用于青枯劳尔氏菌快速检测的靶标内切葡聚糖酶抗体制备与检测试验  

Polyclonal Antibody Preparation and Detection of Endoglucanase as a Rapid Detection Target Against Ralstonia solanacearum

作　　者：白利叶 范晓东[1] 周泽扬[1,2] 王林玲[1] 

机构地区：[1]重庆师范大学生命科学学院,重庆401331 [2]西南大学,家蚕基因组生物学国家重点实验室,重庆400716

出　　处：《蚕业科学》2016年第2期203-209,共7页ACTA SERICOLOGICA SINICA

基　　金：重庆市自然科学基金项目(No.cstc2015jcyj A80014);教育部留学回国人员科研启动基金项目

摘　　要：桑青枯病是一种土传性细菌病害,其病原为青枯劳尔氏菌(Ralstonia solanacearum)。内切葡聚糖酶(EGL)是青枯劳尔氏菌在胞内合成后分泌、吸附于细胞壁周围的一个重要毒力因子,研究以其为靶标的桑青枯病快速检测诊断技术,可以为病害防控提供技术支持。从青枯劳尔氏菌G12-50菌株中克隆了egl基因,将青枯劳尔氏菌与土壤常见的4种细菌以及植物常见的4种病原细菌的内切葡聚糖酶进行氨基酸序列多重比对,其同源性较低。构建重组质粒p ET32a-EGL转化大肠杆菌BL21融合表达EGL蛋白,制备的抗EGL多克隆抗体经ELISA检测其效价达1∶20 000,Western blot分析亦呈现单一的特异性条带。用制备的抗体对培养的青枯劳尔氏菌G12-50菌液和感染桑青枯病的桑茎汁液进行Western blot检测,结果出现明显的特异性条带,而对照大肠杆菌则没有出现条带。研究结果表明,青枯劳尔氏菌的内切葡聚糖酶可以作为桑青枯病早期检测诊断的病原靶标,直接应用于对桑园土壤和桑树样品的快速检测。Mulberry bacterial wilt is a soilborne bacterial disease. Its pathogen is Ralstonia solanacearum. Endoglucanase（ EGL） is an important virulence factor of R. solanacearum,which is synthesized intracellularly and secreted to adsorb around the target cell wall. In this study,a rapid detection technique against mulberry bacterial wilt was established to provide technological support for controlling mulberry bacterial wilt by taking endoglucanase as the target. Multiple sequence alignment of amino acids from cloned egl gene of R. solanacearum G12-50,EGLs of 4 common soil pathogens and 4 common plant pathogenic bacteria showed that the homology between them is low. A recombinant plasmid p ET32a-EGL was constructed and transformed into E. coli BL21 to express fusion EGL enzyme protein,which was used to prepare anti EGL polyclonal antibody. The obtained antibody had a titer of 1 ∶ 20 000 as validated by ELISA and showed a single specific band in Western blot analysis. Western blot detections using the prepared antibody against the R. solanacearum G12-50 bacterial culture liquid and the stalk juice of mulberry infected by bacterial wilt displayed clear specific bands,while no band was observed in the control E. coli. The results show that R. solanacearum endoglucanase can be directly used as the early detection target for rapid detection of mulberry field soil and mulberry samples against mulberry bacterial wilt.

关 键 词：桑青枯病 青枯劳尔氏菌 胶体金免疫层析 内切葡聚糖酶 多克隆抗体 检测靶标 

分 类 号：S888.71[农业科学—特种经济动物饲养]