ABSTRACT
<p><b>OBJECTIVE</b>To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).</p><p><b>METHODS</b>The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.</p><p><b>RESULTS</b>CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.</p><p><b>CONCLUSION</b>The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.</p>
Subject(s)
Bacterial Proteins , Genetics , Reference Standards , Fluoroimmunoassay , Methods , Gene Amplification , Mycobacterium tuberculosis , Reference StandardsABSTRACT
AIM: To investigate the antitumor activity and mechanism of Paecilomyces tenuipes polysaccharide(PTPS).METHODS: PTPS-I was obtained by water extraction and alcohol precipitation,and purified by DEAE-cellulose and Sephadex G-100 chromatography.Human erythroleukemia cell line K562,laryngocarcinoma cell line Hep2 and hepatic carcinoma cell line SMMC-7721were co-cultured with PTPS-I or the conditioned medium which prepared with PTPS-I-stimulated human mononuclear cells(PTPS-I-MNC-CM),and the proliferation of tumor cells was determined.The cell counting kit-8(CCK-8) was used to determine the proliferation of MNCs.The FQ-RT-PCR was applied to investigate the expression of TNF-? and IL-6 mRNA in MNCs.RESULTS: PTPS-I-MNC-CM inhibited the proliferation of K562,Hep2 and SMMC-7721 cells in vitro(P
ABSTRACT
Objective:To develop and apply a method for real-time quantifying IL-6 mRNA.Methods:By Ficoll-Hypaque density gradient centrifugation, human peripheral blood mononuclear cells(PBMC) were isolated from whole blood treated with ethylenediaminetetraacetic acid(EDTA). Total RNA extracted from the PBMC in trizol solution was reverse transcribed to cDNA, which used as templates for fluorecent real-time quantitative PCR amplification of IL-6. PCR system consists of IL-6 primer, SBY Green Ⅰ and so on.Results:The method is wide linear range, sensitive, specific, reproducible and simple.Conclusion:The method can accurately quantify IL-6 mRNA.