Paracoccus luteus sp. nov., isolated from the intestine of grass carp.

A novel Gram-negative bacterium, designated CFH 10530T, was isolated from the intestine of grass carp. The sample was collected from the aquaculture training base at the College of Fisheries, Henan Normal University, Xinxiang, PR China. Cells of strain CFH 10530T were coccoid, ovoid or short-rod-shaped, aerobic, non-spore-forming and non-motile. 16S rRNA gene sequence analysis demonstrated that strain CFH 10530T was closely related to Paracoccus endophyticus SYSUP0003T (97.7 % sequence similarity), Paracoccus halophilus HN-182T (96.5 %) and Paracoccus panacisoli DCY94T (96.1 %). The strain grew optimally at 25-28 °C, at pH 7.0 and with 0-2 % (w/v) NaCl. Cells were positive for catalase and oxidase, nitrate was reduced and H2S was not produced. The isoprenoid quinone was Q-10. Major cellular fatty acids were summed feature 8, C18 : 0 and C18 : 03-OH. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and five unidentified polar lipids. The genome size was 3 331 229 bp with a G+C content of 69.6 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between CFH 10530T and the other species of the genus Paracoccus were found to be below the recommended levels for species delineation (ANIm <85, ANIb <80 and dDDH <24 %). Based on its physiological properties, chemotaxonomic characteristics and low ANI and dDDH results, strain CFH 10530T is considered to represent a novel species for which the name Paracoccus luteus sp. nov., is proposed. The type strain is CFH 10530T (=KCTC 62919T=CGMCC 1.16597T).

Azospirillum thermophilum sp. nov., isolated from a hot spring.

A novel Gram-staining negative, aerobic, motile by flagellum, rod-shaped bacterium, designated CFH 70021T was isolated from a hot spring soil sample collected from Tengchong, Yunnan province, PR China. Growth of CFH 70021T occurred at 15-50 °C (optimum 50 °C), pH 5.0-7.0 (optimum pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum 0 %, w/v). The genome of CFH 70021T consisted of four complete circular chromosomes and five plasmids, the genomic DNA G+C content was 69.3 mol%. Comparison of the 16S rRNA gene sequences indicated that CFH 70021T represented a member of the genus Azospirillum and showed close relationship with the type strains of Azospirillum agricola CC-HIH038T (97.8 %), Azospirillum rugosum IMMIB AFH-6T (97.6 %), Azospirillum doebereinerae GSF71T (97.6 %), Azospirillum thiophilum DSM 21654T (97.4 %) and Azospirillum picis IMMIB TAR-3T (97.2 %). The polar lipids of CFH 70021T contained diphosphatidylglycerol, phosphatidylmehtylethanolamine, phosphatidylglycerol, phosphatidylcholine, two aminolipids and an unidentified phospholipid. The predominant cellular fatty acids (>10 %) included C19:0cyclo ω8c (11.4 %), C16 : 0 (27.6 %) and summed feature 8 (C18:1ω7c/C18:1ω6c, 40.9 %). The major isoprenoid quinone was Q-10. On the basis of the low ANIb result (<78 %) and different phenotypic and chemotaxonomic characters, we conclude that strain CFH 70021T represents a novel member of the genus Azospirillum, for which the name Azospirillum thermophilum sp. nov. is proposed. The type strain is CFH 70021T (=KCTC 62259T= CCTCC AB2018121T).

Antribacter gilvus gen. nov., sp. nov., a new member of the family Promicromonosporaceae from a karstic cavern.

A novel actinobacterium, designated strain CFH 30434T, was isolated from a soil sample collected from a karst cave in Luoyang, Henan Province, PR China. The taxonomic position of the strain was investigated by using a polyphasic approach. Cells of the strain were aerobic, Gram-stain-positive, non-motile and coccoid or short rods. The strain was found to be oxidase-positive and weakly catalase-positive. Strain CFH 30434T grew optimally at 28 °C, pH 7.0-9.0 and in the presence of up to 0-1.5 % NaCl (w/v). The whole-cell sugars were glucose, mannose and rhamnose. The major isoprenoid quinone was MK-9(H8) and the major fatty acids (>10 % of the total fatty acids compositions) were anteiso-C15 : 0, iso-C15:0 and iso-C14 : 0. The polar lipids detected were diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified phosphoglycolipid, an unidentified phospholipid and an unidentified glycolipid. The genomic DNA G+C content was determined to be 72.3 mol%. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that CFH 30434T clustered within the family Promicromonosporaceae, and closely with the type strains of Xylanimicrobium pachnodae DSM 12657T, Myceligenerans crystallogenes DSM 17134T and Promicromonospora xylanilytica CCTCC AA 208046T (97.3 %, 96.2 and 95.9 % sequence similarities, respectively). Phylogenetic analysis showed that strain CFH 30434T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Its phylogenetic distinctiveness and distinguishing phenotypic characteristics supported that strain CFH 30434T represents a novel genus of the family Promicromonosporaceae, for which the name Antribacter gilvus gen. nov., sp. nov. is proposed. The type strain is CFH 30434T (=CGMCC 1.13856T=KCTC 49093T).

