【作者】 陈刚；

【导师】 王中康；

【作者基本信息】 重庆大学 ， 微生物学， 2008， 硕士

【摘要】 柑桔溃疡病（citrus bacterial canker disease, CBCD）是影响全球柑桔种植业发展的重大检疫性病害,其病原菌为地毯草黄单胞柑桔致病变种（Xanthomonas axonopodis pv. citri）,是国内外重大检疫性有害生物。长期以来由于缺乏抗病品种和特效的化学防治药剂,对该病的防治主要采取加强非疫区建设与植物检疫来严防病害的传播,对病树采取挖除并集中烧毁的根除措施。目前国内外正在实施的柑桔非疫生产区建设,强化植物检疫,严防疫害传入的监控策略,迫切需要建立快速、特异、准确的诊断技术及防控措施。基因工程重组单克隆抗体（recombination monoclonal antibody, RMA）是90年代以来,人们利用基因工程重组技术,有目的地在基因水平上对抗体分子进行切割、拼接或修饰,或者直接合成基因序列,再将重组DNA或重组蛋白基因导入细胞表达产生的一类抗体,兼具治疗和诊断双重功能。利用抗原-抗体特异性结合原理,可用于人类疾病、植物病害的诊断、治疗和预防。单链抗体（single chain variable fragment, ScFv）是利用基因工程的方法用一连接肽将抗体的重链可变区（VH）和轻链可变区（VL）拼接在一起的重组蛋白,由于其低或无免疫原性、分子量小、组织穿透力强,成本低,可大规模生产等特点已广泛应用于医学领域。但是,国内外单链抗体ScFv应用于植物病害方面的研究报道寥寥无几,且抗柑桔溃疡病菌的重组基因工程抗体高表达体系的建立以及稳定性改造方面的研究尚未见报道。本研究的目的就是针对现有的柑桔溃疡病快速检测技术体系尚未完善,并且传统上柑桔溃疡病的检测方法如PCR,噬菌体检测法、酶联免疫吸附法（ELISA）和斑点免疫结合法（DIA）等在一定程度上存在着检测周期长、操作复杂、灵敏度不高,不能很好的满足检疫工作的要求的缺点,应用高产重组单链抗体结合胶体金技术为柑桔溃疡病菌XAC的免疫诊断和防治研究提供新的工具和途径。研究内容主要包括:运用PCR方法扩增利用核糖体展示技术筛选的抗柑桔溃疡病菌（Xanthomonas axonopodis pv.citri, XAC）的单链抗体（ScFv 95）基因片段,将单链抗体基因重组到原核表达载体pET30a（+）中,构建单链抗体高效表达载体pET30a（+）-XAC-ScFv。再将pET30a（+）-XAC-ScFv质粒转化进大肠杆菌BL21（DE3）后诱导表达,并对表达产物进行纯化、复性及活性检测,随后对抗体的稳定性进行初步改造从而为柑橘溃疡病菌的血清学诊断提供新型的诊断试剂,而且还可用于进一步研究柑橘溃疡病菌与寄主的初步识别机制,为柑橘溃疡病菌的生物防治提供理论依据。论文主要研究结果如下:①成功扩增抗柑桔溃疡病菌的单链抗体ScFv基因,将其进行酶切后与载体pET30a（+）相连并转化进了大肠杆菌BL21（DE3）中。②采用IPTG诱导的包涵体表达方式进行抗体的表达,将包涵体裂解物进行SDS-PAGE电泳,显示在32 kDa处有一蛋白条带产生。将表达产物采用His-tag蛋白纯化试剂盒纯化后进行SDS-PAGE显示,在32 kDa同样有一蛋白条带产生。为了进一步验证表达的蛋白即目的蛋白,将表达产物进行了Western blot杂交,结果显示,与纯化后的电泳结果一致,在32 kDa处有单一的条带产生,说明表达纯化的蛋白即目的蛋白。③采用凝胶（Sephacryl-200HR）柱上在位复性对蛋白进行活性再生,通过斑点杂交检测复性后蛋白的活性,结果显示蛋白通过凝胶层析复性已成功获得原有的生物功能。提取柑橘溃疡病菌LPS及柑橘溃疡病菌近源种甘蓝黑腐病菌（XCC）LPS、水稻条斑病菌（XOO）LPS、水稻白叶枯（XOOc）LPS,LPS用HEPPES缓冲液（10 mmol/L HEPES,100 mmol/L NaCL pH 7.4）稀释成5μg/ml,以30μL/min注入。用Biacore evauation软件分析单链抗体亲和力,分析结果显示复性后的重组抗体只跟柑橘溃疡病菌LPS具有较高的结合力,亲和常数KA（ka / kd）达到了2.72×10⁷ M^-1。④通过重叠延伸PCR成功将半胱氨酸引入到了ScFv基因的VH和VL链中,为构建抗柑橘溃疡病菌的稳定型重组单链抗体的后续工作做了充足的准备。更多还原

