Eukaryotic Expression And Structural Prediction Of Sialoadhesin V-set Ig-like Domain Derived From Swine And Associated With PRRSV Infection

Porcine reproductive and respiratory syndrome(PRRS)is characterized by reproductive failures in sows and respiratory diseases in pigs of all ages.PRRS virus(PRRSV)is its causative agent and has caused huge economic losses in the swine industry.There are many studies focusing on its infection mechanism,including its host and receptors.Swine are the only known natural host of PRRSV,with the differentiated monocytes,particularly porcine alveolar macrophages(PAMs),being the primary permissive cells.Virus invasion has been further shown to be mediated by the host cell surface receptors.Currently,six putative receptors associated with PRRSV invasion have been identified,including sialoadhesin(Sn),cluster of differentiation 163(CD163)and heparin sulfate(HS).Sn,also referred to CD169 or Siglec-1(sialic acid binding immunoglobulin-type lectin-1),is a macrophage-restricted lectin.Porcine sialoadhesin(p Sn)is a putative receptor of PRRSV.Previous studies have shown that a p Sn V-set Ig-like domain is significant in PRRSV infection.However,its structural details are not fully known.In order to explore the structural information and the specific role of Sn in PRRSV infection,this study firstly cloned,expressed Sn V-set Ig-like domain in Pichia pastoris expression system.However,its low expression level was not helpful for further structural study.Therefore,Sn V-set Ig-like domain was then cloned and expressed in Drosophila S2 cells,and western blotting,mass spectrometry and Ni-sepharose purification were then carried out.Its crystallization and structural prediction were finally performed.The results of the study showed that the sequence of the recombinant protein totally matched the target Sn V-set Ig-like domain.The purity of the biologically active target protein was 99%.This will establish the foundation for the further structural study of p Sn,deepen our understanding of the invasion mechanism of PRRSV and support the structural information for the development of clinical drugs and vaccines against PRRSV.