Nocardia yunnanensis sp. nov., an actinomycete isolated from a soil sample.

A novel actinomycete strain, designated CFH S0054T, was isolated from a soil sample collected from Xiaokongshan in Tengchong, Yunnan Province, in south-west PR China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain CFH S0054T belonged to the genus Nocardia. Based on the results of 16S rRNA gene sequence analysis, strain CFH S0054T was most closely related to Nocardia concava IFM 0354T (98.3 %), Nocardia inohanensis NBRC 100128T (97.9 %), Nocardia otitidiscaviarum NBRC 14405T (97.9 %) and Nocardia heshunensis CFH S0067T (97.8 %). Strain CFH S0054T contained C16 : 0, C18 : 1 ω9c, C18 : 0 10-methyl (TBSA) and summed feature 3 as major cellular fatty acids and MK-8 (H4ω-cycl) as the major respiratory quinone. The polar lipid profile included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and unknown phospholipids. The cell wall contained meso-diaminopimelic acid, and the whole-cell hydrolysates were arabinose, galactose, glucose, fructose, ribose and mannose. The genome of strain CFH S0054T was 7.9 Mbp with a G+C content of 68.4 mol%, the average nucleotide identity (ANI) values between strain CFH S0054T and N. concava IFM0354T were low (ANIm<87.7 % and ANIb=84.1 %). On the basis of phylogenetic, physiological and chemotaxonomic data, strain CFHS0054T is considered to represent a novel species of the genus Nocardia, for which the name Nocardiayunnanensis sp. nov. is proposed, The type strain is CFH S0054T (DSM 46763T=JCM 30082T).

Ornithinimicrobium cavernae sp. nov., an actinobacterium isolated from a karst cave.

A novel actinobacterium, designated strain CFH 30183T, was isolated from a soil sample collected from a karst cave in Luoyang, Henan Province. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of strain CFH 30183T were observed to be Gram-stain positive, motile, asporogenous and coccoid to rod shaped. The strain was found to be aerobic and oxidase positive. On the basis of 16S rRNA gene sequence analysis, strain CFH 30183T was found to be closely related to Ornithinimicrobium murale 01-Gi-040T (97.8% sequence identity). The ANIb/ANIm values between strain CFH 30183T and O. murale DSM 22056T were found to be 80.3%/85.9%. Strain CFH 30183T was found to grow optimally at 28-32 °C, at pH 8.0-9.0 and in the presence of up to 7% NaCl (w/v). Whole cell hydrolysates of strain CFH 30183T contained L-ornithine as the diagnostic diamino acid, and arabinose, glucose, mannose and rhamnose as whole cell sugars. The respiratory quinone was determined to be MK-8(H4), while the major fatty acids were found to consist of iso-C15:0 and iso-C16:0. The polar lipids profile was found to include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified phospholipid, an unidentified phosphoglycolipid and four unidentified lipids. The DNA G + C content of strain CFH 30183T was calculated to be 70.9%. Based on the phenotypic, genotypic and phylogenetic data obtained, strain CFH 30183T is considered to represent a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cavernae sp. nov. is proposed. The type strain is CFH 30183T (= KCTC 49018T = CGMCC 1.16393T).

Microbacterium ureisolvens sp. nov., isolated from a Yellow River sample.

A Gram-stain positive, aerobic, non-motile and short-rod-shaped strain, CFH S00084T, was isolated from a sediment sample of the Yellow River in Henan Province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CFH S00084T clustered within members of Microbacterium and was most closely related to the type strains Microbacterium yannicii JCM 18959T and Microbacterium arthrosphaerae DSM 22421T (98.97 % and 98.36 % similarity, respectively). The strain grew optimally at 25-37 °C, at pH 7.0 and in 0-3 % (w/v) NaCl. The major whole-cell sugars were rhamnose and glucose. The cell-wall peptidoglycan mainly contained glycine, alanine and ornithine. The menaquinones of strain CFH S00084T were MK-13, MK-12 and MK-11. The major fatty acids detected were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genome of strain CFH S00084T was 4.03 Mbp with a G+C content of 70.5 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between CFH S00084T and the other species of the genus Microbacterium were found to be low (ANIm <85 %, ANIb <75 % and dDDH <24 %). The phylogenomic analysis provided evidence for clear phylogenetic divergence between strain CFH S00084T and its closely related type strains. On the basis of the differential physiological properties, chemotaxonomic characteristics and low ANI and dDDH results, strain CFH S00084T is considered to represent a novel species for which the name Microbacteriumureisolvens sp. nov. is proposed. The type strain is CFH S00084T (=KCTC 39802T=DSM 103157T).

