ABSTRACT
Objective To investigate the role of a transcription factor p 53 in dengue virus infec-tion.Methods A plasmid expressing siRNA specific for p 53 gene was constructed and then used to prepare HepG2 cell line with a suppressed expression of p 53 protein.The expression of p53 protein was detected by Western blot assay .A wild type control group and a siRNA group were set up by infecting wildtype HepG 2 cells and p53 low expressing HepG2 cells with type 2 dengue viruses,respectively.The virus titers in two dif-ferent cells were determined by plaque forming assay using Vero cells .Indirect immunofluorescence assay was performed to detect virus multiplication .The apoptosis of virus infected cells were analyzed by flow cytome-try.ELISA was performed to analyze the levels of IFN-βsecreted by infected cells from two groups .Results Compared with wildtype control group ,the cells in siRNA group showed a suppressed expression of p 53 pro-tein,suggesting that the HepG2 cell line with low p53 protein expression was successfully established .The vi-rus titer in supernatants of the cells from siRNA group was about 100-fold higher than that of wildtype control group at 24 hours after viral infection .Fluorescence activated cell sorting analysis showed that the numbers of green fluorescence labeled cells were remarkably increased in siRNA group .We speculated that p53 protein might play a role in the inhibition of dengue virus infection as indicated by the observed results .The numbers of apoptotic cells showed no significant difference between two groups .However,the level of IFN-βsecreted by wildtype HepG2 cells was six times higher than that of the cells in siRNA group .Conclusion p53 pro-tein might inhibit dengue virus infection through the activation of type Ⅰ interferon signaling pathway rather than enhance cell apoptosis .
ABSTRACT
Objective To obtain purified myosin-Vc (Myo5c) protein and prepare its polyclonal antibodies of mouse and rabbit. Methods The fragment encoding Myo5c specific protein (297 amino acids) was amplified with RT-PCR from human gastric mucosa. The product and the prokaryotic expression plasmid pQE-31 containing 6?His were used to construct pQE-31/Myo5c. The recombinant was transformed into E. coli JM109 and induced to express with IPTG. The objective product was purified. BALB/c mice and rabbits were immunized with the purified Myo5c protein and the antiserum was obtained. Titer and specificity of the polyclonal antibodies were determined by ELISA and Western blotting. Results pQE-31/Myo5c was successfully constructed. The fusion protein of Myo5c with molecular weight 42 000 was obtained and purified. The recombined protein showed immunogenicity. Mouse or rabbit antiserum with high level of specific antibodies for Myo5c was obtained. Indirect immunostaining and Western blotting analysis demonstrated that the antibodies could recognize native Myo5c protein. Conclusion Our results suggest that the prepared antibodies might be useful in studying the function of Myo5c in intracellular trafficking of endocytic vesicle, such as viruses.
ABSTRACT
Objective To study the roles of dengue (DEN) virus specific T cells in the pathogenesis of DEN virus infection. Methods 2D42 cells, DEN virus specific CD8 + cell clones, were employed to investigate their in vivo function in DEN virus infection using an animal model. HepG2 was implanted into mice with severe combined immunodeficiency disease (HepG2-SCID) for the establishment of HepG2-SCID model. The animals were divided into 3 groups: Group A: HepG2-SCID mice were inoculated with 2D42 cells and then infected with DEN virus type 2 (DEN2) intraperitoneally; Group B: HepG2-SCID mice were inoculated with normal mouse thymuscytes (NMT) and then intraperitoneally infected with DEN2; Group C: HepG2-SCID mice were intraperitoneally infected with DEN2 alone. The mortality, viremia, and frequency of histopathological changes in the major organs of mice in the three groups were observed after infection. Results After inoculation of 2D42 cells, 80% infected mice showed severe clinical signs and died at the average 12.8 d after infection. The others only had transient manifestations, and then recovered from the disease and survived for more than 3 months. In contrast, after inoculation of NMT and /or DEN2 alone, 100% mortality rate was noted in these two groups. High viremia and frequency of histopathological changes in the major organs were observed in the mice in groups A and B. Conclusion Our data support both protective and pathogenic roles for DEN-specific CD8 + T cells in DEN virus infection.