧光标记的核酸杂交探针，边扩增边检测，步骤简单，不需PCR后处理，可避免假阳性和交叉污染；诱捕PCR-ELISA检测方法只需简单处理的样品就能检测，减少了核酸不纯出现的漏检，由于增加了核酸杂交探针，可不需凝胶电泳EB染色检测，不会出现假阳性问题。     

Production and characterization of a monoclonal antibody specific to the M serotype of plum pox potyvirus

A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.     

Biological and molecular characterization of Tomato spotted wilt Virus in Israel

Received April 24, 1997; received in final form June 29, 1997. Symptoms resembling tomato spotted wilt virus (TSWV) infections were documented among ornamental and vegetable crops in commercial greenhouses and open fields in Israel. Plants exhibiting these symptoms were collected from January 1992 to December 1996. Among cultivated plants analyzed for TSWV by enzyme-linked immunosorbent assay (ELISA), 19 species representing five families were found to be infected; natural infection was also recorded in six plant species of weeds. Virus identity was characterized by host range, serology and electron microscopy. Serological reaction with the isolates, found in Israel, using antisera from different sources as well as the sequence analysis of the nucleocapsid gene, demonstrated that the Israeli isolates of TSWV are a member of tospovirus serogroup I, type I (BR-01 strain). No virus transmission was found in seeds collected from virus-infected vegetable and ornamental crops. A non-radioactive molecular probe derived from the cloned nucleocapsid isolate enables specific detection of the virus in crude sap from infected plants. The detection of TSWV in Israel constitutes a severe potential threat to the ornamental and vegetable industry.     

动物组织中四环素类抗生素残留的ELISA检测研究——土霉素抗体的制备

四环素类抗生素属小分子半抗原物质 ,必须与大分子物质结合才能产生免疫原性。本试验采用重氮化法合成土霉素 牛血清白蛋白 (BSA OTC)及土霉素 鼠血清白蛋白 (RSA OTC)人工抗原 ,用紫外扫描对其进行鉴定。以BSA OTC免疫家兔制备抗血清 ,以RSA OTC为包被抗原 ,采用双向免疫扩散法和ELISA方法对抗血清进行鉴定。结果表明用重氮化法合成的人工抗原对四环素类药物有特异的抗原抗体反应 ,抗血清效价达 1 2 8× 1 0 5,完全能够满足四环素类药物残留的ELISA检测要求