Paracoccus halotolerans sp. nov., isolated from a salt lake.

A novel Gram-stain-negative, aerobic, non-motile, coccus-shaped bacterium, designated CFH 90064T, was isolated from a salt lake sediment sample collected from Yuncheng city, Shanxi province, PR China. 16S rRNA gene sequence comparisons and phylogenetic analyses showed that the strain belonged to the genus Paracoccus and clustered with Paracoccus zeaxanthinifaciens R-1512T (98.2 % similarity), Paracoccus homiensis DD-R11T (97.6 % similarity) and Paracoccus fistulariae 22-5T (96.5 % similarity), respectively. Growth of strain CFH 90064T was observed at 10-37 °C, pH 6.0-9.0 and with NaCl concentrations of up to 6.0 % (w/v). Strain CFH 90064T contained Q-10 as the only isoprenoid quinone, and the major fatty acid was C18 : 1ω7c. Polar lipids of strain CFH 90064T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified glycolipid, an unidentified aminolipid and an unidentified phospholipid. The genome of strain CFH 90008T was 3.75 Mbp with a DNA G+C content of 65.1 %. Based on the phylogenetic analyses, low average nucleotide identity results, chemotaxonomic characteristics and differential physiological properties, strain CFH 90064T could not be classified into any recognized species of the genus Paracoccus, suggesting that this strain represents a novel species, for which the name Paracoccushalotolerans sp. nov. is proposed. The type strain is CFH 90064T (=CCTCC AB 2016131T=DSM 103234T).

Paenibacillus esterisolvens sp. nov., isolated from soil.

A Gram-stain-positive, aerobic, motile with peritrichous flagella, rod-shaped bacterium, designated CFH S0170T, was isolated from a soil sample collected from Catba island in Ha Long Bay, Hai Phong City, Vietnam. Comparison of the 16S rRNA gene sequences showed that strain CFH S0170T belonged to the genus Paenibacillus and showed closest relationship with Paenibacillus vulneris CCUG 53270T (98.1 % similarity). Phylogenetic analysis demonstrated that the novel candidate formed a coherent branch with P. vulneris CCUG 53270T and Paenibacillus yunnanensis YN2T. Furthermore, the novel strain shared 87.2 % rpoB gene sequence similarity with P. vulneris CCUG 53270T. Growth of strain CFH S0170T occurred at 10-40 °C, pH 6.0-8.0 and with 0-2.0 % (w/v) NaCl. Strain CFH S0170T contained mannose, glucose and rhamnose as the major whole-cell sugars. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid, lysine and aspartic acid. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, glycolipids and phospholipids. The dominant cellular fatty acids included anteiso-C15 : 0 and C15 : 0. The genomic DNA G+C content was 50.9 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, strain CFH S0170T is affiliated to the genus Paenibacillus, but could be distinguished from other valid species of this genus. It is concluded that strain CFH S0170T should be considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus esterisolvens sp. nov. is proposed. The type strain is CFH S0170T (=KCTC 33624T=BCRC 80802T).

Nocardia heshunensis sp. nov., an actinomycete isolated from soil.

A novel Gram-stain-positive, aerobic, non-motile and acid-fast actinomycete strain, designated CFH S0067T, was isolated from a soil sample collected from Heshun old town in Tengchong, Yunnan province, in south-west PR China. The taxonomic position of strain CFH S0067T was studied in detail using a polyphasic approach. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain CFH S0067T belongs to the genus Nocardia and is closely related to Nocardia concava JCM 12351T (99.3 % similarity), forming a separated branch with this type strain. However, the strain shared 96.0 % gyrB gene sequence similarity with N. concava JCM 12351T. Furthermore, DNA-DNA hybridization showed 56.5±0.6 % DNA relatedness between the novel strain and N. concava JCM 12351T. The whole-cell hydrolysates contained meso-diaminopimelic acid (type IV) and arabinose, galactose, fructose and mannose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified lipid. Strain CFH S0067T contained MK-8 (H4ω-cycl) as the predominant menaquinone. C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω9c and C18 : 0 10-methyl (TBSA) were the major cellular fatty acids. Mycolic acids were also detected. The G+C content of the genomic DNA was determined to be 66.9 mol%. A combination of the low DNA-DNA hybridization values and phenotypic properties demonstrated that strain CFH S0067Tis clearly distinguishable from its most closely related strain, N. concava JCM 12351T. On the basis of this polyphasic study, it is concluded that strain CFH S0067T should be considered to represent a novel species of the genus Nocardia, for which the name Nocardia heshunensis sp. nov. is proposed. The type strain is CFH S0067T (=DSM 46764T=JCM 30085T).

