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Objective To construct the prokaryotic expression vector carrying Mycobacterium tuberculosis gene mmpL6 (rv1557), overexpress and purify the recombinant MmpL6 (Rv1557) protein in Escherichia coli (E. coli).Methods The DNA fragment of MmpL6 was amplified by polymerase chain reaction using the genomic DNA of Mycobacterium tuberculosis H37Rv strain as a template. The target gene was cloned into pET21b vector to construct the pET21b-MmpL6 expression plasmid, and then transformed into E. coli for protein expression. Recombinant protein expression was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and detected by SDS-PAGE combined with Western blot. The overexpressed MmpL6 protein was purified by Ni-affinity chromatography and gel filtration chromatography. Results The recombinant pET21b-MmpL6 plasmid was successfully constructed and the highest expression level was obtained at 25 ℃ in Rosetta strain induced by IPTG. Conclusion The successful expression and purification of MmpL6 in E. coli lays the foundation for the further structure and function studies of the protein, and also provides clues to the design of anti-tuberculosis drugs targeting efflux pump proteins.
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Objective To investigate Aβ42 and P-tau levels in cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI). Methods CSF of 25 cases of MCI and 14 cases of cognitively normal (CN) were investigated. The CSF levels of Aβ42 and P-tau were detected by ELISA method. Correlation analysis was used to analyze the correlation between P-tau level and MMSE score in MCI. Results The CSF level of Aβ 42 was higher and P-tau was lower in MCI than CN group(P<0.05). P-tau level in MCI was also negatively correlated with MMSE score (P<0.05). Conclusion Aβ42 and P-tau levels in CSF may be valuable biological markers in the diagnosis of MCI. P-tau level in CSF of MCI may be related to the severity of cognitive impairment.
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Objective:To produced mouse monoclonal antibodies against IL 15 that should be a valuable reagent for diagnosis and investigation into the pathological significance of IL 15 protein expression Methods:rhIL 15 was conjugated to naked bacteria of salmonella typhimurium as immunogen to immunize BALB/c mice The spleen cells were fused with SP2/0 cells using polyethlene glycol, suspended in selective growth medium (HAT) Clones secreting specific monoclonal antibody were screened by indirect enzyme linked immunosorbent assay(ELISA) After several cloning,several hybridoma cell clones stably producing anti IL 15 monoclonal antibody were obtainied The McAb was identified by Western blot with goat polyclonal antibody raised against mouse Results:Four hybridoma cell strains which could produce monoclonal antibodies to IL 15 were obtained The chromosome number of hybridoma cells was between 90 108, there were metacentric chromosome and submetacentric chromosome in hybridoma cells Monoclonal antibodies were able to inhibit and/or neutralize the effects of IL 15, while naked bacteria could not The sensitivity of Dot blot was 1 5 ng Conclusion:Monoclonal antibodies to rhIL 15 have been prepared and characterized
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Objective To investigate the expressions of Bcl-2 and Bax protein in the renal tissues of patients with lupus nephritis(LN)and their role in the pathology alteration of LN.Methods During 2003-01～2003-04 immunohistochemical staining was used to detect the expressions of Bcl-2 and Bax in renal tissues of 44 cases of LN in the First Affiliated Hospital of Guangxi Medical University.Their relationship with pathology was also observed.Results (1)The expression of Bcl-2 and Bax was significantly increased and both were positively correlated with the grade of intraglomerular cell proliferation and proliferating cell nuclear antigen(PCNA)positive glomerular cells.(2)The expression of Bcl-2 in interstitial cells was significantly increased and positively correlated with the degree of interstitial infiltration of inflammatory cells.(3)The expression of Bax in the tubule of LN was increased and was positively associated with tubulointerstitial lesions.Conclusion There is abnormal expressions of Bcl-2 and Bax in renal tissues of LN.Overexpression of Bcl-2 in renal tissues of LN might play an important role in the process of glomerular hypercellularity and interstitial infiltration of inflammatory cells.Overexpression of Bax in tubular ducts is correlated with tubulointerstitial lesions.
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Objective:Studies on purification and bioactivity detection of recombinant human IL 15 protein.Methods:The rhIL 15 were purified from rhIL 15 producing DH5? bacteria the inclusion bodies extracted first and followed by gel filtration,ion exchange chromatography and reverse phase high performance liquid chromatography(RP HPLC).The bioactivities were detected by NK cytotoxicity.Results:Recombinant human IL 15(rhIL 15)was obtained with a purity of 99 percent.The N terminal amino acid sequence of the rhIL 15 was as same as the natural IL 15.The specific activity of the rhIL 15 is 1?10 7 U/mg,and the rhIL 15 significantly enhances NK cytotoxicity.Conclusion:Recombinant human IL 15 of high purity and bioactivity could be obtained by three steps of purification.The high quanity and quality of the rhIL 15 is important to both the laboratory research and the clinical trial.