Study On The Resistance Of IRF-1 Transfected Bovine Fetal Fibroblast Cells To BVDV And IBRV

IRF-1(Interferon regulatory factor-1) a member of the interferon regulatory factor family, can active type 1IFN genes and has been reported in regulating the cells' growth, apoptosis, immunization and other biological activities. Bovine viral diarrhea virus (BVDV) is a single-stranded RNA virus, it mainly cause bovine viral diarrhea and can infect many kinds of even-toed animals. Infectious bovine rhinotracheitis virus (IBRV) is a double-stranded DNA virus, it mainly infect cattle, cause respiratory disorders, conjunctivitis, genital infections and other diseases. Because there isn't effective therapeutic method, so these diseases caused huge economic losses to our country's Stockbreeding, the two illnesses should be inspected to import and exit bovine base on the inspection order.In this study we made bovine fetal fibroblast cells, then transfected the cells with the plasmid pIRES2-EGFP-IRF-1 which was with an IRF-1 gene, and then the transgenic cell line integrated with IRF-1 was obtained. Then we treated the cells and the control with BVDV and IBRV to study the related resistance of IRF-1. There are three parts in our research, the concrete of our experiment are as follows:(1)Transfected cells transiently expressed IRF-1 were prepared. Bovine fetal fibroblast cells were isolated by attaching tissue explants and purified by trypsin. The cells were transfected with FuGENE(?) HD and DNA, and then the transfection efficiency was tested by Flow Cytometry and PCR identified the transfected cells. At last we examined the cells' growth curve, apoptosis, the expression level of IRF-1 in transfected cells and control cells. The results showed that cells were spindle shaped eddy-like growth. Cells cultured in 6-wells culture plates transfected with FuGENE(?) HD:DNA 8:2(μL:μg) resulted the highest transfection efficiency, which was 11.27%; PCR proved the transgenic cells; the growth was "latency phase-logarithmic phase-plateau phase", but the transfected cell grew slowly; higher percentage (P<0.05) of apoptosis cells was found in non-transfected cells and the relative expression of IRF-1 in transfected cells was significantly higher (P<0.05).(2)We evaluated the influence of BVDV on the biological characteristics of transfected cells and control. We prepared BVDV on MDBK, and then treated the above cells with BVDV; further we tested the TCID50, the cells'growth curve and apoptosis at 72h. The results showed significant differences in TCID50 (10-6.4/0.1mLVS10-5.6/0.1mL) and apoptosis rate (P<0.05) between control and transfected cells; the growth curve both declined, whereas the transgenic cells proliferated at early time. These results indicated well antiviral effect of IRF-1.(3)The influence of IBRV on the biological characteristics of transfected cells and the control was evaluated. We prepared IBRV on MDBK, then treated the above cells with IBRV, further we tested the TCID50, growth curve and apoptosis at 72h. The results showed that there was no significant difference in TCID50 and the growth curve between control and transfected cells; whereas very significant difference in apoptosis rate was found(P<0.05).So we guess there may be some antiviral effect of IRF-1 on IBRV.