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Hou J.,China Agricultural University | Wang L.,China Agricultural University | He W.,China Agricultural University | Zhang H.,Beijing Entry Exit Inspection and Quarantine Bureau | Feng W.-H.,China Agricultural University
Virus Research | Year: 2012

Atypical porcine reproductive and respiratory syndrome (PRRS) characterized by high morbidity and mortality emerged in China in 2006. The causative agent was confirmed to be a highly pathogenic PRRS virus (HP-PRRSV). However, the pathogenesis of HP-PRRSV is still uncertain. Here, the ability of the highly pathogenic strains (HV and JX) to induce tumor necrosis factor alpha (TNF-α) was studied. Our results showed that HV and JX were weaker inducers of TNF-α than the conventional strain CH-1a. Moreover, HV infection was demonstrated to suppress extracellular signal-regulated kinase (ERK) phosphorylation at the early time points. Pharmacologic inhibition or activation of ERK revealed that TNF-α production in HV-infected macrophages was associated with the activation status of ERK. Furthermore, HV- and JX-infection could potently impair lipopolysaccharide (LPS)- and poly(I:C)-stimulated TNF-α release in a dose dependent manner whereas synergistic effects were observed at mRNA level. The observation suggested the involvement of posttranslational impact of HP-PRRSV on TNF-α production, which might be attributed to the reduced ERK1/2 phosphorylation in response to toll-like receptor (TLR)-ligation. Taken together, our results indicated that HP-PRRSV infection could impair TNF-α production by inhibiting ERK signaling pathway, which might partially contribute to the pathogenesis of HP-PRRSV. © 2012 Elsevier B.V. Source


Cheng J.,Beijing Entry Exit Inspection and Quarantine Bureau
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology | Year: 2013

To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio cholerae and establish the double-antibody sandwich ELISA method for testing Vibrio cholerae from food products. BALB/c mice were immunized with flagellin extracted from Vibrio cholerae Vc75 by differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer in serum reached 1:32 000. The hybridoma cell lines were obtained by regular subcloning and used to generate ascites. And mAbs reacting to Vibrio cholerae flagellin were achieved by purified from the ascites. Six hybridoma cell lines stably secreting mAbs against Vibrio cholerae flagellin were taken and named VcNo.1-VcNo.6. The mAb titer in serum by indirect ELISA was 1:2 × 10(6). SDS-PAGE showed that the flagellin protein molecular weight (Mr) was 44 000 and the purity was high. Double-antibody sandwich ELISA method was set up using VcNo.6 antibody for detecting Vibrio cholerae. The sensitivity reached 10(3) CFU/mL. The ELISA method showed high specificity to Vibrio cholerae through testing 100 Vibrio cholerae (100% positive) and 101 non-Vibrio cholerae strains (100% negative). The detection limit was 1 CFU/g sample in artificial contaminated samples. The mAbs against flagellin core protein of Vibrio cholerae was successfully prepared and used to set up the double-antibody sandwich ELISA. The mAb of VcNo.6 was highly specific to Vibrio cholerae. The sensitivity of the established ELISA was as high as 10(3) CFU/mL. Moreover, it did not react to non-Vibrio cholerae strains. Therefore, the mAbs of VcNo.6 could be widely used in Vibrio cholerae detection from food samples as well as clinical samples. Source


Zeng J.,Beijing Entry Exit Inspection and Quarantine Bureau
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology | Year: 2013

