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Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit (ATP5B) with high purity, and to lay foundation for further study.Methods:The ATP5Bgene was amplified by PCR and cloned into the pET28avector and transformed into E.coli BL21 (DE3) .The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5Bantibody by indirect enzyme-linked immunosorbent assay (ELISA) .The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification, a specific band of 1 455bp was obtained, and the pET28aempty vector was ligated to obtain a recombinant pET28a/ATP5Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1:64 000.In karyotype analysis, the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line, and the highest titer of the antibody was 1:240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5Bprotein is successfully prepared by cloning, expressing and purifying the recombinant protein.
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Objective To evaluate in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against Exophiala dermatitidis (E.dermatitidis).Methods The minimum inhibitory concentrations (MICs) of itraconazole and terbinafine against 12 strains of E.dermatitidis were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M38-A2 Document).A broth microdilution checkerboard method was used to evaluate the in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against E.dermatitidis.Results The MIC ranges of terbinafine and itraconazole against E.dermatitidis were 0.060-0.125 mg/L and 0.5-1 mg/L,respectively.The combination of tacrolimus with terbinafine showed synergistic inhibitory effects against 5 strains of E.dermatitidis,while the combination of tacrolimus with itraconazole revealed synergistic effects against 10 strains of E.dermatitidis.No antagonism was observed in either of the two combinations.Conclusion In vitro combination of tacrolimus with itraconazole or terbinafine can enhance the antifungal activity of itraconazole or terbinafine against E.dermatitidis.
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The peptides,proteins and other biological molecules in transudative pleural effusion correlate directly or indirectly with specific physiological and pathological state,reflecting the information regarding the lungs or other parts of the body.In the present study,the peptide fraction in transudative pleural effusion was isolated by uhrafiltration.After desalted and enriched by C18 tips,the peptide mixture was analyzed by nano LC-MS/MS.The results showed that 314 peptides,which were originated from 52 proteins,in pleural transudate were identified.More than half of the peptides were derived from fibrinogen.Many peptides were characterized as displaying ladder sequences.In addition,a large number of proline oxidation modifications were detected in the peptides derived from collagen and fibrinogen.Gene ontology enrichment analysis showed that the most of the proteins extracellular properties of pleural transudate polypeptide components were protein with exocytosis.The study provided a rapid and efficient separation and analysis methods for lung disease markers related peptide compounds in pleural fluid leakage.Also this research provided a rapid and effective method for screening peptide biomarkers related to lung diseases from transudative pleural effusion.
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BACKGROUND: Chemoresistance remains a significant challenge in chronic myelogenous leukemia (CML) management, which is one of the most critical prognostic factors. Elucidation the molecular mechanisms underlying the resistance to chemoresistance may lead to better clinical outcomes. RESULTS: In order to identify potential protein targets involved in the drug-resistant phenotype of leukemia, especially the chronic myelogenous leukemia (CML), we used a high-resolution "ultra-zoom" 2DE-based proteomics approach to characterize global protein expression patterns in doxorubicin-resistant myelogenous leukemia cells compared with parental control cells. Ultra-high resolution of 2DE was achieved by using a series of slightly overlapping narrow-range IPG strips during isoelectric focusing (IEF) separation. A total number of 44 proteins with altered abundances were detected and identified by MALDI-TOF or LC-MS/MS. Among these proteins, enolase, aldolase, HSP70 and sorcin were up-regulated in doxorubicin-resistant myelogenous leukemia cell line, whereas HSP27 was down-regulated. Some of the results have been validated by Western blotting. Both enolase and aldolase were first reported to be involved in chemoresistance, suggesting that process of glycolysis in doxorubicin-resistant myelogenous leukemia cells was accelerated to some extent to provide more energy to survive chemical stress. Possible roles of most of the identified proteins in development of chemoresistance in myelogenous leukemia cells were fully discussed. The results presented here could provide clues to further study for elucidating the mechanisms underlying drug resistance in leukemia. CONCLUSIONS: As a whole, under the chemical stress, the doxorubicin-resistant myelogenous leukemia cells may employ various protective strategies to survive. These include: (i) pumping the cytotoxic drug out of the cells by P-glycoprotein, (ii) increased storage of fermentable fuel, (iii) sophisticated cellular protection by molecular chaperones, (iv) improved handling of intracellular calcium, (v) increased glucose utilization via increased rates of glycolysis. In the present study, proteomic analysis of leukemia cells and their drug resistant variants revealed multiple alterations in protein expression. Our results indicate that the development of drug resistance in doxorubicin-resistant myelogenous leukemia cells is a complex phenomenon undergoing several mechanisms.
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Objective To study the significance of DNA of Toxoplasma gondii in peripheral blood. Methods DNA of T.gondii in peripheral blood of 50 infected rats was detected by polymerase chain reaction. A pair of primers was designed, according to the sequence P30 gene specific to T.gondii, to amplify DNA from T.gondii by PCR. Results The primers amplified DNA specifically from T.gondii and could not amplify DNA from humans, uninfected rat and mouse and from Trichomonas vaginalis and Entamoeba histolytica. DNA of two Toxoplasma parasites was detected by 35 cycles of amplification, indicating a fair sensitivity of the PCR system. Conclusion PCR may have a value for early diagnosis of T.gondii infection in rat.