Nocardia tengchongensis sp. nov., isolated from a soil sample.

The taxonomic status was determined of two actinomycetes, designated CFH S0057T and CFH S0065, that were isolated from soil samples collected from an extinct volcano in Tengchong county, Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains CFH S0057T and CFH S0065 belong to the genus Nocardia and formed a single clade within this genus. The two isolates were able to grow at 4-45 °C, pH 5.0-7.0 and with a NaCl tolerance up to 5.0% (w/v). The whole-cell hydrolysates were rich in meso-diaminopimelic acid, galactose, arabinose and fructose. Mycolic acids were present. Strains CFH S0057T and CFH S0065 exhibited a menaquinone system with MK-8 (H4, ω cyclo), and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified phospholipid. Major fatty acids were C16:0, Summed features 3, C18:1 ω9c and C18:0 10-methyl (TBSA). The genomic DNA G + C contents of strains CFH S0057T and CFH S0065 were 65.7 and 66.1 mol%, respectively. The combined genotypic, phenotypic and chemotaxonomic results indicated the isolates are considered to represent a novel species of the genus Nocardia, for which the name Nocardia tengchongensis sp. nov. is proposed. The type strain is CFH S0057T (= KCTC 29485T = JCM 30083T).

Amycolatopsis xuchangensis sp. nov. and Amycolatopsis jiguanensis sp. nov., isolated from soil.

Two novel actinomycetes, designated strains CFH S0322T and CFH S01580T, were isolated from soil samples collected from Xuchang city and Ji Guan cave in Henan province, China, respectively. Their taxonomic positions were investigated by a polyphasic approach. The two isolates were Gram-reaction positive, non-motile, aerobic and catalase-positive actinomycetes. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that the two isolates are closely related to the members of the genus Amycolatopsis, sharing high similarities with Amycolatopsis magusensis KCTC 29056T (99.1 %, CFH S0322T) and Amycolatopsis nigrescens DSM 44992T (98.1 %, CFH S01580T), respectively. The major whole cell sugars of strains CFH S0322T and CFH S01580T were arabinose and galactose and the predominant menaquinone was MK-9(H4). The genomic DNA G+C contents of strains CFH S0322T and CFH S01580T were 70.6 and 68.6 mol%, respectively. Morphological, phylogenetic and chemotaxonomic results suggest that strains CFH S0322T and CFH S01580T are representatives of two new species, for which the names Amycolatopsis xuchangensis sp. nov. and Amycolatopsis jiguanensis sp. nov. are proposed. The type strains are CFH S0322T (=NBRC 110769T =KCTC 39516T) and CFH S01580T (=DSM 101526T =CCTCC AA 2015032T).

Cecembia rubra sp. nov., a thermophilic bacterium isolated from a hot spring sediment.

A Gram-staining negative, rod-shaped bacterium, designated strain YIM 78110(T), was isolated from a sediment sample collected from Hehua hot spring in Tengchong, Yunnan province, south-west China. The taxonomic status of strain YIM 78110(T) was confirmed by a polyphasic approach. 16S rRNA gene sequence analysis indicated that strain YIM 78110(T) belongs to the genus Cecembia, displaying 96.8% and 94.7% sequence similarity with the two most closely related type strains, Cecembia calidifontis RQ-33(T) and Cecembia lonarensis LW9T, respectively. The low value of DNA-DNA hybridization (52.3 ± 2.3%) between strain YIM 78110(T) and its closest neighbour, Cecembia calidifontis RQ-33(T), indicated that this new isolate represented a different genomic species in the genus Cecembia. The temperature for growth ranged from 30 to 50 °C. The pH for growth ranged from pH 4.0 to 10.0, with NaCl tolerance of 0.5-6.0% (w/v). The predominant menaquinone of strain YIM 78110(T) was MK-7 and the major polar lipid was phosphatidylethanolamine. The major fatty acids were iso-C15:0 and C15:0. The DNA G+C content was 47.1 mol%. On the basis of physiological, biochemical and phylogenetic analyses, it is proposed that strain YIM 78110(T) represents a novel species of the genus Cecembia, for which the name Cecembia rubra sp. nov. is proposed. The type strain is YIM 78110(T) ( = CCTCC AB2013287(T) = DSM 28057(T)).

