Identification Of Interaction Protein Of Porcine Circovirus Type 2 ORF3 Protein

Porcine circovirus 2(PCV2)which belongs to the Circovirus family causes post-weaning multisystemic wasting syndrome.The study on the pathogenic mechanism of PCV2 and the interaction between host cell and virus is still unclear.The genome is a single-strand,negative-sense DNA of approximately 1.7 kb that contains three mainly open reading frames,ORF1,ORF2 and ORF3.ORF3 encoded a 105 aa protein that was not essential for viral replication but induced apoptosis.Specific antibodies are important tools for studying protein function.In this study,expression vector pET28a-ORF3 was constructed to obtain the recombinant bacteria.Expressed His-tagged protein was purified with Ni-NTA His Bind Resin under denaturing conditions to immunize New Zealand White Rabbits.Polyclonal antibody was successfully prepared.Antibody detection showed that prepared polyclonal antibody specifically recognized the linear epitope of ORF3 protein,but not the conformational epitope.To explore ORF3 interaction protein,PK-15 cell strain that expressed stably recombinant SFB-tagged ORF3 protein was constructed.33 interaction proteins were identified with the tandem affinity purification coupled mass spectrometry(TAP/MS)technology.These proteins were involved in cytoskeleton,chaperone protein and energy metabolism.The interaction between CCT5 and ORF3 was confirmed by co-immunoprecipitation and subcellular colocalization.In this study,CCT5 was identified as an ORF3 interaction protein by TAP/MS,which laid a foundation for further study of the role of ORF3 protein and its interaction proteins in viral replication and pathogenesis of PCV2.