Preparation Of Porcine CD169 Monoclonal Antibody And Preliminary Study On The Mechanism Of CD169 In PRRSV Infection

Porcine Reproductive and Respiratory Syndrome（PRRS）is a highly infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus（PRRSV）.The disease has caused huge economic losses to the swine industry all over the world.With the mutation of viral antigens and the continuous discovery of novel PRRSV receptors,the pathogenic mechanism of the disease is becoming more and more complex.Sialoadhesin（CD169）is one of the major cellular receptors of PRRSV and plays an important role in mediating the attachment and internalization of PRRSV into PAMs.However,pigs with gene knockout of CD169 are still susceptible to PRRSV,so there is no unified conclusion on the role of CD169in PRRSV infection.Therefore,further exploration of the mechanism of CD169 in PRRSV infected susceptible cells will help to deepen the understanding of the CD169 receptor and the pathogenesis of PRRSV.In this study,we explored the role of CD169 in PRRSV infection by preparing monoclonal antibodies that recognize CD169 molecule and constructing CD169 stable cell lines.The main work and results of the research are as follows:1.Expression of porcine CD169 truncated protein and preparation of its monoclonal antibodyThe prokaryotic expression system was used to successfully express and purify the CD169 truncated protein CD169-N540,The protein was immunized in BALB/c mice and a monoclonal antibody that could recognize porcine endogenous CD169 molecule was successfully prepared by cell fusion.The monoclonal antibody subtype is Ig G1,it can recognize the CD169 molecule on the surface of PAMs membrane,which can be used for further research.2.Construction of porcine CD169 stable expression cell lines and the role of CD169molecule in PRRSV infection of susceptible cellsThe CRL-2843^CD169 and CRL-2843^CD163/CD169 cell line were successfully constructed by lentiviral packaging system.The CRL-2843^CD163/CD169 cell line is susceptible to PRRSV,and its sensitivity is close to that of PAMs,which can be used for PRRSV in vitro research;CD169is not a decisive receptor for PRRSV infection,but a co-receptor for PRRSV infection.and the introduction of CD169 greatly increases the susceptibility of cells to PRRSV;Blocking CD169 molecules on CD169 positive PRRSV susceptible cells(CRL-2843^CD163/CD169、PAMs)with m Ab-CD169 can inhibit PRRSV infection,indicating that CD169 plays an important role in promoting PRRSV infection;The expression of CD169 molecule had no effect on virus attachment,but promoted the process of endocytosis into host cells.3.Mechanism of porcine CD169 involved in PRRSV invasion of susceptible cells and screening of viral ligandsPRRSV N protein can bind to CD169 on the surface of PAMs membrane;The results of neutralization test showed that the specific antibody against PRRSV N protein could neutralize the virus only when CD169 molecule was present;Double-antibody sandwich ELISA and virus capture experiments as well as density gradient centrifugation on PRRSV can detect secreted nucleocapsids consisting of viral nucleocapsid proteins and viral nucleic acids from PRRSV infected cell supernatants.The nucleocapsid form of virus particles may be closely related to the CD169-mediated PRRSV infection of target cells.In conclusion,this study successfully prepared a monoclonal antibody（m Ab-CD169）that can specifically recognize the porcine endogenous CD169 molecule.CD169 plays an important role in synergistically promoting PRRSV infection;The introduction of CD169 can increase the susceptibility of cells to PRRSV.CD169 promotes the increase of the susceptibility of cells to PRRSV by promoting the process of PRRSV endocytosis into host cells;PRRSV-N protein can bind to CD169,and in the presence of CD169 molecules,specific antibodies against PRRSV-N protein have neutralizing activity.