Molecular Characteristics Analysis Of Porcine Epidemic Diarrhea Virus Field Strains S Gene In Sichuan And Preliminary Analysis Of The Antigenicity Of S Protein

Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease which is caused by the porcine epidemic diarrhea virus (PEDV). Since 2010, PEDV infection has become a shared concern of global pig industry. It is of great significance to timely grasp the PEDV molecular characteristics of pandemic strain to guide the disease.The S protein of PEDV is a glycoprotein which plays an important role in the induction of the neutralizing antibodies and cell membrane fusion. In order to evade the immune selection pressure of the host, S gene is easy to mutate. Therefore, it is important to carry out the molecular analysis to investigate the genetic diversity of S genes at now. The conservative further genetic analysis is tested for the PEDV strains of the partial and complete S gene popular in Sichuan in 2014 and polyclonal antibodies are prepared by partial S protein which contains 499aa-789aa.This test provides material basis of the antigenic study and the new genetic engineering preparation vaccine. Research results are as follows:(1) Cloning and molecular characteristics of PEDV epidemic strains S gene in Sichuan:102 samples of suspected diarrhea samples (which included the intestinal tissue and fecal samples) of 17 farms in 14 districts in Sichuan Province were detected by RT-PCR. All of 17 farms were PEDV positive and farms positive rate reached 100%.90 samples were diagnosed as PEDV and the positive rate was 88.2%. Mostly PEDV were infection alone. This test designed three pairs of primers according to PEDV CV777 strain complete gene sequence (accession number as AF353511). Part of PEDV epidemic strains in 2014 from different regions of Sichuan were amplificated by RT-PCR, cloned and sequenced.Sequence analysis revealed that S1 fragments of S gene varied greatly, eighteen S1 fragments were 1398bp encoding 466 amino acids and there were a large number of nucleotide mutations, insertions and deletions. Most of the mutations were C→T, T→C, G→T, T→G. Furthermore, eight ninths isolates were a single large ORF of 4,161 nucleotides encoding a protein of 1,386 amino acids (another one was 4,161 nucleotides encoding a protein of 1,374 amino acids). Nine PEDV strains were high with each other both at the deduced amino acid sequence level and nucleotide sequence level. The homology and amino acid sequence homology were higher with 2013-2014 US、Thailand、2014 Korea isolates than early SC-L isolate and CV777 vaccine strains.The S protein had better hydrophilicity, one transmembrance domain and one signal peptide. Phosphorylation sites and N-Glycosylation potential sites had bigger difference with CV777. Codon bias analysis showed that the S gene was biased in some codons, especially those codons encoding amino acids Tip, Met, His, Asp, Cys. It had many flexible regions, α-helices,β-strands and random coils.Phylogenetic analysis suggested that the nine PEDV strains are closely related to each other and belong to the same group.18 Sichuan epidemic strains S1 region had a close relationship with China strains(CH8, YS, CHGD-01, GDMZ/2013), Korea strain CNU-091222-01, American strains(USA/Iowa/18984/2013, USA IA2, USA/ Indiana/17846/2013, USA/Kansas125/2014, USA/Texas128/2014), the latest Korean strains (Korea KUIDL-PED-2014-002 and KUIDL-PED-2014-007), Thailand strains (Thailand PPED0108, Thailand 1-55ST0412, Thailand 6-56ST0413, Thailand SBPED0211-2). The kinship of COE region and whole S gene were similar with S1 region, but COE region has regional differences. It suggested that the strains prevalent in Sichuan are evolving. In addition, all areas of Sichuan PEDV strains were distantly related with CV777, Brl/87, the early Chinese pandemic strains (LZC, SC-L, DX), Korea strains (Chinju99, DR13) and Japanese strain 83p-5.(2) S protein antigenicity preliminary analysis:A pair of primers (SD-F and SD-R) was designed to amplify partial S protein named SD protein containing COE fragment by RT-PCR. The test successfully built two recombinant expression plasmids named pET39b-SC-SD and pET39b-Vaccine-SD.The recombinant plasmids were transformed into the expression host strain E.coil BL21 (DE3) and induced to express by 1.0mmol/L IPTG induction for 4 hours at 37℃. The SDS-PAGE result showed that the target gene fragment was low expression and the molecular weight was about 62kD. The polyclonal antibody was used as coating antigen after the cut plastic purification. Antibody titers of the epidemic strain and vaccine strain were 1:8 and 1:4 which were detected by double agar diffusion method. Western-blotting analysis showed that two recombinant proteins had good immunogenicity.