摘要
位于革兰氏阴性植物病原细菌染色体上的hrp基因簇决定其在非寄主植物上的过敏反应和在寄主植物上的致病性。Hrp基因簇包括编Ⅲ型泌出通道的基因、调节基因和编码效应蛋白的基因。Harpins是由革兰氏阴性植物病原细菌产生并受hrp基因调节的一类非特异性激发子,具有共同的特征：富含甘氨酸、对热稳定对蛋白酶K敏感。它们通过III型泌出通道分泌到细胞膜外并转运到寄主植物的细胞质中,能够在寄主植物上引起致病性,在非寄主植物上引起过敏反应,诱导植物抗病,抗虫,促生长以及提高作物的产量与改善作物品质。Harpinxoo是由水稻白叶枯病菌Xanthomonas oryzae pv. oryzae的hrfl基因编码的蛋白质,大小为139个氨基酸(15.6 kDa),具有与其它harpins类似的生物学活性,但在结构和组成上,它显示一些其它harpins不具有的独特性质,如含有其它harpins不具备的半胱氨酸等。
植物拥有一套天然的免疫系统,使得它们能够抵御外界病原物的侵染。高等植物的HR,快速在病原物侵染点形成坏死斑,死亡细胞可以将侵入寄主体内的病原物限制在侵染点周围,阻止病原物的进一步扩展而实现局部抗病性,是植物针对多种病原物的主动防御,可进一步激活植物抗病防卫信号通路,使植物获得对此后侵入的多种类型病原物的系统抗病性(systemic acquired resistance, SAR)。当harpin被注射到非寄主植物叶片中时,最显著的特征就是引起过敏反应,而harpin的活性位点是如何被植物细胞识别,在什么部位识别,目前还不清楚。在本研究中,我们引入了一种研究植物-病原菌互作的新方法,应用基于Jerne的免疫网络学说制备出的单克隆抗独特型抗体模拟harpin的活性位点,探索harpin在非寄主植物中受体。本论文着重对以下几方面进行了研究。
1 HarpinXoo蛋白的提取与纯化
在本研究中,作者从水稻白叶枯病菌PX099基因组DNA中扩增出hrfl基因,用Ndel和HindⅢ叹酶切,随后插入到到pET30a(+)载体的NdeⅠ和HindⅢ两酶切位点之间,获得了重组质粒pET30a(+)-hrf1,转化宿主菌BL21 (DE3)表达融合蛋白。表达菌株E. coli BL21/pET-hrf1在37℃,225rpm经1mM IPTG诱导培养3小时后收集菌体,在100℃沸水中煮10分钟,提取Harpinxoo蛋白,离心分离菌体,蛋白溶解在提取缓冲液中。SDS-PAGE电泳分析表明,获得了大小为16.8kDa蛋白质。注射烟草叶片,引起过敏反应,说明提取到的蛋白为Harpin。用Ni柱对粗蛋白进行纯化,最终获得了单一条带的Harpin蛋白。
2免源多克隆抗-Harpinxoo抗体(Abl)的制备与纯化
500μg纯化的Harpinxoo溶解于灭菌的PBS中,与等体积的福氏完全佐剂混合乳化后皮下多点注射与肌肉注射交叉免疫新西兰大白兔,随后的加强免疫用200μg抗原与福氏不完全佐剂混合乳化,每两周免疫一次。六周后,采集兔血清用间接ELISA法测定效价,以免疫前三天采集的阴性血清作对照。Protein A/G亲合层析柱纯化兔源多克隆抗血清,获得多克隆抗-Harpinxoo IgG。HarpinXoo注射烟草引起的过敏反应能被兔源多克隆抗体Ab1抑制,甚至完全阻止,说明Ab1结合harpin的一个或多个活性位点,而这些活性位点可能是非寄主植物中受体的结合位点。
3体外、体内都具有抑制活性的单克隆抗-Harpinxoo抗体的制备
Balb/c小鼠接受四次免疫注射纯化的Harpinxoo后,选取效价最高的一只小鼠做融合,制备杂交瘤细胞。间接ELISA法筛选,有限稀释法亚克隆,两轮筛选后获得6株单克隆抗Harpinxoo杂交瘤细胞(2A10、3H12、4B2、4F7.6G4、7F11)。ELISA叠加实验表明,Harpinxoo上的四个表位被6株单克隆细胞株所识别,其中被6G4识别的表位为Harpinxoo引起烟草过敏反应活性位点。体外研究表明,6G4能够与烟草叶片总蛋白竞争性地结合Harpinxoo,最重要的是在体内,6G4能够部分抑制Harpinxoo引起的烟草过敏反应。6G4显然为一个非常合适的免疫原来制备具有Harpinxoo活性位点“内影像”特征的抗独特型抗体。阐明6G4在体外、体内的反应特性将有助于鉴定引起烟草过敏反应的harpin的关键区域。
4具有Harpinxoo"内影像”特征的抗独特型抗体的制备与特性分析
用固定化的胃蛋白酶消解纯的兔源多克隆抗体Ab1获得F(ab')2片段,具有抗原结合活性的F(ab')2被用作免疫原来制备具有Harpinxoo“内影像”特征的抗独特型抗体。为制备单克隆抗独特型抗体Ab2, F(ab')2片段和福氏佐剂乳化后免疫小鼠。随着免疫次数的增加,小鼠血清中抗独特型抗体的浓度也在增加。Ab2能够抑制Ab1与harpin的结合,当抑制率达到50%时,三只小鼠血清的稀释倍数分别为：鼠1(917)<鼠2(1094)<鼠3(2540)。因此我们选取竞争抑制效果最好的小鼠3进行细胞融合。
应用间接ELISA法和竞争抑制ELSIA法筛选具有Harpinxoo“内影像”特征的抗独特型抗体。用间接ELISA法初步筛选,获得224株抗Ab1的阳性克隆,进一步用竞争ELSIA法筛选,抑制率达到50%以上的细胞株仅获得23株。经过第一轮筛选,具有全部抗独特型抗体的特征并能稳定分泌抗体的细胞株6B2被筛选出来。竞争ELISA结果表明,6B2与Ab1的结合受harpin抑制,但不受其它无关蛋白如：BSA抑制,这进一步可以说明6B2与Ab1的结合位点可能为独特型结合位点。6B2与Harpinxoo竞争性地结合非寄主植物中推定的harpin受体,这是本研究的关键所在。6B2与受体的结合位点和harpin与受体的结合位点可能为同一个活性位点或非常相近的活性位点。6B2与非寄主植物中推定受体的结合可能是交叉反应的结果,或者受体构象发生改变结果,然而,也有可能是两种机制共同作用的结果。6B2免疫新西兰大白兔进一步诱导出与兔源多克隆抗体Ab1相同或相似活性的Ab3,这就佐证了我们制备的抗独特型抗体6B2确实具有Harpinxoo的“内影像”特征。这些结果表明,6B2具有抗原相似性,即,抗独特型抗体6B2可以充当Harpinxoo抗原的“内影像”,因此,根据Jerne的系统命名法,6B2可以归类为Ab2β。
总之,应用抗独特型抗体进行抗原模拟是探索生物体内含量非常低的受体的一种可行方法。这种方法涉及到多种学科,如：蛋白质化学、酶学、免疫学以及蛋白质-蛋白质互作,其应用也是非常广泛的,如：功能模拟,用作探针鉴定受体和免疫组织化学定位。
