ABSTRACT
Objective:To screen and identify the protein that interacts with the adhesion protein of Mycoplasma genitalium (MgPa)from T7-phage display cDNA library of human uroepithelial cells(SV-HUC-1).Methods:Recombinant adhesion protein of My-coplasma genitalium(rMgPa)was used as target molecule to biopan the T7 phage display cDNA library of SV-HUC-1 cell,the selected positive clones were analysed using DNA sequencing and BLAST analysis and identified by means of indirect ELISA,Dot immunoblot and Far-western blot.Results:After four rounds of biopanning,positive phages were obviously enriched.According to the results of DNA sequencing and BLAST analysis,the selected randomly 32 positive clones included 7 kinds different sequences,of which the number of RPL35 repeats was the most.The results of indirect ELISA,Dot immunoblot and Far-western blot showed that 7 representative phages could bind specifically with rMgPa.Conclusion:60S ribosomal protein L35(RPL35) may be the interacting protein of MgPa,which lays the experimental foundation for understanding the function of MgPa and the pathogenesis of Mycoplasma genitalium.
ABSTRACT
We preliminarily investigated the role of NLRP3 inflammasome in Helicobacter pylori (H.pylori) Tipa-induced pro-inflammatory cytokines secretion in PMA-differentiated human acute monocytic leukemia cell line THP-1.PMA-differentiated THP-1 cells were treated with pure recombinant Tips protein.The secretion levels of TNF-α,IL-1β and IL-18 in supernatant of culture medium were detected by ELISA.Then we blocked MyD88/NF-κB and NLRP3/Caspase-1 pathways by PDTC or the general ROS scavenger,NAC,respectively,and determined the secretion levels of proinflammatory cytokines and the expression levels of NLRP3 and Caspase-1.The results showed that Tips protein can significantly induced the secretion of TNF-α,IL-1β and IL-18 in THP-1 cells in a time and dose-dependent manner.Levels of TNF-α,IL-1β and IL-18 approached their peaks at 6 h post-treatment by 40 μg/mL of Tipα protein (P＜0.05).Moreover,the blockade of NF-κB signaling pathway by PDTC can inhibit the secretion of proinflammatory cytokines and the expression of NLRP3 and Caspase-1.When THP-1 cells were pre-treated with ROS scavenger NAC,the Tipα-induced increased IL-1β and IL-18 secretion was obviously eliminated (P＜0.05),while TNF-α level had no significant difference,and the expression levels of Caspase-1 and NLRP3 also have a significant decrease.Our results demonstrated that Tipα can promote THP-1-drived macrophages to secrete proinflammatory cytokines TNF-α,IL-1β and IL-18,and the NLRP3/Caspase-1 pathway may be involved in the Tipα protein-induced IL-1β and IL-18 secretion.
ABSTRACT
Objective To construct the Neisseria surface protein A (NspA) DNA vaccine of Neisseria gonorrhoeae and evaluate the humoral and cellular immune responses induced by this vaccine in mice model.Methods The recombinant expression vector pcDNA3.1 (+)/NspA was constructed by inserting NspA gene into the eukaryotic expression vector pcDNA3.1 (+) and confirmed by poly merase chain reaction (PCR),restriction enzymes HindⅢ,XbaⅠand DNA sequencing.NspA mR- NA in transfected RAW264.7 cells and NspA protein expression in transfected COS-7 cells were de- tected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical stai- ning,respectively.Forty-five male BALB/c mice were immunized with pcDNA3.1 (+)/NspA recom binant plasmid.The level of serum anti-Neisseria gonorrhoeae antibody of the immunized mice was detected by tube agglutination test,and the level of interieron (IFN)-?was assayed by enzyme-linked immunosorbent assay (ELISA).The proliferation of splenocytes was determined by methyl thiazolyl tetrazolium (MTT) colormetry.The NspA gene in BALB/c mice was identified by PCR with the total DNA extracted from quadriceps femoris in immunized sites.Results Restriction enzymes digestion a- nalysis and DNA sequencing results revealed that the pcDNA3.1 (+)/NspA had been constructed successfully.NspA gene had been transcripted and expressed in mammalian cells.The peak titer of specific antibody was 1:640 in pcDNA3.1(+)/NspA immunized group and there was no specific an- tibody detected in both pcDNA3.1 (+) immunized group and PBS group.The IFN-?level in pcD NA3.1 (+) immunized group was (23.79?11.85)pg/mL and that in pcDNA3.1 (+)/NspA immu- nized group was(169.71?30.52)pg/mL (P
ABSTRACT
<p><b>BACKGROUND</b>Daxx has been identified as a nuclear protein that involves in apoptosis and transcriptional repression. Daxx co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription. Human Daxx (hDaxx) is a protein that functions as a transcriptional regulation through its interaction with some DNA-associated proteins. The aim of this study was to explore the transcriptional regulatory effect of hDaxx interacting with adenovirus (Ad) 12 E1B (Ad12E1B) 55-kDa oncoprotein.</p><p><b>METHODS</b>The co-localization of hDaxx-Ad12E1B or hDaxx-PML protein in the nucleus was observed under a confocal microscope. Interaction of hDaxx and Ad12E1B was analyzed by yeast two-hybrid assay. Direct binding of hDaxx and Ad12E1B was analyzed using coimmunoprecipitation and Western blot in vivo and in vitro. The activity of a luciferase reporter gene, which was regulated by an hDaxx modulated thymidine kinase (TK) promoter, was detected in an automat luminometer.</p><p><b>RESULTS</b>Ad12E1B, which co-localized with hDaxx in the nuclei of G401-CC3 cells, disrupted the co-localization of hDaxx and PML in the PML oncogenic domains (PODs). hDaxx bound directly to Ad12E1B in vivo and in vitro. hDaxx interacted with Ad12E1B along its full length. Ad12E1B enhanced transcriptional repression activity of hDaxx.</p><p><b>CONCLUSION</b>Ad12E1B disrupts the co-localization of hDaxx with PML in PODs and enhances transcriptional repression activity of hDaxx.</p>
Subject(s)
Humans , Adaptor Proteins, Signal Transducing , Adenovirus E1B Proteins , Physiology , Carrier Proteins , Genetics , Cell Line, Tumor , Intracellular Signaling Peptides and Proteins , Neoplasm Proteins , Nuclear Proteins , Genetics , Promyelocytic Leukemia Protein , Repressor Proteins , Physiology , Transcription Factors , Transcription, Genetic , Tumor Suppressor ProteinsABSTRACT
<p><b>BACKGROUND</b>This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.</p><p><b>METHODS</b>Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor kappaB (NF-kappaB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.</p><p><b>RESULTS</b>M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-kappaB activation, a possible mechanism for the induction of iNOS expression.</p><p><b>CONCLUSION</b>This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-kappaB.</p>