【作者】 梁焯；

【导师】 孟小林；

【作者基本信息】 武汉大学 ， 微生物学， 2010， 博士

【摘要】 Harpin蛋白是植物病原菌通过Ⅲ型分泌途径(typeⅢsecretion system, TTS)向胞外分泌的重要致病因子,控制病原菌在敏感植物上的增殖和致病力。Harpin蛋白引起寄主或非寄主发生过敏性反应(hypersensitive response, HR),从而使植物产生系统获得性抗性(systemic acquired resistance, SAR),增强植物抗病能力并促进生长,已被开发成为新一代生物农药。但在农业实际应用中,其生物利用度较低,多次施药还会造成药效下降。近20年来纳米药物发展迅速,形式、尺寸不同的纳米药物能够产生透皮吸收、体内长循环、基因传导以及缓控释放等多种功效,是现代药学领域的研究热点之一。本研究中,为了获得良好的试验平行性,用发酵罐发酵、亲和层析纯化得到了高纯度的rHrpZ (recombinant HarpinZ)。并通过3种不同给药方式在烟草叶片上测定rHrpZ的生物学活性,发现至少需要喷洒5μg/mL rHrpZ才能使苯丙氨酸解氨酶(Phenylalanine ammonia lyase, PAL)活性升高一倍,而通过叶片穿刺小孔向叶肉组织内渗透,至少需要10μL 8mg/mL rHrpZ才能使穿刺孔周围产生可视的HR；而向叶柄中注射rHrpZ使其通过维管束传输,仅需rHrpZ就能使重约1g的叶片发生弥散性HR,全叶萎败。说明rHrpZ的生物学活性很高,但难以通过叶片表皮层渗透进入叶肉组织,即使通过伤口进入叶肉,rHrpZ也难以在叶肉组织中扩散。而进一步对多次施药后rHrpZ的药效研究发现,第二次施药rHrpZ对PAL和病程相关蛋白基因PR5-dB的转录水平促进作用均明显降低,其原因是由于PAL活性增强导致木质素加快积累,细胞壁渗透能力下降,导致rHrpZ的生物利用度(bioavailability)降低。利用具有高度生物安全性的合成高分子PLGA (poly d,l-lactide-co-glycolide),用乳化-溶剂挥发法(emulsification/solvent evaporation)制备了含有rHrpZ的纳米颗粒rHrpZ-PLGANP,通过动态光散射颗粒直径分布仪和扫描探针显微镜测定了其尺寸范围,结果显示其尺寸符合哺乳动物中透皮吸收和长循环的范围,并且在体外释放试验中体现了良好的药物释放速度。喷施同等剂量的rHrpZ-PLGA NP和rHrpZ后,测定了PAL活性和PR5-dB转录水平变化,结果显示,rHrpZ-PLGANP能明显延长rHrpZ作用时效,将药效时间从不足2天延长至超过2周。用PR5-dB转录水平评价药效,rHrpZ-PLGANP能使同等剂量的rHrpZ药效提高5倍,其原因可能是由于纳米颗粒能产生类似于哺乳动物中的透皮吸收效果,帮助rHrpZ通过植物气孔进入叶肉组织并进入细胞壁,在原位释放(in situ release)出rHrpZ,同时达到被动靶向和缓释功能。本研究证明了Harpin蛋白在农业应用时生物利用度低下,且多次施药后药效下降,并在国内外首次将纳米药物用于研究、开发新型蛋白质生物农药,增强了rHrpZ的生物利用度,提高了药效。本研究是纳米药物在生物农药中应用基础,为开发新型生物农药提供了新思路。对虾白斑综合症病毒(White spot syndrome virus,WSSV)是水产养殖业中危害最大的病毒之一,感染虾群在3-10天内死亡,死亡率可达100%,自上世纪发现以来,已造成世界范围内水产养殖业不可计量的损失,至今尚未有特效药问世。对虾抗菌肽penaeidins是一种既含有二硫键,又富含脯氨酸的新型抗菌肽,对于革兰氏阴性、阳性细菌、丝状真菌均有抑制作用,是对虾先天性免疫系统中的重要组成部分。本研究在大肠杆菌系统中表达了南美白对虾抗菌肽Pen-2融合蛋白,经过金属螯合亲和层析、肠激酶切得到了Pen-2成熟肽。将不同浓度的Pen-2成熟肽与WSSV提取液混合后感染克氏原鳌虾,结果显示,Pen-2能降低WSSV的感染能力,60μg/mL的Pen-2成熟肽能使WSSV致死率下降82.7%。用编码Pen-2的真核表达载体pcDNA3.1(-)-SP通过注射的方式给药后,发现注射质粒浓度超过20μg时,10%克氏原鳌虾发生蜕壳困难死亡现象,推测其原因可能是由于Pen-2蛋白参与调控虾蜕壳循环。用10μg质粒剂量注射克氏原鳌虾后,RT-PCR检测Pen-2基因在一周内持续转录,且对多酚氧化酶(prophenoloxidase,PPO)活性没有影响。注射后第3天用WSSV攻击,发现pcDNA3.1(-)-SP能抑制WSSV侵染,其相对保护率为48.3%。一个月后RT-PCR检测幸存虾的血淋巴中WSSV复制酶基因Swssvgp514转录水平发现,对照组用半数致死量WSSV攻击后的幸存虾血淋巴中全部检测到Swssvgp514转录,而质粒注射组有30%检测不到Swssvgp514基因转录。该试验结果说明pcDNA3.1(-)-SP对WSSV侵染具有抑制作用,但是注射给药方式每次仅能提供一周的保护效果,作用效果较弱。为延长Pen-2在虾体内的转录时间,加强药效,用来源于甲壳类动物甲壳的天然高分子壳聚糖与pcDNA3.1(-)-SP质粒以离子凝胶化方法制备了直径约80nm的纳米药物SP-CS NP(pcDNA3.1(-)-SP chitosan nanoparticle)。克氏原鳌虾分三次通过口服SP-CS NP,每次质粒剂量为10μg后,RT-PCR能在2个月内检测到Pen-2转录。最后一次口服SP-CS NP后第3天用WSSV攻击,结果显示SP-CS NP对WSSV攻击的相对保护率达到62.0%。且口服壳聚糖溶液对虾壳聚糖酶(chitosanase)、几丁质酶(chitinase)活性均没有明显影响,排除了壳聚糖对虾免疫机制的影响。一个月后RT-PCR检测克氏原鳌虾血淋巴,仅10%幸存虾中能检测Swssvgp514转录。说明SP-CS NP能长时间对WSSV起到抑制作用,并帮助虾清除体内病毒。由于白斑综合症是由WSSV感染引起虾免疫机能下降,从而多种病原物共同作用造成虾大量死亡。本研究还在病死南美白对虾中分离到一株溶藻弧菌(Vibrio alginolyticus)并命名为VA0712。用VA0712攻击注射pcDNA3.1(-)-SP和口服SP-CS NP的克氏原鳌虾,取血淋巴直接涂布TCBS平板,菌落计数结果显示给药方式均能加快虾对溶藻弧菌的清除速率,证明了Pen-2能从多角度抵抗对虾疾病。该研究首次证明体外表达的南美白对虾抗菌肽Pen-2具有对WSSV的抑制作用,并用真核表达载体pcDNA3.1(-)-SP在体内表达Pen-2抑制WSSV侵染,为开发利用对虾抗菌肽基因Pen-2防治抗对虾白斑综合症提供了基础。进而制备了pcDNA3.1(-)-SP-壳聚糖口服纳米颗粒,通过口服给药方式加强了Pen-2在体内的表达时效和对WSSV的抑制作用,初步找到了应用Pen-2抑制WSSV侵染的可行性方法,为开发抗WSSV药物提供了新的思路。更多还原

