| Breeding for disease resistant can improve the disease resistance, and has a very important role in the healthy development of the pig industry. The study found that the disease resistance on porcine respiratory and reproductive syndrome virus(PRRSV) were significantly different between different pig breeds. Porcine interferon gamma inducible protein 30(IFI30) can constitutively expressed in professional antigen presenting cells and also can be induced expression or expression up-regulation by some inflammatory cytokines in other cells, such as IFN-γ and so on. The main function of IFI30 is to catalyze the reduction of two sulfur bonds. It plays a key role in the process of MHC- II restricted antigen processing. Some studies have indicated that the expression of IFI30 in the body is affected by antiviral immunity, tumor immunity and response status. Preliminary experimental study found that when the pigs infected with different doses of porcine respiratory and reproductive syndrome virus(PRRSV), the expression of IFI30 had changed in porcine pulmonary alveolar macrophages(PAMs). The main purpose of this study was to investigate the effects of porcine IFI30 on the proliferation PRRSV in vitro and the effect on the infection of the host cells.In this study, the length of 741 bp CDS region of IFI30 was amplified from the porcine lung tissue, cloned into the eukaryotic expression p EF1a-IRES-Ds RedExpress2 with red fluorescence to construct the p EF1a-IRES-Ds Red-IFI30 expression vector. And the expression vector p EF1a-IRES-Ds Red-IFI30 was transfected into monkey kidney epithelial cells(Marc145 cells) and porcine testis cell(ST cells), the transfection efficiency was observed by fluorescence microscopy, and the expression vector was verified by q RT-PCR and Western Blot, The experimental results showed that the transfection efficiency of IFI30 expression rate reached more than 80% in Marc145 cells and ST cells, the expression level of m RNA and protein of IFI30 was significantly higher after transfected with experssion vector than that of control group(P< 0.01).Secondly, the p EF1a-IRES-Ds Red-IFI30 expression vector and the empty vector were transfected into Marc145. After transfection 24 h, the transfection efficiency reached 80 ~ 90%, and using1 MOI PRRSV infected Marc145 cells. After 24 hours of infection, the cell supernatant was collected to extract the virus RNA, and then the absolute fluorescence quantitative PCR was performed to detect the change of RNA level. And the virus total protein was extracted, and the expression level of M protein was detected by Western Blot. The apoptosis of Marc145 cells was detected by Annexin V-FITC/PI flow cytometry. Experimental results show that: When expression of porcine IFI30 in Marc145 cells, the copy number of PRRSV in host cells was significantly lower than control group(p<0.01), and the expression level of M in PRRSV was lower than that in the control group(p<0.01). Cell apoptosis rate of IFI30 expression group(20.60%) was significantly decreased compared with empty group(66.0%) and control group(64.66%) by flow cytometry.Finally, The expression of porcine IFI30 gene expression vector, the empty vector and interference vector, interference empty vector four kinds plasmids were transfected into porcine testis cells(ST cells), untreated cells as negative control group. After transfection 24 h, using 1MOI PRRSV infected ST cells, and the cell supernatant was collected in each group after infected 24 hours, the virus RNA was extracted, and the changes of viral RNA were detected by absolute fluorescence quantitative PCR, And the virus total protein was extracted, and the expression level of M protein was detected by Western Blot. The apoptosis of ST cells was detected by Annexin V-FITC/PI flow cytometry. Experimental results show that: Compared with the negative control group, PRRSV copy number and virus M protein expression levels of the empty vector group had no significant change, ST cell apoptosis levels had no significant changes. When transfected into p EF1a-IRES-Ds Red-IFI30 expression vector, the IFI30 gene expression in ST cells was significantly increased, the number of viral copies of PRRSV and M protein decreased. It proved IFI30 could inhibit PRRSV replication. Cell apoptosis rate of IFI30 expression group(9.73%) was significantly decreased compared with empty group(10.38%) by flow cytometry. When inhibiting the expression of IFI30 gene, the number of viral copies of PRRSV rose. The apoptosis rate of IFI30 expression group(25.48%) was significantly increased compared with empty group(11.46%) in ST cells.Conclusion: Porcine IFI30 inhibited PRRSV proliferation in Marc145 cells and ST cells in vitro, and reduced the apoptosis induced by inoculation with PRRSV... |
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