To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio (V.) vulnificus and establish the double-antibody sandwich ELISA for testing V. vulnificus from food products. BALB/c mice were immunized by flagellin which was extracted by differential centrifugation method from V. vulnificus ATCC 1.1758. The splenocytes taken from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer reached 1:32 000 in serum. The hybridoma cell lines were prepared and screened by hybridoma technique and ELISA. The cells secreting mAbs were cloned through the limited dilution. The hybridoma cell lines were used to generate ascites. The mAbs were obtained by purification from the ascites. Five hybridoma cell lines which stably secreted mAbs against flagellin were isolated and named VVNo.1-VVNo.5. The mAb titer in serum reached 1:(2×10(6);). SDS-PAGE showed that the relative molecular mass (Mr;) of flagellin protein was 44 000, and that the purity was high. Double-antibody sandwich ELISA was set up using VVNo.5 antibody, and the sensitivity reached 10(3); CFU/mL culture broth. The ELISA showed that VVNo.5 antibody was highly specific to V. vulnificus. The detection limit was 2 CFU/25 g culture broth in artificial contaminated samples. The mAbs were obtained against flagellin core protein of V. vulnificus. The double-antibody sandwich ELISA was established using VVNo.5 mAb. The monoclonal antibody of VVNo.5 was highly specific to V. vulnificus, without cross reaction with non-target bacteria. Therefore the monoclonal antibodies of VVNo.5 could be widely used in detecting V. vulnificus from food samples as well as the clinical samples. Source


Zhang L.,Beijing Entry Exit Inspection and Quarantine Bureau
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology | Year: 2013

To prepare monoclonal antibodies (mAbs) against flagellin core protein of Vibrio (V.) parahaemolyticus and establish the double-sandwich ELISA for testing V.parahaemolyticus from food products. BALB/c mice were immunized with flagellin which was extracted from V.parahaemolyticus ATCC17802 through differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells in log-phase growth when the antibody titer in serum was 1:32 000. The hybridoma cell lines were screened by regular subcloning approach and used to generate ascites. And mAbs reacting to V.parahaemolyticus flagellin were obtained by purifying from the ascites. Six hybridoma cell lines secreting mAbs against V.parahaemolyticus flagellin were prepared and named VpNo.1-VpNo.6. The mAb titer in serum by indirect ELISA was 1:2×10(6);. The mAbs were subjected to Ig classes and subclasses detection and Western blotting. The Ig subclasses of these mAbs were IgG1, IgG1, IgM, IgG1, IgG2a and IgM, respectively. SDS-PAGE showed that the flagellin protein molecular weight (Mr;) was 42 000. Using VpNo.6, Double-sandwich ELISA kit was set up and applied in detecting V.parahaemolyticus, and its sensitivity was 10(3); CFU/mL. The ELISA showed VpNo.6 had a desirable specificity to V.parahaemolyticus in testing 134 V.parahaemolyticus and 74 non-V.parahaemolyticus strains. The lowest limit of detection was 21 CFU/g sample in the base-material addition test. The monoclonal antibodies against flagellin core protein of V.parahaemolyticus were successfully prepared. The double-sandwich ELISA we established using VpNo.6 mAb had a sensitivity as high as 10(3); CFU/mL. The monoclonal antibody of VpNo.6 was highly specific to V.parahaemolyticus. Source


Wu H.,Peking Union Medical College | Guo J.,Astrazeneca | Chen S.,Peking Union Medical College | Liu X.,Beijing Entry Exit Inspection and Quarantine Bureau | And 3 more authors.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2013

Over the past few years, the applications of liquid chromatography coupled with mass spectrometry (LC-MS) in natural product analysis have been dramatically growing because of the increasingly improved separation and detection capabilities of LC-MS instruments. In particular, novel high-resolution hybrid instruments linked to ultra-high-performance LC and the hyphenations of LC-MS with other separation or analytical techniques greatly aid unequivocal identification and highly sensitive quantification of natural products at trace concentrations in complex matrices. With the aim of providing an up-to-date overview of LC-MS applications on the analysis of plant-derived compounds, papers published within the latest years (2007-2012) involving qualitative and quantitative analysis of phytochemical constituents and their metabolites are summarized in the present review. After briefly describing the general characteristics of natural products analysis, the most remarkable features of LC-MS and sample preparation techniques, the present paper mainly focuses on screening and characterization of phenols (including flavonoids), alkaloids, terpenoids, steroids, coumarins, lignans, and miscellaneous compounds in respective herbs and biological samples, as well as traditional Chinese medicine (TCM) prescriptions using tandem mass spectrometer. Chemical fingerprinting analysis using LC-MS is also described. Meanwhile, instrumental peculiarities and methodological details are accentuated. © 2012 Elsevier B.V. Source

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