Design, preparation and characterization of octopus-like self-releasing intracellular protein transporter LEB5 based on Escherichia coli heat-labile enterotoxin.

Despite the great potential of protein drugs as intracellular therapeutic agents, the unmet challenge in breaking through the cell membrane barrier and delivering them to intracellular targets remains. Therefore, developing safe and effective delivery vehicles is critical for fundamental biomedical research and clinical applications. In this study, we designed an octopus-like self-releasing intracellular protein transporter, the LEB5, based on the heat-labile enterotoxin. This carrier comprises five identical units, each of which has three main components: a linker, a self-releasing enzyme sensitivity loop, and the LTB transport domain. The LEB5 comprises five purified monomers that self-assemble to create a pentamer with ganglioside GM1 binding capacity. The fluorescent protein EGFP was used as a reporter system to identify the LEB5 features. The high-purity fusion protein ELEB monomer was produced from modified bacteria carrying pET24a(+)-eleb recombinant plasmids. EGFP protein could effectively detach from LEB5 by low dosage trypsin, according to electrophoresis analysis. The transmission electron microscopy results indicate that both LEB5 and ELEB5 pentamers exhibit a relatively regularly spherical shape, and the differential scanning calorimetry measurements further suggest that these proteins possess excellent thermal stability. Fluorescence microscopy revealed that LEB5 translocated EGFP into different cell types. Flow cytometry showed cellular differences in the transport capacity of LEB5. According to the confocal microscopy, fluorescence analysis and western blotting data, EGFP was transferred to the endoplasmic reticulum by the LEB5 carrier, detached from LEB5 by cleavage of the enzyme-sensitive loop, and released into the cytoplasm. Within the dosage range of LEB5 10-80 µg/mL, cell counting kit-8 assay revealed no significant changes in cell viability. These results demonstrated that LEB5 is a safe and effective intracellular self-releasing delivery vehicle capable of transporting and releasing protein medicines into cells.

The preventive effect of Glycyrrhiza polysaccharide on lipopolysaccharide-induced acute colitis in mice by modulating gut microbial communities.

Acute colitis is characterised by an unpredictable onset and causes intestinal flora imbalance together with microbial migration, which leads to complex parenteral diseases. Dexamethasone, a classic drug, has side effects, so it is necessary to use natural products without side effects to prevent enteritis. Glycyrrhiza polysaccharide (GPS) is an α-d-pyranoid polysaccharide with anti-inflammatory effects; however, its anti-inflammatory mechanism in the colon remains unknown. This study investigated whether GPS reduces the lipopolysaccharide (LPS)-induced inflammatory response in acute colitis. The results revealed that GPS attenuated the upregulation of tumour necrosis factor-α, interleukin (IL)-1ß, and IL-6 in the serum and colon tissues and significantly reduced the malondialdehyde content in colon tissues. In addition, the 400 mg/kg GPS group showed higher relative expressions of occludin, claudin-1, and zona occludens-1 in colon tissues and lower concentrations of diamine oxidase, D-lactate, and endotoxin in the serum than the LPS group did, indicating that GPS improved the physical and chemical barrier functions of colon tissues. GPS increased the abundance of beneficial bacteria, such as Lactobacillus, Bacteroides, and Akkermansia, whereas pathogenic bacteria, such as Oscillospira and Ruminococcus were inhibited. Our findings indicate that GPS can effectively prevent LPS-induced acute colitis and exert beneficial effects on the intestinal health.

[Preparation and characterization of HBc virus like particles with site-directed coupling function].

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.

[Effects of methyl bromide fumigation on community structure of denitrifying bacteria with nitrousoxide reductase gene (nosZ) in soil].

OBJECTIVE: We studied the effect of methyl bromide fumigation on soil edaphic denitrification. METHODS: We adopted nosZ-PCR-RFLP (restriction fragment length polymorphism) method, nosZ-MPN-PCR (Most-Probable-Number- PCR) counting method and soil nitrate elimination rate method, to explore the effect of methyl bromide fumigation on community structure, quantity and activity of denitrifying bacteria in soil. RESULT: After methyl bromide fumigating soil for 100 d, soil denitrification did not change obviously (P > 0. 05). Margalef index, Shannon-wiener index and Evenness index had no significant difference (P > 0.05) in nosZ denitrifying bacterial communities between fumigated soil and the control. There were Rhodopsendomonas, Pseudomonas fluorescens, Herbacspirillum, uncultured bacterium partial in both of them. However, Azospirillum, Rhizobium melibei, Nitrosospira multiformis were exclusively found in the control, and Uncultured Azospirillum sp, Mesorhizobium sp were in fumigated one. Moreover, the number of denitrifying bacteria in the control resolved by nosZ-MPN-PCR (Most-Probable-Number-PCR) was 1.4 times higher than that of the fumigated one. CONCLUSION: After 100 d fumigating soil, the composition of nosZ denitrifying microbial community and the population of denitrifying bacteria changed. Furthermore, there was no difference in denitrification between the fumigated soil and the control.

[Screen, identification and optimized fermentation condition of an actinomycete strain against pathogenic fungus Fulvia fulva].

An actinomycete strain xjy was isolated from the soil of cotton field in Xinjiang against pathogenic fungus Fulvia fulva. The growth of 23 plant pathogens and 6 bacteria were strongly inhibitded by strain xjy on PDA plate. Antimicrobial spectrum of fermentation filtrate of strain xjy is extensive and selective to different pathogens. The morphology, cultural characteristics, physiological and biochemical properties, chemotaxonomy and 16S rDNA sequences of this strain were studied. The strain showed faint yellow vegetative mycelium, spiral spore-bearing filaments and column spores with smooth surface. No pigment was produced in culture. The cell wall type I and sugar type C showed the strain with streptomyces character. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related species and showed 99.6% identity of nucleotide sequence of 16S rDNA with Streptomyces lavendulae. From the polyphasic taxonomical view, the strain xjy falls into Streptomyces lavendulae. The optimum fermentation condition of strain xjy for producing the most effective ferment filtrate were cultured in 2% soybean flour, 2% glucose, 0.8% NaCl, 0.2% CaCO3, 0.32% (NH4)2 SO4, the initial pH of 7.0, at 28 degrees C and shaked at 180r/mim for 6d. These results are valuable to strain application, antibiotics purification and its industrialization.

[Endophytic bacteria isolated from wheat and their antifungal activities to soil-borne disease pathogens].

In this paper, endophytic bacteria (EB) were isolated from the roots, stems, leaves and immature seeds of wheat at its different growth stages. The EB populations in fresh wheat tissues reached 5.0 x 10(5) CFU x g(-1) on average, with a significant difference among different tissues, growth stages and fields. The EB count was 7.8 x 10(5) in wheat roots, 4.8 x 10(5) in stems, 3.2 x 10(5) in leaves, and 2.8 x 10(5) CFU x g(-1) in immature seeds, and was estimated as 3.1 x 10(5), 5.7 x 10(5) and 7.0 x 10(5) CFU x g(-1) at seedling, elongation and filling stages, respectively. A total of 51 antifungal EB isolates were trapped by the wheat pathogenic fungus Gaeumannomyces graminis, and 45 by Rhizoctonia cerealis. Among them, 78 isolates showed antifungal activities in vitro. A total of 23 isolates from roots (6), stems (7), leaves (9) and immature seeds (1) were highly inhibitory to the mycelial growth of G. graminis var. tritici, with the diameters of their inhibition zone exceeding 10 mm. The other twenty isolates from different plant parts were also active against R. cereali. It was revealed that higher ratios of EB isolates with high antifungal activities were found in leaves, as compared with stems, roots and immature seeds.