Role of IFNLR1 gene in PRRSV infection of PAM cells

Background@#Interferon lambda receptor 1 (IFNLR1) is a type II cytokine receptor that clings to interleukins IL-28A, IL29B, and IL-29 referred to as type III IFNs (IFN-λs). IFN-λs act through the JAK-STAT signaling pathway to exert antiviral effects related to preventing and curing an infection. Although the immune function of IFN-λs in virus invasion has been described, the molecular mechanism of IFNLR1 in that process is unclear. @*Objectives@#The purpose of this study was to elucidate the role of IFNLR1 in the pathogenesis and treatment of porcine reproductive and respiratory syndrome virus (PRRSV). @*Methods@#The effects of IFNLR1 on the proliferation of porcine alveolar macrophages (PAMs) during PRRSV infection were investigated using interference and overexpression methods. @*Results@#In this study, the expressions of the IFNLR1 gene in the liver, large intestine, small intestine, kidney, and lung tissues of Dapulian pigs were significantly higher than those in Landrace pigs. It was determined that porcine IFNLR1 overexpression suppresses PRRSV replication. The qRT-PCR results revealed that overexpression of IFNLR1 upregulated antiviral and IFN-stimulated genes. IFNLR1 overexpression inhibits the proliferation of PAMs and upregulation of p-STAT1. By contrast, knockdown of IFNLR1 expression promotes PAMs proliferation. The G0/G1 phase proportion in IFNLR1-overexpressing cells increased, and the opposite change was observed in IFNLR1-underexpressing cells. After inhibition of the JAK/STAT signaling pathway, the G2/M phase proportion in the IFNLR1-overexpressing cells showed a significant increasing trend. In conclusion, overexpression of IFNLR1 induces activation of the JAK/STAT pathway, thereby inhibiting the proliferation of PAMs infected with PRRSV. @*Conclusion@#Expression of the IFNLR1 gene has an important regulatory role in PRRSVinfected PAMs, indicating it has potential as a molecular target in developing a new strategy for the treatment of PRRSV.

Role of IFNLR1 gene in PRRSV infection of PAM cells

Background@#Interferon lambda receptor 1 (IFNLR1) is a type II cytokine receptor that clings to interleukins IL-28A, IL29B, and IL-29 referred to as type III IFNs (IFN-λs). IFN-λs act through the JAK-STAT signaling pathway to exert antiviral effects related to preventing and curing an infection. Although the immune function of IFN-λs in virus invasion has been described, the molecular mechanism of IFNLR1 in that process is unclear. @*Objectives@#The purpose of this study was to elucidate the role of IFNLR1 in the pathogenesis and treatment of porcine reproductive and respiratory syndrome virus (PRRSV). @*Methods@#The effects of IFNLR1 on the proliferation of porcine alveolar macrophages (PAMs) during PRRSV infection were investigated using interference and overexpression methods. @*Results@#In this study, the expressions of the IFNLR1 gene in the liver, large intestine, small intestine, kidney, and lung tissues of Dapulian pigs were significantly higher than those in Landrace pigs. It was determined that porcine IFNLR1 overexpression suppresses PRRSV replication. The qRT-PCR results revealed that overexpression of IFNLR1 upregulated antiviral and IFN-stimulated genes. IFNLR1 overexpression inhibits the proliferation of PAMs and upregulation of p-STAT1. By contrast, knockdown of IFNLR1 expression promotes PAMs proliferation. The G0/G1 phase proportion in IFNLR1-overexpressing cells increased, and the opposite change was observed in IFNLR1-underexpressing cells. After inhibition of the JAK/STAT signaling pathway, the G2/M phase proportion in the IFNLR1-overexpressing cells showed a significant increasing trend. In conclusion, overexpression of IFNLR1 induces activation of the JAK/STAT pathway, thereby inhibiting the proliferation of PAMs infected with PRRSV. @*Conclusion@#Expression of the IFNLR1 gene has an important regulatory role in PRRSVinfected PAMs, indicating it has potential as a molecular target in developing a new strategy for the treatment of PRRSV.

Construction of miR-331-3p overexpression vector and its effect on cell proliferation / 生物工程学报

To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.

Cloning and analysis of promoter of pig copper zinc superoxide dismutase gene (CuZnSOD) / 生物工程学报

Pig copper zinc superoxide dismutase (CuZnSOD) is an important antioxidant enzyme. Some studies focused on the function of CuZnSOD gene, but the transcriptional regulation of the CuZnSOD gene is not yet fully elucidated. Therefore, the aims of the study were to determine the core promoter region and to explore its mechanism of transcriptional regulation. The 853 bp DNA sequence of 5'-flanking promoter was amplified by performing PCR. A series of CuZnSOD promoter fragments with gradually truncated 5'-end were produced by nested PCR and inserted into pGL3-Basic vector. The activities of the promoters were measured by the dual-luciferase assay system after transient transfection into the NIH/3T3 cells. The results demonstrated that there were 2 potential transcription start sites in the regions from initiation codon to -87 bp and -266 bp, respectively. The region from -383 bp to +67 bp in CuZnSOD gene promoter showed higher activity than other regions, and further deletion analysis demonstrated that the region from -75 bp to -32 bp contained an essential promoter sequence for pig CuZnSOD gene transcription. In addition, several potential transcription factor binding sites were predicted with bioinformatics method. These results suggest that these transcription factor binding sites may be involved in the transcriptional regulation of CuZnSOD gene.

Generation of transgenic rabbits expressing swine PID1 by testicular injection / 中国实验动物学报

Objective The aim of this study was to investigate the relationship between phosphotyrosine interac-tion domain containing 1 ( PID1 ) gene and variation in intramuscular fat ( IMF ) content and the possibility to generate transgenic animals by testicular injection .Methods Expression vector pIRES2-acGFP-PID1 carrying pig PID1 gene was incubated with transfection reagents and injected into the testes of male New Zealand rabbits .We examined the F1 genera-tion by fluorescence detection , PCR, Western blotting and measuring the IMF content .The F1 generation gave birth to the F2 generation.Then we examined the F2 generation through detecting the positive rate and the IMF content .Results The exogenous PID1 gene and fluorescent protein gene were expressed at different levels both in the F 1 generation and the F2 generation, and the positive rates were 35.88%and 34.33%, respectively.The IMF content was significantly elevated (P<0.05) in the transgenic positive individuals compared with the negative ones and the control group , and the PID1 protein expression was similarly higher .Conclusions The results of this study demonstrate that PID 1 gene affects intramuscular fat content significantly .Moreover, the results of our analysis provide further evidence that transgenic animals can be gener -ated by testicular injection , and the exogenous gene can be inherited steadily .