【Abstract】 Harpin is a kind of important factors secreted by type III secretion system of plant pathogen, which is important for pathogens proliferation and pathogenicity. Harpins can cause hypersensitive response(HR) on both host and non-host plants, thereby generate systemic acquired resistance(SAR) on plants, enhance growth and pathogen-resistance ability. But in agriculture application, the bioavailability is poor and the drug effect decreases with the multi-application. In the past two decades, great advances have been made in the field of nanomedicine. Various forms of nanomedicines can bring different effects, such as transdermal absorption, in vivo long circulation, gene transfection and controlled release. So that nanomedicines have been one of the hotspots of pharmaceutics research.In this study, recombinant HarpinZ(rHrpZ) was expressed using a fermentor and then purified by affinity chromatography. To test the HR, various dosages of purified rHrpZ were administrated on tobacco by three methods, sprayed on leaves, infiltrated from micro pore on leaves and injected into petiole. Sprayed on leaves, at least 5μg/mL rHrpZ could double the activity of phenylalanine ammonia lyase(PAL). Infiltrated into tobacco leaves, no less than 80μg rHrpZ (10μL of 8mg/mL rHrpZ) elicited HR, local symptoms were observed 16 hours after administration. While injected into petiole, rHrpZ could be transported though vascular bundle, only 20ng of purified rHrpZ (2μL of 10μg/mL rHrpZ) elicited a diffusive HR, the whole piece of leaf damped off in 2 hours. These results indicate that rHrpZ has strong biological activity, but only few rHrpZ could pass epidermis without any wound on leaves. And even when rHrpZ entered mesophyll through wounds on leaves by infiltration, there were still difficulties on pervasion. Furthermore, we investigated the drug effect after multi-administration, the effects of rHrpZ on both the PAL activity and the PR-5dB transcription level were decreased in the second administration. PAL controls the rate of lignin synthesis. With lignin accumulated, plant cells osmosis decreased, and then fewer rHrpZ can pass cell wall to interact with acceptors. So the stimulation effect of the second administration of rHrpZ was slighter than the first time.We prepared poly d,l-lactide-co-glycolide nanoparticles containing rHrpZ (rHrpZ-PLGA NP) by emulsion/solvent evaporation technique. rHrpZ-PLGA NP were characterized for size distribution using both dynamic light scattering particle size analyzer and scanning probe microscope. The result showed that the mean diameter of rHrpZ-PLGA NP fitted the range of long-circulating and transdermal in mammalians. It also showed good drug release rate in in vitro release test.After spraying the same dose of rHrpZ-PLGA