Preparation Of EHV-1 Polyclonal Antibody And Prokaryotic Expression Of Tegument Protein UL16

Equine rhinopneumonitis is a highly infectious disease caused by equine herpesvirus type ?(EHV-1)and equine herpesvirus type IV(EHV-4),causing different degrees of respiratory disease.EHV-1 can even cause abortion,and fatal horse spinal encephalitis,which is a serious threat to the horse breeding industry.The UL16 protein is one of the tegument proteins of EHV-1,encoded by ORF46,which is highly conserved and plays an important role in the replication and maturation of herpesviruses.The main purpose of this experiment is to prepare the polyclonal antibody of EHV-1 against rabbits and to express UL16 protein through a prokaryotic expression system.EHV-1 438/77 strain was purified by sucrose gradient centrifugation and emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant.These were used for the first immunization and booster immunization in rabbits to prepare immune serum,respectively.Western Blot assay showed that the polyclonal antibody in rabbit-derived EHV-1 immune serum had good reactogenicity with the purified viral protein.A pair of specific primers was designed according to the ORF46 gene sequence of EHV-1 in GenBank.Two restriction sites—EcoRI and XhoI were added to the end of primers.A fragment of this gene was obtained by PCR amplification.The ORF46 gene was obtained by PCR amplification and cloned into expression vector pET-32a.The recombinant prokaryotic expression plasmid pET-32a-UL 16 was constructed and transformed into E.coli BL21(DE3),and the positive recombinant bacteria was induced overnight at 16? or 37?,1 mM IPTG.SDS-PAGE assay showed a protein sized about 59 KD was successfully expressed that same as expected size.Western blot analysis indicated that the expressed protein UL16 could be efficiently binded by the prepared EHV-1 rabbit polyclonal antibody.Summary,we successfully prepared the EHV-1 rabbit polyclonal antibody which has a good antigen binding and obtained UL16 protein which has good reactivity.This provides a technical support for the research of the UL16 protein function and development of new diagnostic methods in the laboratory.