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The Up-Regulation of CAR and DAF during CVB3 Invading into HeLa Cells

Author: PengYing
Tutor: ZhouYaBin
School: Shandong University
Course: Pathogen Biology
Keywords: Coxsackie virus type B3 Coxsackie virus and adenovirus receptor Decay accelerating factor Acceptor HeLa cells RT-PCR
CLC: R373
Type: Master's thesis
Year: 2009
Downloads: 47
Quote: 1
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Abstract


Purpose of Coxsackie virus (Coxsachievirus, CV) is a virus that is widespread in nature, is a small RNA virus the Branch (Picornaviridas), enterovirus (Enterovirus). 30 known serotypes of Coxsackie virus, according pathogenic characteristics of the virus to suckling mice and cell sensitivity to different virus divided into Group A and Group B, six serotypes B1-B6 B group of viruses can cause the the characteristic infectious Sternocostal pain, merge meningoencephalitis, myocarditis, hepatitis, hemolytic anemia. Coxsackie virus type B3 (CVB3) after it enters the body, the major cause of viral myocarditis (viral myocarditis, VMC), meningitis. CVB3 infection dynamics that CVB3-infected cells spectrum is very wide, and one of the main factors for infection and extent depends on the cell surface coxsackie virus receptor and virus interactions; through the analysis Coxsackie differences virus susceptible cells with non-infected cells, have been discovered Coxsackie virus - adenovirus receptor (CAR), decay accelerating factor (DAF) receptor. CAR almost expressing the surface of all body cells, it is possible with the cell adhesion and proliferation, but the exact biological function is still unclear, the recent discovery CAR as common Coxsackie virus and adenovirus virus receptor before widespread concern by researchers. The DAF also widely expressed in a variety of cells in the body, early detection of DAF can be used as a complement regulatory proteins regulate complement now clear, but it is little studied as a function of a viral receptor. , CVB3 specific pathogenic mechanism is unclear, the binding sites positioning in the the CVB3 receptor binding, dynamic conformation, intracellular signal transduction, cytoskeletal changes and so worthy of further study. HeLa cells in the present experiment using the CAR antibody and DAF antibody intervention detecting CVB3 infection CAR antibody and DAF antibody on cell protective effect of indirectly reflects the size of the role in the invasion of HeLa cells during the CAR and DAF CVB3; and variation of time and concentration of CAR and DAF are infected with the virus were detected RNA and protein levels. Method 1, the protective effect of different concentrations of CAR antibody and DAF antibody in HeLa cells: experiment were divided into five groups: ① V (virus) groups: the virus group join CVB3 interaction with HeLa cells as a positive control; ② D (DAF antibody ) Group: DAF antibody action group, before joining the CVB3 first interaction with DAF antibody with HeLa cells. ③ C (CAR antibody) Group: CAR antibody treated group, before joining the CVB3 first interaction with the the CAR antibody with HeLa cells. ④ C D (CAR antibody and DAF antibody) Group: CAR antibody and DAF antibody interaction group, before joining the CVB3 first CAR antibody and DAF antibody interaction with HeLa cells. ⑤ NOR (normal) group: normal HeLa cells control group with normal HeLa cells as a negative control group. (1) MTT assay of cell activity: 490nm wavelength detection groups cell optical density (OD) of each group calculated the mean cell relative survival rate (RSR) virus and antibody inhibition rate ( IR). (2) plaque counting method detects the influence of the antibody on viral virulence: the counter counts for each group virus plaques, and calculate plaque-forming units of each group. 2, reverse transcription-polymerase chain reaction (RT-PCR) detection CARmRNA the DAFmRNA expression changes in the process of invasion of HeLa cells in CVB3: Total cellular RNA was extracted using Trizol, reverse transcribed cDNA, PCR amplification after agarose gel gel electrophoresis, ethidium electrophoresis results and take photographs, scan. Gel electrophoresis band density computer analysis, amplified bands optical density values ??calculated electrophoretic bands and β-actin as the relative expression intensity. 3, the flow cytometry CAR and DAF expression changes in the process of virus invasion of HeLa cells: CVB3 HeLa cells after interaction, the CAR and DAF fluorescence antibody, expression of both by flow cytometry to detect . Results 1 MTT assay results: cell survival and antibody concentration was positively correlated with the survival rate of the cells in the D group and V group compared with no significant difference between C group and V group compared the difference was statistically significant ( P <0.05), CD group compared with the V group, the difference was statistically significant (P <0.01). The rate of inhibition of virus CD set of the best inhibitory effect on the virus, and the inhibitory effect on C Group D

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