| Porcine reproductive and respiratory syndrome(PRRS)is an immunosuppressive disease caused by porcine reproductive and respiratory syndrome virus(PRRSV)infection,which mainly causes death of piglets,slow growth of fattening pigs,late abortion and stillbirth of sows.PRRSV infection has strict cellular tropism and receptor dependence,and it mainly infects monocyte-macrophage lineage represented by porcine alveolar macrophages(PAMs)in vivo.PRRSV infection makes the expression of many kinds of cytokines imbalance in vivo,and the infection of different virulence PRRSV strains leads to some differences in the expression of cytokines in PAMs.Granulocyte-macrophage colony-stimulating factor(GM-CSF)was initially found to play an important role in the development and differentiation of hematopoietic cells.Recent studies have shown that under the influence of different signal stimulation and immune microenvironment,GM-CSF shows two-way regulation in disease development and inflammatory response:on the one hand,it can recruit inflammatory macrophages into infection or inflammatory local tissue,and further aggravate the inflammatory response;on the other hand,it can also negatively regulate the immune response by recruiting immunomodulatory dendritic cells.Although it has been reported that the fusion expression of pGM-CSF and PRRSV vaccine strain or PRRSV structural protein can enhance the immunoprotective effect of vaccine,there is no report on the role of this cytokine in the process of PRRSV infection.Therefore,in this study,a double antibody sandwich ELISA method for quantitative detection of porcine granulocyte-macrophage colony stimulating factor(pGM-CSF)was established,and then the content of pGM-CSF protein secreted by cells after PAMs infection by different PRRSV strains and the content of pGM-CSF protein in animal serum of PRRSV challenge group and antibody treatment group were determined respectively,and the application of this method was preliminarily established.The work and results of this study are as follows:1. Preparation and activity identification of anti-pGM-CSF monoclonal antibodyThe recombinant expression vector p ET28a-pGM-CSF,was constructed and the recombinant pGM-CSF protein was expressed and purified.The recombinant protein was immunized with BALB/c mice and New Zealand rabbits.After the titer of antibody in mouse serum reached 10~5 or above,cell fusion was carried out.After three rounds of subcloning,the positive clones obtained two monoclonal antibodies stably secreting anti-pGM-CSF,named Mab-1B7E10 and Mab-2A4H11,respectively,which were proved to have good activity.When the titer of rabbit serum reached 10~6 or above,the positive serum was collected.The recombinant pGM-CSF protein coupled with agarose 4B activated by CNBr was used to purify rabbit polyantibodies.After affinity chromatography,the polyclonal antibodies against pGM-CSF with high purity were obtained and proved to have good activity.3. Establishment of a sandwich ELISA method for detection of anti-pGM-CSF double antibodiesUsing Mab-2A4H11 as capture antibody(coating concentration is 10?g/m L)and rabbit polyclonal antibody as detection antibody(concentration is 1.6?g/m L),a double antibody sandwich ELISA method for quantitative detection of pGM-CSF was established.The conditions were as follows:capture antibody was coated overnight at 4?,5%skimmed milk powder was sealed at 37?for 1 h,and the samples to be tested,detected antibody and enzyme-labeled second antibody were incubated at 37?for 1 h respectively.The gradient diluted recombinant pGM-CSF protein was used as the standard to draw the standard curve,and the sensitivity of the detection method was 42.5 pg/m L.The specificity of the detection method was determined by the detection of irrelevant cytokines.4. Application of established double antibody sandwich ELISA detection methodThe double antibody sandwich ELISA detection method established in this study was used to determine the content of pGM-CSF protein secreted by different PRRSV strains infected with PAMs and the content of pGM-CSF protein in the serum of animals in PRRSV attack group and PRRSV challenge+antibody treatment group.After different strains of PRRSV were infected with PAMs,the m RNA level was significantly higher than that of the control group.Then the pGM-CSF protein in the culture supernatant of PAMs cells infected with PRRSV was detected by the above established ELISA detection method and commercial kit,respectively.The results showed that no pGM-CSF protein was detected by the two methods,suggesting that there was post-transcriptional inhibition in the process of PRRSV infection,but the reason is not clear.The detection results of clinical samples showed that pGM-CSF protein could be detected in the animal serum samples of PRRSV challenge+antibody treatment group,and its content was between 1075 and 5025 pg/m L,while pGM-CSF protein was not detected in PRRSV challenge group,suggesting that the secretion of pGM-CSF protein may have the effect of cell repair,but the mechanism needs to be further studied.The double antibody sandwich ELISA method established in this paper can realize the quantitative detection of pGM-CSF protein in the sample.In summary,two mouse monoclonal antibodies against pGM-CSF were successfully prepared,named Mab-1B7E10 and Mab-2A4H11,respectively,and rabbit polyclonal antibodies against pGM-CSF were prepared and purified at the same time.A double antibody sandwich ELISA method for quantitative detection of pGM-CSF was successfully established by using mouse monoclonal antibody as coating antibody and rabbit polyantibody as detection antibody,and the specificity and sensitivity of the method were determined.This method was used to determine the content of pGM-CSF protein secreted by different PRRSV strains infected with PAMs and the content of pGM-CSF protein in serum of animals in PRRSV challenge group and PRRSV challenge+antibody treatment group.It was found that the transcriptional level of pGM-CSF in different strains of PRRSV infected with PAMs increased significantly,and the expression of pGM-CSF protein in the supernatant of cell culture was not detected,which may be the mechanism of inhibition of post-transcriptional expression by PRRSV infected cells.PGM-CSF protein was detected from the animal serum samples of PRRSV challenge+antibody treatment group,and its content was between 1075 and 5025 pg/m L,while it was not detected in PRRSV challenge group,suggesting that the secretion of pGM-CSF may have the effect of cell repair,but the mechanism needs to be further studied. |
PDF Full Text Request