Severe fever with thrombocytopenia syndrome virus (SFTSV), a newly discovered member of the Bunyaviridae family, is the causative agent of an emerging hemorrhagic fever, SFTS, in China. Currently, there are no vaccines or effective therapies against SFTS. In this study, a combinatorial human antibody library was constructed from the peripheral lymphocytes of 5 patients who had recovered from SFTS. The library was screened against purified virions for the production of single-chain variable-region fragments (ScFv). Of the 6 positive clones, one clone (monoclonal antibody [MAb] 4-5) showed neutralizing activity against SFTSV infection in Vero cells. MAb 4-5 was found to effectively neutralize all of the clinical isolates of SFTSV tested, which were isolated from patients in China from 2010 to 2012. MAb 4-5 was found to bind a linear epitope in the ectodomain of glycoprotein Gn. Its neutralizing activity is attributed to blockage of the interactions between the Gn protein and the cellular receptor, indicating that inhibition of virus-cell attachment is its main mechanism. These data suggest that MAb 4-5 can be used as a promising candidate molecule for immunotherapy against SFTSV infection.
Neuroinflammation is considered to have a prominent role in the pathogenesis of Alzheimer's disease (AD). Microglia are the resident macrophages of the central nervous system, and modulating microglia activation is a promising strategy to prevent AD. Essential oil of Jasminum grandiflorum L. flowers is commonly used in folk medicine for the relief of mental pressure and disorders, and analyzing the volatile compound profiles and evaluating the inhibitory effects of J. grandiflorum L. essential oil (JGEO) on the excessive activation of microglia are valuable for its application. This study aims to explore the potential active compounds in JGEO for treating AD by inhibiting microglia activation-integrated network pharmacology, molecular docking, and the microglia model. A headspace solid-phase microextraction combined with the gas chromatography-mass spectrometry procedure was used to analyze the volatile characteristics of the compounds in J. grandiflorum L. flowers at 50°C, 70°C, 90°C, and 100°C for 50 min, respectively. A network pharmacological analysis and molecular docking were used to predict the key compounds, key targets, and binding energies based on the detected compounds in JGEO. In the lipopolysaccharide (LPS)-induced BV-2 cell model, the cells were treated with 100 ng/mL of LPS and JGEO at 7.5, 15.0, and 30 μg/mL, and then, the morphological changes, the production of nitric oxide (NO) and reactive oxygen species, and the expressions of tumor necrosis factor-α, interleukin-1β, and ionized calcium-binding adapter molecule 1 of BV-2 cells were analyzed. A total of 34 compounds with significantly different volatilities were identified. α-Hexylcinnamaldehyde, nerolidol, hexahydrofarnesyl acetone, dodecanal, and decanal were predicted as the top five key compounds, and SRC, EGFR, VEGFA, HSP90AA1, and ESR1 were the top five key targets. In addition, the binding energies between them were less than -3.9 kcal/mol. BV-2 cells were activated by LPS with morphological changes, and JGEO not only could clearly reverse the changes but also significantly inhibited the production of NO and reactive oxygen species and suppressed the expressions of tumor necrosis factor-α, interleukin-1β, and ionized calcium-binding adapter molecule 1. The findings indicate that JGEO could inhibit the overactivation of microglia characterized by decreasing the neuroinflammatory and oxidative stress responses through the multi-compound and multi-target action modes, which support the traditional use of JGEO in treating neuroinflammation-related disorders.
Objective In order to evaluate anti-H7 serum agar medium assay for flagellar antigen of Enterohemorrhagic Escherichia coli O157∶H7.Methods 109 suspicious stains of O157∶H7 derived from feces of human,animal,food and water of enviroment were detected by anti-H7 serum agar medium assay for H7 flagellar antigen,in compare with tube agglutination test and the detection of species gene for H7, flagellor antigen positive and negative as control. Results The results showed that the three assay for detecting H7 flagellar antigen were good for the strains of positive and negative marker.The rate of sensitivity,specificity and coincident of anti-H7 serum agar medium assay were 100% at all in compare with tube agglutination test, and were 93.75%, 95.31%, 97.25% respectively in compare with detection of specific gene for H7. Conclusion The data showed that anti-H7 serum agar medium assay was a rapid,convenient and reliable method for detecting H7 flagellar antigen, so it was worthy to be used.