NP and rHrpZ protein, PAL activity and PR5-dB transcription had been tested to investigate the drug effect. The results showed that rHrpZ-PLGA NP extended the drug effect duration from about 2 days to more than 2 weeks. Evaluating the drug effect by PR5-dB transcription level, the drug effect of rHrpZ-PLGA NP was five folds more than that of rHrpZ protein. It probably due to nanoparticles could produce transdermal effect similar with that in mammalian organ. The rHrpZ-PLGA NP could help rHrpZ pervade through epidermis and cell wall mainly from stoma, and released rHrpZ in situ, achieved both passive targeting and sustained release functions.This study proved that the bioavailability of Harpin was quite low in agriculture applications, the drug effect even decrease after multi-administration. In home and abroad, for the first time, we applied the nanomedicine technology in studying and developing new protein biopesticide. The nanomedicine caused enhancement on bioavailability and drug effect of rHrpZ. This study is basic work of apply nanomedicine technology in biopesticides development, and provide a new idea for biopesticides development. White spot syndrome virus(WSSV) is considered to be one of the most critical virus of shrimp, which is responsible for 100% mortality within 3-10 days after onset of the infection and has caused incalculable loss to the shrimp culture industry worldwide since it first broke out last century. There is no specific medicine for the white spot syndrome so far. Penaeidins are a kind of new antimicrobial peptides (AMP) which contain both disulfide bonds and proline-rich domain. As an important immune factor of the penaeid’s innate immune system, penaeidins exhibit a broad spectrum of activity against Gram-positive and Gram-negative bacteria, yeasts, fungi, parasites, enveloped viruses, and even tumor cells.In this study, Penaeus vannamei antimicrobial peptide Pen-2 fusion protein was expressed in E. coli system and purified with metal affinity chromatography. Then the fusion protein was digested by enterokinase to remove the fusion tag resulting the mature peptide Pen-2. In vitro anti-virus experiment was processed by mixing WSSV suspension together with various concentrations of mature peptide Pen-2, the mixture was injected into the second abdominal segment to infect cray fish,Procambarus clarkia. The result showed that Pen-2 could inactivate WSSV by membrane attack mechanism.60μg/mL of Pen-2 reduced 82.7% of the mortality by WSSV infection. Administrated by intramuscular injection, when the dose of pcDNA3.1(-)-SP up to 20μg,10% of the crayfish died due to molting difficulties. It suggested that Pen-2 probably involved into the molting cycle of the shellfish. Injected 10μg pcDNA3.1(-)-SP, Pen-2 transcription could be detected in the haemolymph for about a week and did not shown any effect on prophenloxidase activity. The