Preliminary Identification Of Receptors Of Porcine Hemagglutinating Encephalomyelitis Virus Of S1Protein

The hemagglutinating encephalomyelitis virus (HEV) is the pathogen of Porcinehemagglutinating encephalomyelitis. This is a kind of acute and highly contagious infectiousdisease, which causes vomiting, exhaustion and obvious neurological symptoms of piglets. HEVcould infect--s1-3week-old pigs, the mortality rate as high as20%to100%to those who areinfected. Most countries have reported about this disease since the first outbreak of HEV inCanada in1958. HEV is belong to Coronavirus, There are three different functional proteinsanchored in the surface of the virus envelope: Spike glycoprotein (S) forms the large glycosylatedpeplomers that are characteristic of coronaviruses. M, the transmembrane protein, is highlyhydrophobic and spans the membrane three times, and hemagglutinin-esterase (HE), which formssmaller spikes on virions. Currently, the pathogenic mechanism of HEV is not clear, but the Spikeglycoprotein is relative to the mechanism of virus-infected cells in recently research. Base on thistheory, we use the S1protein, which be expressed by the eukaryotic expression, interacts with themembrane protein of the susceptible cells. Searching for HEV receptors in susceptible cells,by theway of co-immunoprecipitation and mass spectrometry technology.HEV proliferates in the Neuro-2a cells(N2a), that is shown that the N2a membranecontaining the virus receptor. In this study, we built eukaryotic expression vectorpcDNA3.1-HEV-S1at first; and transfected the vector into Hela cells. The expression efficiencyof the recombinant transfected cell lines is28.4%after identification. Secondly, extracted totalmembrane protein of N2a, interacted with S1protein which was expressed by eukaryoticexpression. The hybrid protein precipitated by co-immunoprecipitation method, the proteinprecipitation separated by SDS-PAGE. There was a special strip in the gel which size was65kDafter electrophoresis and this protein was unknown which came from total membrane protein ofN2a. The strip was cut down and analyzed by mass spectrometry. The result of massspectrometry was shown that the unknown protein was pyruvate kinase. According to the resultspyruvate kinase could be the receptor of HEV on N2a cells.