ABSTRACT
To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.
Subject(s)
Animals , Bacteriophage T7 , Genetics , Cell Line , DNA-Directed RNA Polymerases , Genetics , Gene Targeting , Promoter Regions, Genetic , Genetics , Viral Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate effects of anti-dsDNA autoantibodies on growth of tumor in vitro and in vivo.</p><p><b>METHODS</b>BALB/c mice were inoculated with inactivated tumor cells and challenged s.c. with SP 2/0 and Wehi 164 tumor cells four weeks after the last inoculation. The naïve mice were inoculated with SP 2/0 tumor cells immediately after incubating with sera derived from the immunized mice at week 6. Then the tumor size was examined. In vitro, the cytotoxicity of anti-dsDNA autoantibodies to tumor cells was analysed. Furthermore, apoptosis of SP 2/0 and Wehi 164 tumor cells induced by anti-dsDNA autoantibodies was examined by FACS.</p><p><b>RESULTS</b>In vivo study showed that the growth of SP 2/0 and Wehi 164 tumors were inhibited in mice with anti-dsDNA autoantibodies, but not in mice lack of anti-dsDNA autoantibodies. In vitro, apoptosis of SP 2/0 and Wehi 164 tumor cells was induced when the tumor cells were incubated with the sera containing anti-dsDNA autoantibodies. Statistical analysis showed that the ability of anti-dsDNA autoantibodies to induce apoptosis of SP 2/0 and Wehi 164 tumor cells was significantly correlated with affinity (r = 0.990, P < 0.01 and r = 0.901, P < 0.05).</p><p><b>CONCLUSION</b>Anti-dsDNA autoantibodies have inhibitory effect on tumor cells via inducing apoptosis.</p>
Subject(s)
Animals , Mice , Antibodies, Neoplasm , Allergy and Immunology , Apoptosis , Autoantibodies , Allergy and Immunology , Cell Line, Tumor , DNA , Allergy and Immunology , Fibrosarcoma , Pathology , Immune Sera , Allergy and Immunology , Mice, Inbred BALB C , Mice, Inbred DBA , Multiple Myeloma , Pathology , Neoplasm TransplantationABSTRACT
T cell anergy has been successfully induced under different conditions in cloned CD4(+) T cells, but induction of T cell anergy in vivo has been difficult and controversial. Due to the low frequency of naturally occurring T cell population with specificity to a defined antigen, it is very difficult to study anergy of naïve T cells without prior in vivo priming which complicates the interpretation of experimental data. To solve this problem, we adopted the HNT-TCR transgenic mice which have homogeneous antigen specific CD4(+) T cell population. In this study, we generated an influenza virus hemagglutinin (HA) peptide-specific CD4(+) T cell clone from the HNT-TCR transgenic mice and induced anergy using APCs which were treated with the crosslinker, ECDI (1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide). The proliferative response of the cloned or freshly purified naïve CD4(+) transgenic T cells after treatment with ECDI-treated APCs and the HA peptide antigen was monitored as the index of anergy induction. The results showed that anergy was successfully induced in the cloned HNT-TCR transgenic CD4(+) T cells. It was determined that the induced anergy was antigen- and MHC-specific. By contrast, anergy was not observed in freshly purified naïve CD4(+) transgenic T cells under the same conditions. The results suggest that naïve CD4(+) T cells may have different anergy inducing requirements, or that cloned CD4(+) T cells may have certain priming or in vitro cloning artifact which makes them more susceptible to anergy induction. We propose that induction of T cell anergy may depend on the T cell growth, activation and differentiation state or cloning conditions. The results from the present study may have important implications for the study of the mechanism(s) underlying T cell anergy induction in vivo and for applications of immune tolerance based therapy.