甘蔗宿根矮化病病原菌一推测膜蛋白基因(Lxx18460)单克隆抗体的制备和检测  

Preparation and detection for monoclonal antibody of speculative membrane protein gene(Lxx18460)from Leifsonia xyli subsp. xyli

作　　者：邵敏[1] 张小秋[1] 张琨琨[1] 杨丽涛[1,2] 李杨瑞[1,2] 

机构地区：[1]广西大学农学院/亚热带农业生物资源保护与利用国家重点实验室,南宁530005 [2]中国农业科学院甘蔗研究中心/广西农业科学院/农业部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室,南宁530007

出　　处：《南方农业学报》2016年第3期382-388,共7页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31360293);国家国际科技交流与合作项目(2013DFA31600);广西自然科学基金项目(2012GXNSFDA053011)

摘　　要：【目的】对甘蔗宿根矮化病病原菌(Leifsonia xyli subsp.xyli,Lxx)一推测为抗K因子的膜蛋白基因(Lxx18460)进行研究,为今后研究该基因在感病甘蔗体内表达打下基础。【方法】诱导表达并纯化含有推测为抗K因子的膜蛋白Lxx18460基因的p ET-30a质粒菌液,委托南京金斯瑞生物科技有限公司制备单克隆抗体;用Western blotting对细菌及感病和健康甘蔗样品总蛋白进行检测。【结果】Lxx18460基因具有特异性,只存在于甘蔗Lxx中。ELISA单克隆抗体效价大于1∶512000,能够灵敏地检测细菌总蛋白和甘蔗样品蛋白。Western blotting检测结果表明,Lxx18460基因在细菌中和感病甘蔗样品中表达,在健康甘蔗样品中几乎不表达。【结论】Lxx18460基因具有特异性,是Lxx中的抗K因子的膜蛋白基因,能够在细菌和感病甘蔗样品体外进行表达。【Objective】The present experiment was conducted to investigate a speculative membrane protein（Lxx18460）gene of anti-Kfactor in Leifsonia xyli subsp. xyli（Lxx）, in order to lay foundation for future studying in vivo expression of Lxx18460 gene in diseased sugarcane. 【Method】The bacterium suspension of p ET-30 plasmid containing Lxx18460 gene of anti-sigma K（K） factor were induced and purified, and the monoclonal antibody were prepared by Nanjing Genscript Biotechnology Company. Then, the total bacterial proteins and proteins from diseased and healthy sugarcane plants were detected by Western blotting test. 【Result】The results showed that, Lxx18460 gene was specific because it only existed in Lxx. The ELISA results showed that, monoclonal antibody titer was more than 1 ∶512000, and the total bacterial proteins and proteins from sugarcane plants could be detected sensitively. Western blotting test results showed that Lxx18460 gene could be expressed in bacteria and diseased sugarcane samples, but wasn＇t expressed in healthy sugarcane samples. 【Conclusion】The Lxx18460 gene has specificity, and is membrane protein gene of anti-sigma K（K） factor in Lxx, and can be expressed in bacteria and diseased sugarcane in vitro.

关 键 词：甘蔗宿根矮化病 Lxx18460基因 单克隆抗体 ELISA检测 WESTERN blotting检测 

分 类 号：Q-331[生物学]