柑橘黄龙病亚洲种病原( Cadidatus Liberibacter assiaticus)重组抗体的研究

Study on the diversity of Huanglongbing(HLB) and pathogenesis mechanism as well the interation relationship between the pathogen and its host is very important for the control of the disease. Citrus huanglongbing is a destructive disease of citrus and caused by endogenous, sieve tube-restricted bacteria which is belong to a member of uncultured bacteria. There are still no curative methods to control HLB. It is very important to study the recombinant antibody against HLB bacterium and to constructure the express system of its single chain fragments, which will not only provide a large number of high sensitive and specific antibody to be used in studying the diversity of the pathogen, pathogenesis mechanism and interaction relationship between the pathogen and its host, the detection technique of the pathogen such as basing on immunological technique, but also a large number of the antibody which can be used to study the control of HLB disease. In this project, it will ba studied the best form of the antigen of HLB pathogen which is used to immunize mice, the synthesis of first strand cDNA, PCR of VH and k strain, and constructure of VH/ k strain, affinity selection of the ScFv by ribosome display, Cloning and expression of the ScFv, as well as Specificity and affinity analysis of the Soluble recombinant antibodies.

研究病原菌多态性、致病机理、探讨病原菌与寄主互作关系是病害控制的基础性工作，抗体为蛋白质水平研究这方面工作提供了很好的技术方法。黄龙病是柑橘生产上一种毁灭性病害，由韧皮部杆菌引起，限韧皮部筛管细胞生长，属难培养细菌。目前没有治疗黄龙病的有效药物。开展黄龙病菌重组抗体研究，构建其表达体系，能够为黄龙病应用基础研究，以及如基于免疫层析技术的胶体金试纸条等检测技术的研发提供提供高特异性和高灵敏度抗体，同时由于重组抗体技术能够提供大量特异性抗体的优点，还能够为抗体用于黄龙病防治的探讨提供大量抗体。本项目将研究：免疫小鼠的黄龙病菌最适抗原形式；小鼠脾脏细胞总RNA RT-PCR合成cDNA第一链、 PCR扩增获得linker DNA、VH和 k链；VH/k重组子的构建；核糖体展示技术筛选黄龙病菌特异ScFv，以及ScFv的克隆和表达，分析表达抗体的亲和性和特异性，获得黄龙病菌重组抗体的表达体系。