Mimotope ELISA for Detection of Broad Spectrum Antibody against Avian H5N1 Influenza Virus

Science and Technology Foundation of Fujian Province [2009YZ0002]; National Natural Science Foundation of China [30901077]; National High Technology Research and Development Program [2010AA022801]Background: We have raised a panel of broad spectrum neutralizing monoclonal antibodies against the highly pathogenic H5N1 avian influenza virus, which neutralize the infectivity of, and afford protection against infection by, most of the major genetic groups of the virus evolved since 1997. Peptide mimics reactive with one of these broad spectrum H5N1 neutralizing antibodies, 8H5, were identified from random phage display libraries. Method: The amino acid residues of the most reactive 12mer peptide, p125 (DTPLTTAALRLV), were randomly substituted to improve its mimicry of the natural 8H5 epitope. Result: 133 reactive peptides with unique amino acid sequences were identified from 5 sub-libraries of p125. Four residues (2,4,5.9) of the parental peptide were preserved among all the derived peptides and probably essential for 8H5 binding. These are interspersed among four other residues (1,3,8,10), which exhibit restricted substitution and probably could contribute to binding, and another four (6,7,11,12) which could be randomly substituted and probably are not essential for binding. One peptide, V-1b, derived by substituting 5 of the latter residues is the most reactive and has a binding constant of 3.16x10(-9) M, which is 38 fold higher than the affinity of the parental p125. Immunoassay produced with this peptide is specifically reactive with 8H5 but not also the other related broad spectrum H5N1 avian influenza virus neutralizing antibodies. Serum samples from 29 chickens infected with H5N1 avian influenza virus gave a positive result by this assay and those from 12 uninfected animals gave a negative test result. Conclusion: The immunoassay produced with the 12 mer peptide, V1-b, is specific for the natural 8H5 epitope and can be used for detection of antibody against the broad spectrum neutralization site of H5N1 avian influenza virus

Chemical Composition and Antimicrobial Activity of Three Essential Oils from <i>Curcuma wenyujin</i>

Curcuma wenyujin is a traditional medicinal plant in China. The non-steamed rhizomes, steamed rhizomes and steamed roots of this plant are used as herbal medicines in three clinics, namely Pian-jiang-huang (PJH), Wen-e-zhu (WEZ), and Wen-yu-jin (WYJ), and are officially listed in the Chinese Pharmacopoeia. The purpose of this study was to conduct a comparative analysis of the three essential oils extracted from the C. wenyujin rhizomes and roots using GC-MS, and in doing so thirty compounds were identified. Principal component analysis (PCA) effectively distinguished the samples taken from the three different groups. Monoterpenoids, including camphene, linalool, camphor, isoborneol, borneol and eucalyptol, were characteristic components of the PJH oil, while β-elemene, β-elemenone, γ-elemene and δ-elemene were typical components of the WEZ oil, and propanenitrile, caryophyllene oxide, (-)-caryophyllene, germacrene B, pogostol and α-humulene were representative ingredients of the WYJ oil. The ratio of sesquiterpenoids to monoterpenoids in PJH, WEZ, and WYJ were 2:1, 5:1 and 7:1, respectively. The antimicrobial activities of the three essential oils and of the six main ingredients were tested against two bacterial and one fungal strains using agar diffusion and broth dilution methods. The essential oil of PJH was shown to present a higher antimicrobial activity than that of WEZ and WYJ. Based on the Partial Least Square Model (PLS), the correlation between the antimicrobial activity of the tested oils and the identified chemical components was discussed and potential components of the antimicrobial activity were predicted according to Variable Importance in the Project (VIP) Value. The tested monoterpenes eucalyptol and isoborneol demonstrated a higher inhibitory activity than the sesquiterpenes germacrone, curdione and β-elemene. Therefore, the potent inhibitory effect of the PJH oil might be attributed to its higher content of monoterpenes. The MIC values for the essential oils and their ingredients ranged from 62.5 to 500 μg/mL. </jats:p

Tight controlled expression and secretion of Lactobacillus brevis SlpA in Lactococcus lactis

© Springer Science+Business Media B.V. 2012Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xyloseinducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle