ABSTRACT
A possible ?-Galactosidase gene(pwtsA) was discovered from soil metagenomic DNA of Taishan Mountain.PwtsA gene was inserted into the expression vector pET30a and transferred into E.coli BL21(DE3).Recombinant protein PWTSA was expressed as a soluble form at high level through IPTG induction,with a molecular mass of 57 kD analyzed by SDS-PAGE.PWTSA can produce o-nitrophenol from o-nitrophenol-?-D-galactopyranoside(ONPG),and its specific activity was determined as 13.6 U/mg.The enzymatic studies demonstrated that the recombinant protein PWTSA was a thermostable ?-Galactosidase,its optimum temperature and pH were 85?C～95?C and 6.5 respectively.In standard assays,the Km for ONPG was 0.83 mmol/L.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the induction of antitumor immune responses and therapeutic effects of 10-hydroxycamptothecinc-treated (HCPT) DC-Hepa fusion vaccines by DC fused with hepal-6 cell from hepatoma.</p><p><b>METHODS</b>The fused cells were isolated by magnetic cell sorting and adherent culture. Cell apoptosis was detected by Rhodamine123/PI double-labeled assay, CTL activity by 4 h (51)Cr releasing assay. Protective and therapeutic effects of the fusion vaccine to the tumor-bearing mice was also observed.</p><p><b>RESULTS</b>The apoptosis rate was 29.7%+/-4.1% when DC-Hepa fusion vaccine was treated with 50 microg/ml HCPT for 24 h. After treatment with the HCPT-DC-Hepa fusion vaccine, the tumor grew obviously slowly, survival period of the mice was prolonged, induced more potent CTL cytotoxicity, and resisted against the rechallenge of Hepal-6 cells.</p><p><b>CONCLUSION</b>Vaccination with HCPT-DC-Hepa fusion vaccine could elicit potent antitumor responses, which will provide a new approach to the DC-mediated therapeutic antitumor immunity.</p>
Subject(s)
Animals , Female , Mice , Rats , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Camptothecin , Pharmacology , Cancer Vaccines , Genetics , Allergy and Immunology , Cell Fusion , Cytotoxicity, Immunologic , Dendritic Cells , Allergy and Immunology , Transplantation , Liver Neoplasms , Allergy and Immunology , Pathology , Mice, Inbred C57BL , Neoplasm Transplantation , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Tumor Cells, CulturedABSTRACT
To explore a simple and effective method to determinate the volume of CD34(+) cells in the peripheral blood of donors received drug mobilization for stem cell transplantation by using flow cytometry, the mobilized peripheral blood from donors and 100 micro l fresh whole blood were labeled with monoclonal antibodies Anti-CD34-PE and Anti-CD45-FITC, after lying the red blood cells, and assessed with flow cytometer FL2 (log) vs SSC (log) and FL1 (log) vs SSC (log) were mainly used for analysis windows. The results showed that a level of CD34(+) cells in whole nucleated cells as low as 0.05% - 0.1% can be detected effectively using this method when 10(5) nucleated cells were counted. At day 5 or day 6, the level of CD34(+) cells in most samples of patients reached a peak volume, some of samples and the levels were more than one percent in. It was concluded that CD34(+) cells can be effectively determined by using this method. According to the relative rate of CD34(+) cells, the time to harvest the stem cells in blood can be determined.