Analysis Of The Antigenic Difference On GC Protein Between Different Pseudorabies Virus

Pseudorabies(PR)is an infectious disease which made a great damage to pig industry.Since the second half of 2011,the PRV variants is widespread in vaccinated herds in China.Our laboratory isolated a PRV variant strain firstly,named it as HeN1 strain.The study showed that existing vaccine could not provide adequate protection against PRV variants infection.The serum neutralization test implied antigenic differences between vaccine strain Bartha-K61 and HeN1 strain.gC protein is an important neutralizing antigen of PRV.Neutralizing antibody which produced by vaccination could not provide adequate protection against PRV variants infection due to the variation of gC.Therefore,we use yeast surface display technology to obtain the antigen domains of gC protein of Bartha-K61 and HeN1 strain to explore the antigenic differences.To further understand the genetic variation and prevalence of PRV.During 2016 to 2017,we use PCR detection on PRV-like swine tissues which collected from large-scale swine farm of Anhui,Hebei,Shandong province.We identified sixteen PRV positive samples,gC and gE gene were sequenced and analyzed.The amino acids homology of gC and gE gene of the sixteen samples were 98.2%~100% and99.3~100% compared with PRV strains isolated after 2012,respectively.The amino acids homology compared with classic strains were 92.5%~94.8% and 95.3%~99.1%,respectively.The PRV variants all have two insertions of Asp at sites of 48 and 492 in gE,seven amino acids continuous insertions at sites64~70 in gC protein.These were considered to be the molecular marker to identify the PRV variants.Phylogenetic analysis showed that the sixteen PRV isolates were located in the same branch of the evolutionary tree compared with PRV strains which was reported in GenBank recent years and different branch with the classic strains.It's suggested that HeN1 is still the representative isolate of PRV prevalent strains.In this study,the gC gene of Bartha-K61 and He N1 was randomly digested into 100~250 bp DNA fragments in length with DNase I,and the small fragments were inserted into pCTCON2 vector and transformed into DH5? to construct a random yeast display library.The recombinant plasmid of two library was transformed into Saccharomyces cerevisiae EBY-100 strains for culture and induction.Two library expressed on the yeast cells were stained with positive serum against Bartha-K61 and HeN1 strain sorted by FACS for two rounds.The positive plasmid in the sorted yeast clones were extracted and sequenced.Draw the antigen map by analyzing the quantity and distribution of clones.The result shows that the main antigen domain of HeN1-gC located on the A1(23-152 aa),secondary antigen domain located on the A3(337-487 aa),meanwhile A2(153-336 aa)does not detected any visiable antigenic responses.The main antigen domain of Bartha-K61 located on the B1(23-130 aa),secondary antigen domain located on the B2(131-222 aa),B3(223-480 aa)does not detected any visiable antigenic responses.These gC antigenic domains of two isolates were expressed on the yeast surface and E.coli.The results verified the characters of the antigen domains of two isolates.Across analysis based on antigendomains suggested the difference in gC primary antigenic domains of two isolates is little.The antigen characters of two isolates' s secondary antigen domains have differences.A2 and B3 have reaction in across analysis.Our study speculated the difference between gC of two isolates could have a smaller relationships with the amino acid homology and bear on the regulation of the virus itself.In summary,this study has displayed the gC peptides of variant HeN1-gC and vaccine Bartha-K61 on the yeast surface using yeast surface display technology.It is screened the two isolates' major dominants and secondary antigenic domains of gC which are confirmed by FACS and ELISA assays.The work will underlay the foundation for studying the variations between PRV isolates and the mechanism of immune evasion.