| Porcine reproductive and respiratory syndrome(PRRS)is widely prevalent all over the world,which seriously affects the development of pig industry.At present,there is no specific drug to control porcine reproductive and respiratory syndrome virus(PRRSV).Antibody-dependent enhancement(ADE)of PRRSV infection has the potential to enhance the severity of disease and possibly the susceptibility to PRRSV infection in pigs with low-titer antibodies induced by exposure to wild-type or vaccine strains of PRRSV.In recent years,various types of materials or products from traditional Chinese medicine have been widely studied for producing anti-PRRSV objective,in MA-104 or Marc-145 cells.These two cell lines derived from monkey kidney cells are different from porcine alveolar macrophages(PAM)which are the target cells of PRRSV.In this study,immortalized PAM 3D4/21/CD163 was used for determining the main mechanism of IRPS anti-PRRSV1 Establishment of real-time quantitative PCR detection method for PRRSVIn order to detect the content of PRRSV simply and quickly in the experiment,a real-time fluorescence quantitative PCR detection method of PRRSV was established.According to the PRRSV-N gene,a specific primer of about 221 bp length was designed,and the standard plasmid concentration was 1 ×1010copies/μL.Then the reaction procedure was optimized,including primer concentration,primer volume and annealing denaturation temperature,and the standard curve was drawn.Then different concentrations of standard plasmids were used to evaluate the specificity,sensitivity and repeatability for the detection method.The results show that this method has good specificity,and the lowest plasmid concentration detected by this method is 1 ×10copies/μL,which is about 10 times higher than that of ordinary PCR,indicating that it has good sensitivity.The establishment of the fluorescence detection method can monitor the copy number of PRRSV in the subsequent test cells in real time.2 Study on the effect of Isatidis Radix polysaccharide(IRPS)on PRRSV in vitro.In this study,immortalized porcine alveolar macrophages(3D4/21/CD163)were used as target cells to study the antiviral effect of Isatidis Radix polysaccharide(IRPS)on PRRSV in vitro.In order to study the best administration mode of IRPS and the link of inhibiting virus proliferation,the best action mode and concentration of IRPS inhibiting PRRSV proliferation were determined by preventive effect,direct killing effect and therapeutic effect,respectively;From the influence of IRPS on the adsorption and invasion of PRRSV,the specific links of IRPS inhibiting virus replication are determined.The anti-PRRSV effect of IRPS was verified by Quantitative Real-time PCR and Indirect immunofluo-rescence assay(IFA).The results showed that when the concentration of the IRPS was 0.0625 mg/mL,the direct killing effect and preventive effect on PRRSV were the best,and the copy number of PRRSV in cells was significantly reduced.IRPS mainly acts on the intrusion link of virus replication.3 Effect of Isatidis Radix polysaccharides on Cytokines by PRRSV infected cellsIn this experiment,blank control group,virus control group and treatment group were set up.The cells were treated with the optimal mode and link of action of IRPS screened out in the second experiment.Then fluorescence quantitative PCR and ELISA kit were used to determine the transcription level of cytokines and protein secretion between different treatment groups.The results showed that the transcription level of cytokines had the same trend as the protein expression leveLThe expression level of inflammatory factors in virus control group was significantly higher than that in blank control group(P<0.0001).The expression levels of IL-1β,IL-6,IL-8 and TNF-α in IRPStreatment group were significantly lower than those in virus control group(P<0.0001),indicating that IRPS treatment group could reduce the secretion of inflammatory factors in 3D4/21/CD163 cells infected with PRRSV.The results of IL10 secretion and other inflammatory factors were opposite,and the virus control group was significantly lower than the blank control group(P<0.0001).The expression of IL10 in IRPS treatment group was significantly higher than that in virus control group(P<0.0001)but lower than that in blank control group(P<0.001),which was between them.These results indicated that the high expression of IL-10 could inhibit the secretion of inflammatory factors such as IL-1β,IL-6,IL-8 and TNF-α.The results showed that IRPs had a biaxially immunomodulatory effect on cytokines secreted by porcine alveolar macrophages 3D4/21/CD163 infected with PRRSV4 Effect of Isatidis Radix Polysaccharide on TLR expression by PRRSV infected cellsIn the same way as in experiment 3,the mRNA transcription levels of TLR3,TLR4,TLR7 and TLR9 in cells at different times were detected by fluorescence quantitative PCR.The results showed that the expressions of TLR3,TLR4,TLR7 and TLR9 were changed diferently with time by infected cells.The expression of TLR in PRRSVinfected cells with IPRS treatment could be regulated in bidirection,that is IRPS will be down-regulate,if one is in high expression,and vice versa.Compared with the virus control group,the transcription level of TLR3 and TLR4 was significantly up-regulated in the IRPS treatment group at 24 h(p<0.05).The protein expression results of TLR4 further verified its transcription level Because these two receptors are involved in the expression of IL-10,they may activate the downstream signal pathway and cause the increase of IL-10 secretion.The IL-10 expression level of the IPRS treatment group was also significantly higher than that of the virus control group,but the TLR7 and TLR9 transcription levels were no significant difference in the transcription level between the IPRS treatment group and the virus control group at 24 h.It is suggested that IRPS may promote the secretion of IL-10 and inhibit the secretion of other inflammatory cytokines by up-regulating the transcription of TLR3 and TLR4,thus exerting bidirectional immunomodulatory effect. |
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