Effect Of GFP-HCMV IE1 Fusion Protein On Cytokines Secretion And Apoptosis Of Macrophages

Objective: To study the effect of over-expression of GFP-IE1 fusion protein on secretion and apoptosis of THP-1-macrophage , and to provide the experimental basis for the further studies on the pathogenesis of IE1 protein from Human cytomegalovirus (HCMV).Methods:(1) HCMV IE1 gene was excised from pNEB193-IE1 with BamHâ… /Hindâ…¢and subcloned into pCDNA3.1(+). The reconstructed plasmid was confirmed by restriction enzyme and DNA sequencing respectively. Then the pCDNA3.1(+)/IE1 was digested with Salâ… /BamHâ… and IE1 gene product was subcloned into pEGFP-C1 to reconstruct the pEGFP-C1/IE1.(2) The plasmid pEGFP-C1/IE1 or pEGFP-C1 was transfected into THP-1-macrophage respectively. The experiment was divided into three groups, namely, control group, GFP group, GFP-IE1 group. Forty-eight hours after transfection, the expression and localization of GFP or GFP-IE1 was observed under the fluorescent microscope.(3) The level of TNF-αor IL-1βin the cells media was examined by ELISA at 12h, 24h, 48h, respectively. The mRNA expression of them was analyzed by RT-PCR. Cells undergoing apoptosis were determined by flow cytometry using the propidium iodide staining method (PI).(4) All of data were analyzed by SPSS13.0.Results:(1) The size and sequence of IE1 gene in pCDNA3.1(+)/IE1 was identical with that in GenBank (Pubmed NC₀01347), using restriction enzyme analysis and sequencing. Then the eukaryotic expression vector pEGFP-C1/IE1 was successfully constructed as described above. At 48 h after transfection of THP-1-macrophage with pEGFP-C1/IE1 or pEGFP-C1, GFP could be detected in whole cells using fluorescent microscope, Whereas GFP-IE1 fusion protein only located in nuclei. Western blot result showed that the bands with molecular weight of about 101.4KD were consistent with GFP-IE1. (2) Twelve hours after transfection, the level of TNF-αor IL-1βof GFP-IE1 group obviously increased. As time increases, two kinds of cytokines secretion is the rising trend. GFP-IE1 group had an obvious difference compared with the control group or GFP group(P<0.01), but no significant difference existed between GFP group and the control group(P>0.05). TNF-αor IL-1βmRNA expression enhanced in macrophages transfected with pEGFP-C1/IE1.(3) At 48h after transfection, flow cytometry assays demonstrated that GFP had not remarkable difference compared with control group (P>0.05). While there was remarkable difference on the apoptosis rates of macrophages between GFP-IE1 and control group or GFP group (P <0.01).Conclusion:(1) The recombinant eukaryotic expression vector pEGFP-C1/IE1 was successfully constructed, and GFP-IE1 fusion protein could express correctly in THP-1-macrophage. GFP-IE1 fusion protein only located in nuclei.(2) Transient expression of GFP-IE1 fusion protein up-regulated TNF-αand IL-1βsecretion of THP-1-macrophage.(3) Transient expression of GFP-IE1 fusion protein induced the apoptosis of THP-1 -macrophage.