Antitumor activity of recombinant antimicrobial peptide penaeidin-2 against kidney cancer cells.

Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 µg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

De novo characterization of Japanese scallop Mizuhopecten yessoensis transcriptome and analysis of its gene expression following cadmium exposure.

BACKGROUND: Japanese scallop has been cultured on a large-scale in China for many years. However, serious marine pollution in recent years has resulted in considerable loss to this industry. Moreover, due to the lack of genomic resources, limited research has been carried out on this species. To facilitate the understanding at molecular level immune and stress response mechanism, an extensive transcriptomic profiling and digital gene expression (DGE) database of Japanese scallop upon cadmium exposure was carried out using the Illumina sequencing platform. RESULTS: RNA-seq produced about 112 million sequencing reads from the tissues of adult Japanese scallops. These reads were assembled into 194,839 non-redundant sequences with open reading frame (ORF), of which 14,240 putative amino acid sequences were assigned biological function annotation and were annotated with gene ontology and eukaryotic orthologous group terms. In addition, we identified 720 genes involved in response to stimulus and 302 genes involved in immune-response pathways. Furthermore, we investigated the transcriptomic changes in the gill and digestive gland of Japanese scallops following cadmium exposure using a tag-based DGE system. A total of 7,556 and 3,002 differentially expressed genes were detected, respectively, and functionally annotated with KEGG pathway annotations. CONCLUSION: This study provides a comprehensive transcripts sequence resource for the Japanese scallop and presents a survey of gene expression in response to heavy metal exposure in a non-model marine invertebrate via the Illumina sequencing platform. These results may contribute to the in-depth elucidation of the molecular mechanisms involved in bivalve responses to marine pollutants.

TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish.

White spot syndrome virus is a highly pathogenic virus that infects crayfish and other crustaceans. VP28 is one of its major envelope proteins, and plays a crucial role in viral infection. Cell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargoes, and one well known example is TAT peptide from HIV-1 TAT protein. In this study, recombinant plasmids were constructed and transformed into Escherichia coli strain BL21 (DE3) to express TAT-VP28, VP28, TAT-VP28-EGFP and VP28-EGFP fusion proteins. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry methods were used to confirm that TAT fusion proteins can translocate from the intestine to the hemolymph of the crayfish Cambarus clarkii. After immunization, activities of phenoloxidase and superoxide dismutase were analyzed, and it was found that rTAT-VP28 produced the most pronounced increase in both C. clarkii were vaccinated by oral administration of rTAT-VP28 and rVP28 for 7 and 14 days, and rTAT-VP28 resulted in the highest relative percent survival (RPS) (63.3% at 7 days, and 67.8% at 14 days), compared with rVP28 (44.4% at 7 days, and 53.6% at 14 days) following challenge with WSSV after the last day of feeding. This study reports the use of TAT-derived peptide as an oral delivery method of a subunit vaccine against WSSV in C. clarkii.

Improve bioavailability of Harpin protein on plant use PLGA based nanoparticle.

Harpins can induce systemic acquired resistance (SAR) pathway on scores of non-host plant, provide protection against a range of pathogens. In this study, we demonstrated that applied recombinant HarpinZ Pseudomonas syringae pv. tomato (rHrpZ) on tobacco with three kinds of methods: infiltrating from micro-pore into leaf; injecting into petiole, and spraying on leaf, there is great difference in assimilation of protein because of the poor osmosis of tobacco leaves, and with multi-application of rHrpZ, the stimulation effect decreased. We prepared poly d,l-lactide-co-glycolide nanoparticles containing rHrpZ (rHrpZ PLGA NPs). To study the drug effect, we analyzed the change ratio of phenylalanine ammonia lyase (PAL) activity and PR-5dB gene expression after administration of rHrpZ and rHrpZ PLGA NPs on tobacco leaves. The results show that rHrpZ could elicit a rapid and transient increase in both PAL activity and PR-5dB expression, but the effect decreased after multi-application. While sprayed rHrpZ PLGA NPs on leaves, both the change ratio of PAL activity and PR-5dB expression were comparatively smooth and durable. Our study suggested that rHrpZ NPs could help protein enter leaf epidermis and cell wall, release rHrpZ in situ continuously, and enhance the bioavailability of rHrpZ.

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium.

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.