The hrp (hypersensitive response and pathogenicity) gene clusters in Gram-negative phytopathogenic bacteria determine hypersensitive response (HR) in nonhost plants and pathogenicity in host plants of the bacteria. A hrp gene cluster usually contains genes coding for the components of the typeⅢsecretion pathway, effectors and the proteins that regulate the productions and transportations of effectors. Harpins are non-specific elicitors produced from Xanthomonas oryzae pv. oryzae and regulated by hrp genes. They are exported and delivered from the bacteria into the cytosol of host plant cells and have common characters:glycine-rich, heat stable and protease K-sensitive. They function at the bacterial-plant cell surface interface and have the ability to induce pathogenicity in susceptible plants and to elicit a hypersensitive reaction (HR) in nonhost plants or resistant cultivars of host plants. The application of harpins in many plants is to induce disease and insect resistance, trigger many plant-reaction phenotypes, promote plant growth, and enhance yield and quality by activating distinct signaling pathways. HarpinXoo, identified recently, is encoded by hrfl gene from Xanthomonas oryzae pv. oryzae and contains 139 amino acids (15.6kDa). They have similar biological activities with other harpins, while show some unque natures, such as containing cysteine.
Plants possess an innate immune system enabling them to defend themselves against attacking pathogens. Using a large set of receptors, the immune system has developed different strategies to realize disease resistance. The HR of higher plants, characterized by rapid, localized necrosis of plant cells at the sites of pathogen infection, is an active defense of plants against many pathogens. The dead cells limit pathogen around the sites of infection, and prevent the further expansion of pathogen realizing local disease-resistance. The signaling pathway of pant disease-resistance was further activated, making plants acquire system resistance to many types of pathogens invaded later. The most prominent feature is the event of eliciting a hypersensitive response, when harpin was infiltrated into nonhost plants. To respond to harpin, the presence of harpin must be recognized by the plant cell. How and where harpin's active site is recognized has not been clear completely. Here, we introduced a new strategy to study the problem of plant-pathogen, empolying an anti-idiotypic antibody based on Jerne's immune network to probe harpin receptor(s) in nonhost plant.
1 Extraction and purification of HarpinXoo protein
Hrf1 gene amplified from genomic DNA of Xanthomonas oryzae pv. oryzae strain PXO99 was digested with NdeⅠand HindⅢ, subsequently purified, inserted between the NdeⅠand HindⅢsites of the pET30(a+) vector containing an IPTG promoter. The resulting plasmids, named as pET30a(+)-hrf1, were analyzed by restriction enzyme digestion and DNA sequencing, and transformed into E. coli BL21 (DE3) for expression of the fusion proteins. The recombinant E. coli BL21/pET-hrf1 was incubated at 37℃for 3h at 225 rpm under inducing withlmM IPTG, and then the E. coli cells were harvested by centrifugation at 4℃. HarpinXoo fusion protein was extracted from the harvested cells by boiling them for 10 min in 100℃, purified through Ni affinity chromatography and analyzed on SDS-PAGE. The molecular massed of Harpinxoo is 15.6kDa, in consistence with the prediction by a pertinent software program. This result is an essential basis for further studies on functions of the gene and protein in plants.
