Study Of Efficacy Of Harpin Protein Nanomedicine And Pen-2 DNA Nanomedicine Against WSSV

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.