Mastitis caused by Escherichia coli ( E. coli ) remains a threat to dairy animals and impacts animal welfare and causes great economic loss. Furthermore, antibiotic resistance and the lagged development of novel antibacterial drugs greatly challenge the livestock industry. Phage therapy has regained attention. In this study, three lytic phages, termed vB_EcoM_SYGD1 (SYGD1), vB_EcoP_SYGE1 (SYGE1), and vB_EcoM_SYGMH1 (SYGMH1), were isolated from sewage of dairy farm. The three phages showed a broad host range and high bacteriolytic efficiency against E. coli from different sources. Genome sequence and transmission electron microscope analysis revealed that SYGD1 and SYGMH1 belong to the Myoviridae , and SYGE1 belong to the Autographiviridae of the order Caudovirales . All three phages remained stable under a wide range of temperatures or pH and were almost unaffected in chloroform. Specially, a mastitis infected cow model, which challenged by a drug resistant E. coli , was used to evaluate the efficacy of phages. The results showed that the cocktails consists of three phages significantly reduced the number of bacteria, somatic cells, and inflammatory factors, alleviated the symptoms of mastitis in cattle, and achieved the same effect as antibiotic treatment. Overall, our study demonstrated that phage cocktail may be a promising alternative therapy against mastitis caused by drug resistant E. coli .
Background: Numerous studies have shown that mitochondrial damage induces inflammation and activates inflammatory cells, leading to sepsis, while sepsis, a systemic inflammatory response syndrome, also exacerbates mitochondrial damage and hyperactivation. Mitochondrial autophagy eliminates aged, abnormal or damaged mitochondria to reduce intracellular mitochondrial stress and the release of mitochondria-associated molecules, thereby reducing the inflammatory response and cellular damage caused by sepsis. In addition, mitochondrial autophagy may also influence the onset and progression of sepsis, but the exact mechanisms are unclear. Methods: In this study, we mined the available publicly available microarray data in the GEO database (Home - GEO - NCBI (nih.gov)) with the aim of identifying key genes associated with mitochondrial autophagy in sepsis. Results: We identified four mitophagy-related genes in sepsis, TOMM20, TOMM22, TOMM40, and MFN1. Conclusion: This study provides preliminary evidence for the treatment of sepsis and may provide a solid foundation for subsequent biological studies.
In this work, we have used a new method to predict the epitopes of HA1 protein of influenza virus to several antibodies HC19, CR9114, BH151 and 4F5. While our results reproduced the binding epitopes of H3N2 or H5N1 for the neutralizing antibodies HC19, CR9114, and BH151 as revealed from the available crystal structures, additional epitopes for these antibodies were also suggested. Moreover, the predicted epitopes of H5N1 HA1 for the newly developed antibody 4F5 are located at the receptor binding domain, while previous study identified a region 76-WLLGNP-81 as the epitope. The possibility of antibody recognition of influenza virus via different mechanism by binding to different epitopes of an antigen is also discussed.
To establish a rapid eukaryotic expression system of hemagglutinin (HA) gene of novel avian influenza H7N9 using lentiviral vector, express the recombinant protein and study its functions in human embryonic kidney HEK293T cells.The full-length HA gene was amplified from H7N9 genomic RNA by reverse transcription PCR (RT-PCR) and linked with pMD18-T vector to generate pMD18-T-HA plasmid. Blunt-end HA gene with Kozak sequence was amplified from pMD18-T-HA vector, and then pLenti-HA-V5 expression vector was constructed by Topo cloning for transient expression in HEK293T cells. Expression of HA-V5 recombinant protein was confirmed by immunofluorescence assay (IFA) and Western blotting. Hemagglutination test was performed to evaluate the biological activity of the recombinant protein.The full-length HA gene (1 683 bp) was obtained and eukaryotic expression plasmid was constructed successfully. A recombinant protein with relative molecular mass (Mr) 70 000 was expressed and the antigenicity and binding specificity to positive serum were demonstrated by IFA and Western blotting. The hemagglutination activity was proved by hemagglutination test. IFA and Western blotting showed that the Mr 70 000 recombinant protein had an immuoreactivity to positive serum. The hemagglutination activity was confirmed by hemagglutination test.The rapid eukaryotic expression system of HA gene was successfully constructed, which laid a solid foundation for further research on subunit vaccine development, neutralizing epitope mapping and packaging pseudovirus.
Abstract A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu 3+ ) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.
Objective
To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ (StxⅡ) in shiga toxin-producing Escherichia coli (STEC) infection.
Methods
A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-antibody sandwich ELISA. The established ELISA system was used to detect StxⅡ in the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated.
Results
Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-antibody sandwich ELISA was set up. StxⅡ and its variants rather than StxⅠ was detected in the culture supernatants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commercial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity.
Conclusion
The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential candidate on the diagnosis and therapy of STEC infection.
Key words:
Shiga toxin-producing Escherichia coli (STEC); Shiga toxin Ⅱ (StxⅡ); Monoclonal antibody; Double-antibody sandwich ELISA
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