The Establishment Of The Jurkat Cell Model Of Enterovirus 71 Infection And Its Effect On IFN-γ Secretion

PART ONE THE ESTABLISHMENT OF THE JURKAT CELL MODEL OF ENTEROVIRUS 71 INFECTIONOBJECTIVE To establish a model of human Jurkat cell for enterovirus 71(EV71)in vitro.METHODS The EV71 strain was isolated and preserved by the Guangxi CDC in 2008 from the secretions of HFMD patients.Jurkat cells were incubated with EV71 at an MOI of 5 for 24 h at 37℃.Unbounded virus were removed by washing the cells with medium.Infected cells and culture supernatants were collected for total RNA extraction using Trizol reagent,and Reverse transcription-PCR was performed using a One Step RT-PCR Kit.The PCR products were analyzed in ethidium bromide stained agarose gels.The washed cells were fixed with 4%methanol solution and stained with mouse anti-EV71 monoclonal antibody.After being washed,the cells were incubated with DAPI and Alexa Fluor 488-conjugated goat anti-mouse Ig G antibody and observed under a fluorescent microscope.RESULTS1.EV71 was detected in EV71-infected Jurkat cells by reverse transcription-PCR and agarose gel electrophoresis.2.EV71 antigen in the cytoplasmic region of EV71-infected Jurkat cells was detected by suspension cell immunofluorescence staining with anti-EV71 antibody.3.Virus-mediated cytopathic effect(CPE)was observed in EV71-infected Jurkat cells under an inverted microscope.CONCLUSION The Jurkat cell model of EV71 infection was successfully established.PART TWO THE EFFECT OF ENTEROVIRUS 71 INFECTION ON HUMAN JURKAT CELL SECRETION OF IFN-γOBJECTIVE To investigate the effect of EV71 infection on the secretion of IFN-γ of human Jurkat cell.METHODS Jurkat cells were incubated with EV71 at an MOI of 5 at 37℃.Unbounded virus were removed by washing the cells with medium.Infected cells and culture supernatants were collected at different time intervals.Total RNA was extracted using an isolation Trizol reagent and quantified at 260 nm.The m RNA expression levels of IFN-γ was measured by relative Real-time PCR,which were conducted using the Two-Step SYBR-Green method.One microgram of total RNA was reverse transcribed into c DNA using a Prime Script RT reagent Kit with g DNA Eraser kit.Two microliters of c DNA were combined with a PCR mixture of SYBR Premix Ex Taq Ⅱ Kit in a PCR thermal cycler.A housekeeping gene encoding GAPDH was used as an internal control.The secretion level of IFNgamma was detected by ELISA.Data were expressed as the means±standard deviations(SD).Analyze of variance(ANOVA)was used to compare the significance of the difference among the EV71-infected groups and the control groups.Statistical significance was set at P<0.05.Values were considered significantly different at P<0.01.RESULTS1.The m RNA expression of IFN-γ remained similar in human Jurkat cells after incubation with EV71 for 6h and 24h(P>0.05).Neither of them had significant difference compared with the control group(P>0.05).The m RNA expression of IFN-γ in human Jurkat cells after incubation with EV71 for 48 h and 72 h were both significantly higher than the group incubated for 6h and the control group(P<0.01).There was no difference between the group incubated for 24 h and 48h(P>0.05).The m RNA expression of the group incubated for 72 h was higher than that incubated for 24h(P<0.05),but had no difference compared with the group incubated for 48h(P>0.05).2.There was no significant expression of IFN-γ in both EV71 infected and uninfected Jurkat cells detected by ELISA.There was no difference among the EV71-infected groups and the control groups.CONCLUSION1.EV71 infection could increase the m RNA expression of IFN-γ of human Jurkat cell,which was related to the time of incubation.2.There was no significant expression of IFN-γ in the EV71 infected Jurkat cells detected by ELISA.