2 Preparation and purification of rabbit polyclonal anti-HarpinXoo antibodies (Ab1)
New Zealand white rabbits were immunized subcutaneously at multiple sites and intramuscularly with 500μg of purified HarpinXoo emulsified with an equal volume of Freund complete adjuvant and boosted at bi-weekly intervals with 200μg of antigen emulsified with Freund incomplete adjuvant. Six weeks later, the rabbits were bled and the anti-HarpinXoo serum titer was determined by the ELISA method with pre-immune sera analyzed in parallel. Protein A/G affinity chromatography was performed on a 2-mL settled gel pre-packed column to purify Ab1. HR induced by Harpinxoo can be inhibited and even completely blocked by Ab1, which further demonstrates that Ab1 binds to one or more harpin's active sites, which are likely to be combining sites of receptor. This makes it an interesting candidate for generating anti-idotypes.
3 Preparation of monoclonal anti-HarpinXoo antibody with inhibitory activities in vitro and in vivo
Female Balb/c mice received four times of immunization with purified harpinXoo. The mice with the highest serum antibody titer were selected to produce hybridomas. Six monoclonal harpinXoo hybridomas were selected, subcloned and identified. ELISA additivity tests revealed that four epitopes in harpinXoo are defined by six monoclonal antibodies, of which one epitope recognized by the mAb designated 6G4 was found to part of the functional sites of harpinXoo, depending upon their immunoinactivation properties. In vitro studies showed that 6G4 was able to bind to harpinXoo competing with total protein of tobacco leaves. Most importantly, in vivo,6G4 blocked partially tobacco HR elicited by HaprinXoo.6G4 appear to be a suitable antigen to produce aniti-iditypic antibodies against harpinXoo, which have the properties of the internal images of harpinXoo actives sites. Elucidating the effect of 6G4 in vitro and in vivo will provide more insight into identifying key functional regions in Harpins that induce HR in nonhost plant.
4 Preparation and characterization of monoclonal Ab2 having the properties of an internal image of HarpinXoo
F(ab')2 fragments retaining antigen binding activity, which were generated by digesting the purified rabbit polyclonal anti-harpin antibodies (Ab1) using immobilized pepsin, were used to produce mouse anti-idiotypic antibodies having the properties of "internal image" of HarpinXoo- For the preparation of mAb Ab2, mice were immunized with different doses of F(ab')2 fragments in the presence of adjuvant. The concentration of Ab2 in mouse sera increased with an increase in the number of immunizations. When the inhibition rate reached 50%, the order of the three mouse sera dilutions were:mouse 1 (917)< mouse 2 (1094)< mouse 3 (2540). So we selected mouse 3 having the best inhibitory effect on the binding of Ab1 to HarpinXoo to generate hybridomas.
The anti-idiotypic antibody that has the properties of an internal image of the protein HarpinXoo was screened by using standard ELISA and competitive inhibition ELISA. Clones were screened by indirect ELISA first,224 were positive against Ab1 polyclonal antibody, but only 23 were able to block more than 50% of the binding of Ab1 to Harpinxoo. After a first selection round,6B2 was selected to be fully characterized and to be propagated as a stable cell line. The results from the competition ELISA demonstrate that the binding of 6B2 mAb to the fragments of Ab1 was inhibited by HarpinXoo but not by other proteins, such as BSA, indicating that the binding probably occurred close to or on the combining site of the idiotype. In addition, we showed that 6B2 competes with Harpinxoo for binding to the harpin putative receptor in nonhost plants, which was the crucial point of this study. This fact provides strong evidence that 6B2 binds to the putative receptor at the harpin binding site. The binding of 6B2 to the harpin putative receptor could be the result of cross-linking to the receptor or of the induction of a conformational change in the receptor. However, it is also possible that both mechanisms are active together. Additional evidence for the anti-idiotype nature of 6B2 was provided by the induction of an Ab3 response.6B2 mAb induced a strong response against harpin in rabbits and elicited Ab3 antibodies with the same specificity as the rabbit polyclonal antibodies Ab1. These results implied that 6B2 mAb has ligand-like properties, i.e. that it behaves as the internal image of harpin protein and therefore 6B2 can be classified as Ab2βaccording to Jerne's nomenclature.
In conclusion, Antigen mimicry by anti-idiotypic antibodies is employed as a reliable strategy to probe receptors that are present in very low concentrations in the organism. This approach calls on knowledge at the crossroads of various fields such as general protein chemistry, enzymology, and immunology, as well as protein-protein interactions, for which the scope of applications is wide, for example:functional mimicry, using as a probe to identify cell surface receptors and immunohistochemical localization.
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