【Abstract】 Citrus canker caused by the bacterial pathogen Xanthomonas axonopodis pv. citri （Xac）, a gram-negative bacterium, is a severe bacterial disease of most commercial citrus species and cultivars around the world, as well as some citrus relatives. For the lack of resistant cultivars and specific chemicals, the following measures have been adopted to control CBCD for a long time: enhanced construction of non-quarantine area and phytosanitary system against the spread of Xac, eradication and mass destruction methods dealing with infected plants. The pathogen is the target of quarantine efforts abroad and domestics, then the development of rapid and reliable procedures for diagnosis and control of this pathogen has been an important priority.Recombination monoclonal antibodies （RMA） are artificial constructions produced by recombining the antibody molecule or synthesizing the gene sequence with gene recombination technology and then transforming the antibody DNA into cells for expression, which hold dual function between therapy and diagnosis. Using antigen-antibodies specific binding principle, it can be used for human disease, plant disease diagnosis, treatment and prevention. Single chain variable fragment （ScFv） is artificial construction small molecular antibody produced by genetic engineering, whose heavy chain variable region and light chain variable region are spliced together with a connecting peptide using genetic engineering methods. Because of its low or no immunogenicity, small molecular weight, strong organizations penetrating power, low cost, large-scale production, and other characteristics has been widely used in the medical field, which may be used to identify target pathogens and prevent plant diseases including XAC. But there have been few reports about recombination monoclonal antibodies against phytopathogen except several reports about selection of ScFvs for diagnosing research by phage display. There have been no reports about high efficient expression and stability reconstruction of recombinant antibody for citrus bacterial canker disease.The purpose of this study is to pave a new way for immunization diagnosis and exploration of integrated control of citrus bacterial canker disease, which combines high yield recombinant single-chain antibody technology with colloidal gold, aiming at the existing citrus canker rapid detection technology system has not been perfect, and conventional detection methods for CBCD include PCR detection, phage detection, enzyme-linked immunosorbent assay （ELISA） and dot immunobinding assay （DIA） are all time-consuming, complex and less sensitive in a certain extent, which can not fulfill the requirements of quarantine adequately.The main content in this study are as follows: single-chain antibody fragments （ScFv 95） selected by ribosome display was amplified using an assembly of polymerase chain reaction （PCR）, and a recombined plasmid pET30a（+）-XAC-ScFv was constructed by inserting the single chain Fv gene into bacterial expression vector pET30a（+）. PET30a（+）-XAC-ScFv was transformed into Escherichia coli BL21 （DE3） and expressed which was induced by IPTG. Products were purified though Ni-NTA His Bind resin. The collected antibodies were refolded by gel filtration chromatography, and activity assaying process and the stability of the initial antibody transformation was done, which can be further applied to development of diagnostic reagent of Xac and research on the interaction between Xac pathogen and citrus during the phase of initial attachment-infection process. The main results are as follows:①Succeed in amplify single-chain antibody fragments for citrus bacterial canker disease and transformed into Escherichia coli BL21 （DE3） linking with vector pET30a（+） after digestion.②Antibody was expressed as inclusion bodies induced by IPTG. Inclusion extract was run on a denaturing polyacrylamide gel and about 32 kDa protein band was produced. Products purified though Ni-NTA His Bind resin on the denaturing polyacrylamide gel were transferred to PVDF membrane for western blot analysis. A band at 32 kDa was apparent.③The collected antibodies were refolded by gel filtration chromatography （Sephacryl-200HR）, and activity was tested by Dot-blot. The results showed the functional ScFv was obtained through renaturation. The LPS of Xac and other bacteria （Xanthomonas. oryzae pv. oryzae, Xooc; Xanthomonas. campestris pv. campestri, Xcc; Xanthomonas. oryzae pv. oryzicola, Xoc） was extracted and diluted by HEPPES buffer （10 mmol/L HEPES, 100 mmol/L NaCL pH7.4）, than was injected into HPA chip. The appetency of single-chain antibody was tested by Biacore evauation software. The Biacore analysis indicates that XAC-ScFv-95 showed significant affinity to LPS of Xac. The equilibrium constant （KA） determined by Biacore analysis for ScFv was 2.72×10⁷ M^-1.④Cysteine was imported into VH and VL of ScFv gene successfully by overlap and extension PCR, which lays the foundation for follow-up stability reconstruction of recombinant antibody.更多还原