crayfish was infected with WSSV 3 days post injection, the result shown that Pen-2 can inhibit WSSV infection in vivo. The relative protection ratio by injecting pcDNA3.1(-)-SP to WSSV was about 48.3%. A month after infection of WSSV, the core subunit gene of WSSV DNA replicase Swssvgp514 transcription was detected by RT-PCR. The result showed that 30% survivors of the pcDNA3.1(-)-SP injection group was negative in detection, while all the survivors of the group infected with half lethal dose of WSSV were positive in RT-PCR detection. The results showed that pcDNA3.1(-)-SP could inhibit WSSV infection, but the drug effect last too short to provide enough protection against WSSV.To extend the drug effect, nanoparticles with mean diameter about 80nm were formed with pcDNA3.1(-)-SP plasmid and natural macromolecule chitosan by ionic gelation technology. The SP-CS NP (pcDNA3.1(-)-SP chitosan nanoparticle) suspension was fed to crayfish directly for three times, each dose contained 10μg pcDNA3.1(-)-SP. The Pen-2 expressed in crayfish last about 2 month by oral administration of SP-CS NP contain 30μg pcDNA3.1(-)-SP, none of these crayfishes died without other process. WSSV infection was proceed 3days after the last administration. The final relative protection ratio of SP-CS NP was about 62.0%, and 70% of the survivors in WSSV attack was negative in Swssvgp514 transcription detection. In addition, oral administration of chitosan showed no effect on chitinase and chitosanase, so the effect of chitosan on shellfish immunity can be ignored. These result showed SP-CS NP could provide long term protection against WSSV, help shellfish clear virus in vivo.When shrimp infected by WSSV, the innate immune system was suppressed by virus genes, and then the shrimp died for co-infection by multiple pathogens. In this study, a strain of Vibrio alginolyticus called VA0712 was isolated and identified from Penaeus vannamei corpse. Crayfishes were infected by VA0712 after administration of pcDNA3.1(-)-SP and SP-CS NP respectively. The haemolymph was collected and spreaded on TCBS agar plate. After cultured for 12 hours, VA0712 colonies were counted to evaluate VA0712 clearance speed. In this experiment, both pcDNA3.1(-)-SP and SP-CS NP accelerated the clearance speed of VA0712 in vivo. This result indicated that Pen-2 could provide protection against white spot syndrome in multiple aspects.Our study here demonstrated that Pen-2 could eliminate the activity of WSSV, express Pen-2 in vivo using eukaryotic expression vector could provide protection against WSSV infection. It is the basal work to develop antimicrobial peptide Pen-2 for the medicine against WSSV. In addition, we prepared oral pcDNA3.1(-)-SP-chitosan nanoparticles preparation, it extended the drug effect of pcDNA3.1(-)-SP and enhanced antivirus effect. It provides a new way to apply AMP Pen-2 gene against WSSV.更多还原