ABSTRACT
Objective: The incidence of breast cancer is approximately one million which makes this cancer one of the most common among women worldwide. Breast cancer comprises 7% of the total death rate caused by cancers. Several strategies that use tumor-associated antigen [TAA] vaccination and early detection of breast cancer are clinically being developed. Breast cancer is caused by increased over expression of certain genes. HER-2 is a tyrosine kinase receptor in the epidermal growth factor family. The role of HER-2 in breast cancer has been extensively studied. HER-2 is found in 25%-30% of breast cancer patients. Herceptin, a human antibody, is used as a therapeutic target for HER-2. The purpose of this study is to produce recombinant protein HER-2 for early detection of breast cancer cells
Methods: We used specific primers to amplify the HER-2 gene. The amplified gene was cloned into pET28a as an expression vector. Cloning was confirmed by restriction analysis and sequencing. Expression was induced using IPTG and the recombinant protein was analyzed by SDS-PAGE
Results: Cloning of the HER-2 gene was confirmed by enzyme digestion and sequencing. The gene was expressed in E.coli BL21 DE3. The pET-28a vector which contained the HER-2 gene showed a high level of expression. The recombinant protein was confirmed by Western blot analysis
Conclusions: A portion of the HER-2 gene was expressed as a recombinant in E.coli. This could be a good diagnostic test for breast cancer
ABSTRACT
OBJECTIVE@#To investigate the caspase-1 dependent inflammatory pathway activity and interleukin-1β (IL-1β) secretion in murine macrophage cell lines J774G8 infected with Leishmania major (L. major) using caspase-1 activity assay and ELISA.@*METHODS@#Novy-MacNeal-Nicolle biphasic medium was applied to produce promastigote form of L. major. Metacyclic promastigotes in the stationary phase were applied to infect macrophage. Caspase-1 activity and IL-1β secretion were assessed by the CPP32/caspase-1 fluorometric protease assay and ELISA IL-1β kits, respectively, with time intervals of 6, 18 and 30 h.@*RESULTS@#Our study showed an increase in caspase-1 activity and IL-1β secretion in infected samples compared to non-infected macrophages. The highest increase in IL-1β production was observed after 6 h of infection.@*CONCLUSIONS@#These results arise that the activation of inflammasome pathway could be one of the innate immunity pathways against L. major.
ABSTRACT
Objective: To investigate the caspase-1 dependent inflammatory pathway activity and interleukin-1β (IL-1β) secretion in murine macrophage cell lines J774G8 infected with Leishmania major (L. major) using caspase-1 activity assay and ELISA. Methods: Novy-MacNeal-Nicolle biphasic medium was applied to produce promastigote form of L. major. Metacyclic promastigotes in the stationary phase were applied to infect macrophage. Caspase-1 activity and IL-1β secretion were assessed by the CPP32/caspase-1 fluorometric protease assay and ELISA IL-1β kits, respectively, with time intervals of 6, 18 and 30 h. Results: Our study showed an increase in caspase-1 activity and IL-1β secretion in infected samples compared to non-infected macrophages. The highest increase in IL-1β production was observed after 6 h of infection. Conclusions: These results arise that the activation of inflammasome pathway could be one of the innate immunity pathways against L. major.